structure and biological function of human 3-alpha hydroxysteroid

I

STRUCTURE AND BIOLOGICAL FUNCTION OF HUMAN 3-ALPHA HYDROXYSTEROID DEHYDROGENASE TYPE

3 IN BREAST CANCER

Thèse

BO ZHANG

Doctorat en Physiologie-Endocrinologie

Philosophiae Doctor (Ph.D.)

QUÉBEC, CANADA

© Bo Zhang, 2014

III

Résumé Court

RÉSUMÉ COURT

La 3-alpha hydroxystéroïde déshydrogénase de type 3 humaine (3α-HSD3) a un rôle

essentiel dans l’inactivation de la 5α-dihydrotestostérone (5α-DHT). Par une combinaison

de la mutagenèse, de la cinétique et de la cristallographie par rayon-X, nous démontrons

que la mutation de la Valine54 à la Leucine54 dans la 3α-HSD3 réduit sa capacité

d’inactivation de la 5α-DHT et améliore une activité 20-alpha hydroxystéroïde

déshydrogénase. Par ailleurs, la structure cristalline de la 3α-HSD3 en complexe avec la

5α-androstane-3,17-dione/épi-androstérone (A-dione/epi-ADT) a été obtenue par la co-

cristallisation, avec la 5α-DHT, ce qui implique que la 5α-DHT a des modes de liaison

alternative avec la 3α-HSD3. La suppression de l’expression de la 3α-HSD3 par des ARNsi

ne fait pas seulement qu’augmenter la concentration de la 5α-DHT dans les cellules MCF7,

mais elle diminue aussi la prolifération cellulaire des cellules MCF7 en présence de 5α-

DHT.

Le récepteur des estrogènes de type alpha (ERα) contrôle le développement sexuel et les

fonctions reproduction. Nous avons mis en place une méthode de purification du fragment

de l’ERα incluant son domaine de liaison à l’ADN et de son domaine de liaison au ligand

(DBD-LBD). La cristallogenèse préliminaire de l’ERα DBD-LBD en complexe avec les

éléments de réponse à l’estrogène a été effectuée. Par ailleurs, nous avons rapporté une

méthode de purification simple et efficace pour le domaine de liaison au ligand de l’ERα.

V

Abstract

ABSTRACT

Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) has an essential role in

the inactivation of androgen 5α-dihydrotestosterone (5α-DHT). By a combination of

mutagenesis, kinetics and X-ray crystallography, we demonstrate that the mutation of

Valine54 to Leucine54 in 3α-HSD3 reduces its 5α-DHT inactivation ability and enhances a

20-alpha hydroxysteroid dehydrogenase activity. Furthermore, the crystal structure of 3α-

HSD3 in complex with 5α-androstane-3,17-dione/epi-androsterone (A-dione/epi-ADT) is

obtained by co-crystallization with 5α-DHT, which implies that 5α-DHT has the alternative

binding mode within 3α-HSD3. Suppression of 3α-HSD3 expression by specific siRNA not

only increases 5α-DHT concentration in MCF7 cells, but also decreases MCF7 cell

proliferation in the presence of 5α-DHT.

Estrogen receptor alpha (ERα) controls sexual development and reproductive functions. We

establish a purification protocol of ERα fragment including its DNA binding domain and

ligand binding domain (DBD-LBD). Preliminary crystallogenesis of ERα DBD-LBD in

complex with estrogen response elements is carried out. Additionally, we report a simple

and efficient purification method for ERα LBD.

VII

Résumé

RÉSUMÉ

L’estrogène 17β-estradiol (E2) stimule la prolifération des cellules du cancer du sein, alors

que la 5α-dihydrotestosterone (5α-DHT), qui est un androgène, prévient la croissance des

cellules du cancer du sein. La 3-alpha hydroxystéroïde déshydrogénase de type 3 humaine

(3α-HSD3) a un rôle essentiel dans le métabolisme des hormones stéroïdiennes,

particulièrement dans l’inactivation de l’androgène le plus actif, soit la 5α-DHT, ce qui fait

que la 3α-HSD3 agit comme un régulateur de pré-récepteur du récepteur des androgènes

(AR) dans le cancer de la prostate. Cependant, le rôle de la 3α-HSD3 dans les cellules

cancéreuses du cancer du sein n’a pas été assez étudié.

En combinant la mutagénèse et l’étude de la cinétique, nous démontrons que la mutation à

un seul point de la Valine54 à la Leucine54 (V54L), dans la 3α-HSD3 réduit

considérablement sa capacité de désactivation de la 5α-DHT et l’enzyme mutante acquiert

une activité de 20-alpha hydrostéroïde déshydrogénase accrue pour inactiver la

progestérone. De plus, nous avons analysé les structures cristallines des complexes 3α-

HSD3·NADP+·progestérone et 3α-HSD3 V54L·NADP

+·progestérone. Fait intéressant, la

progestérone dans l’enzyme sauvage possède deux différents modes de liaison, ce qui

indique que la site de liaison du stéroïde a une flexibilité considérable pour accommoder

différentes orientations du stéroïde. Cependant, la chaine latérale volumineuse de la Leu54

dans l’enzyme mutante restreint significativement le mouvement spatial du stéroïde et donc

permet seulement une orientation de liaison dans le site du substrat.

Par ailleurs, la structure cristalline de la 3α-HSD3 en complexe avec la 5α-androstane-3,17-

dione/épi-androstérone (A-dione/epi-ADT) a été obtenue par co-cristallisation avec la 5α-

DHT en présence de NADP+. Durant le processus de cristallisation, l’oxydoréduction de la

5α-DHT a produit un mélange de stéroïdes. L’A-dione et l’épi-ADT occupent la cavité de

deux monomères dans une unité asymétrique du cristal. Dans la cavité de l’enzyme, les

deux stéroïdes sont orientés l’un envers l’autre et les plans des deux stéroïdes sont

VIII

renversés d’environ 145°, ce qui démontre que la 5α-DHT a différentes façons de liaison

avec la 3α-HSD3 pour générer des produits. De plus, la suppression de l’expression de la

3α-HSD3 par des ARNsi spécifiques augmente la concentration de la 5α-DHT dans les

cellules MCF7 et, en conséquence, la suppression de l’expression de la 3α-HSD3 diminue

la prolifération des cellules MCF7 en présence de la 5α-DHT.

Le récepteur des estrogènes de type alpha (ERα) est un membre bien connu de la

superfamille des Récepteur Nucléaires (NRs). Par la liaison à la 17β-estradiol, l’ERα

contrôle le développement sexuel et les fonctions de reproduction. À cause des difficultés

rencontrées lors de l’expression de la protéine, de la purification et de la cristallisation, la

structure du ERα pleine longueur reste une tâche non tenues. Dans ce travail, nous avons

mis en place un protocole d’expression et de purification du fragment de l’ERα comprenant

son domaine de liaison à l’ADN et son domaine de liaison au ligand (DBD-LBD). La

cristallogenèse préliminaire de l’ERα DBD-LBD en complexe avec les éléments de réponse

à l’estrogène (EREs) a été effectuée, ce qui a généré des connaissances pour faciliter

l’acquisition de fins cristaux du complexe ERα DBD-LBD avec l’EREs. D’ailleurs, nous

avons rapporté une méthode simple et efficace pour la purification du domaine LBD de

l’ERα.

IX

Summary

SUMMARY

Estrogen 17β-estradiol stimulates the proliferation of breast cancer cells, whereas androgen

5α-dihydrotestosterone (5α-DHT) decreases breast cancer cell growth. Human 3-alpha

hydroxysteroid dehydrogenase type 3 (3α-HSD3) has an essential role in the steroid

metabolism, especially in the inactivation of the most potent androgen 5α-DHT, which

makes 3α-HSD3 acting as a pre-receptor regulator of androgen receptor (AR) in prostate

cancer. However, the role of 3α-HSD3 in breast cancer cells is short of investigations.

By combining mutagenesis and kinetic studies, we demonstrate that the mutation of

Valine54 to Leucine54 (V54L) in 3α-HSD3 significantly reduces its 5α-DHT inactivation

ability and the mutant enzyme acquires an enhanced 20-alpha hydroxysteroid

dehydrogenase activity to inactivate progesterone. Moreover, the crystal structures of the

3α-HSD3·NADP+·progesterone complex and the 3α-HSD3 V54L·NADP

+·progesterone

complex are hereby presented. Interestingly, progesterone in the wild type enzyme

possesses two different binding modes, which indicates the steroid binding pocket having

considerable flexibility to accommodate different orientations of a steroid. However, the

bulky side chain of Leu54 in the mutant enzyme significantly confines the spatial

movement of the steroid and thus allows only one binding orientation in the steroid binding

site.

Furthermore, the crystal structure of 3α-HSD3 in complex with 5α-androstane-3,17-

dione/epi-androsterone (A-dione/epi-ADT) is obtained by co-crystallization with 5α-DHT

in the presence of NADP+. During the crystallization process, the oxidoreduction of 5α-

DHT occurred and outputted a mixture of steroid metabolites. A-dione and epi-ADT are

found to occupy the cavities of two monomers in a crystal asymmetric unit, respectively. In

the steroid binding sites, two steroids are oriented upside down to each other and the planes

of two steroids are flipped about 145°, which shows the structural clues of 5α-DHT having

the alternative binding modes within 3α-HSD3 to generate products. In addition,

X

suppression of 3α-HSD3 expression by specific siRNA not only increases 5α-DHT

concentration in MCF7 cells, but also decreases MCF7 cell proliferation in the presence of

5α-DHT.

Estrogen receptor alpha (ERα) is a well-defined member of Nuclear Receptor superfamily

(NRs). Through binding to 17β-estradiol, ERα controls sexual development and

reproductive functions. Due to difficulties in protein expression, purification and

crystallization, the full-length ERα structure remains an unfulfilled task. In this work, we

establish an expression and purification protocol of ERα fragment including its DNA

binding domain and ligand binding domain (DBD-LBD). Preliminary crystallogenesis of

ERα DBD-LBD in complex with estrogen response elements (EREs) is carried out, which

provides basic knowledge to facilitate the acquisition of the fine crystals for ERα DBD-

LBD in complex with EREs. In addition, we report a simple and efficient purification

method for ERα LBD domain.

XI

TABLE OF CONTENTS

RÉSUMÉ COURT ............................................................................................................. III

ABSTRACT………………………………………………………………………………..V

RÉSUMÉ………………………………………………………………………...……….VII

SUMMARY………………………………………………………………………..……...IX

TABLE OF CONTENTS .................................................................................................. XI

LIST OF PUBLICATIONS .......................................................................................... XVII

LIST OF FIGURES ........................................................................................................ XIX

LIST OF TABLES .......................................................................................................... XXI

LIST OF ABBREVIATIONS ..................................................................................... XXIII

FOREWORD .............................................................................................................. XXVII

ACKNOWLEDGEMENTS ........................................................................................ XXIX

CHAPTER 1 INTRODUCTION ......................................................................................... 1

1.1 STEROID HORMONES AND HUMAN STEROIDOGENESIS ............................... 2

1.2 STEROID-RELATED CANCERS ............................................................................... 5

1.3 HUMAN 3ΑLPHA-HSD FAMILY .............................................................................. 7

1.4 HUMAN 3ΑLPHA-HSD3 ............................................................................................ 9

1.4.1 The biological functions of human 3α-HSD3 ..................................................... 9

1.4.2 The crystal structures of human 3α-HSD3 ........................................................ 10

1.4.3 The conserved NADP+ binding site .................................................................. 12

1.4.4 The steroid binding site ..................................................................................... 14

1.4.5 Catalytic mechanism ......................................................................................... 15

1.4.6 Kinetic features of human 3α-HSD3 ................................................................. 15

1.5 HUMAN NUCLEAR RECEPTORS .......................................................................... 16

1.6 HUMAN ESTROGEN RECEPTOR ALPHA ............................................................ 16

1.7 THE TOPICS OF THE RESEARCH PROJECTS ..................................................... 21

XII

CHAPTER 2 STRUCTURE-FUNCTION STUDY OF HUMAN 3-ALPHA

HYDROXYSTEROID DEHYDROGENASE TYPE 3 (3ΑLPHA-HSD3)

V54L MUTATION ..................................................................................... 23

2.0 PREFACE ................................................................................................................... 24

2.1 ABSTRACT ............................................................................................................... 26

2.2 INTRODUCTION ...................................................................................................... 27

2.3 MATERIALS AND METHODS ............................................................................... 28

2.3.1 Materials ........................................................................................................... 28

2.3.2 Site-directed mutagenesis ................................................................................. 29

2.3.3 Protein expression and purification .................................................................. 29

2.3.4 Crystallization ................................................................................................... 29

2.3.5 Data collection and structure determination ..................................................... 30

2.3.6 Kinetic analysis of the wild type and the mutant 3α-HSD3 ............................. 30

2.4 RESULTS ................................................................................................................... 31

2.4.1 Overall structure of the wild type and the mutant 3α-HSD3 ............................ 31

2.4.2 Conserved NADP+ binding ............................................................................... 31

2.4.3 The ternary complex of human 3α-HSD3·NADP+·progesterone ..................... 32

2.4.4 The ternary complex of human 3α-HSD3 V54L·NADP+·progesterone .......... 33

2.4.5 Kinetic study of the wild type and the mutant 3α-HSD3 .................................. 34

2.5 DISCUSSION ............................................................................................................. 34

2.6 FUNDING SOURCES ............................................................................................... 39

2.7 ACKNOWLEDGEMENTS........................................................................................ 39

2.8 REFERENCES ........................................................................................................... 40

CHAPTER 3 STRUCTURE CLUES OF 5ΑLPHA-DHT ALTERNATIVE BINDING

WITHIN HUMAN 3ΑLPHA-HSD3 AND THE ROLE OF THE

ENZYME IN BREAST CANCER ............................................................ 59

3.0 PREFACE ................................................................................................................... 60

3.1 ABSTRACT ............................................................................................................... 62

3.2 INTRODUCTION ...................................................................................................... 63

3.3 MATERIALS AND METHODS ............................................................................... 64

3.3.1 Materials ........................................................................................................... 64

3.3.2 Protein purification and crystallization ............................................................. 65

XIII

3.3.3 Data collection and structure determination ...................................................... 65

3.3.4 The oxidoreduction assay of steroids and the GC/MS analysis ........................ 66

3.3.5 Cell culture ........................................................................................................ 66

3.3.6 siRNA synthesis and transfection ...................................................................... 66

3.3.7 Quantitative real-time RT-PCR ......................................................................... 67

3.3.8 Determination of 5α-DHT levels by Elisa assay ............................................... 67

3.3.9 Cell proliferation assay ...................................................................................... 67

3.4 RESULTS ................................................................................................................... 68

3.4.1 Crystal growth of human 3α -HSD3 in complex with A-dione/epi-ADT and 4-

dione .................................................................................................................. 68

3.4.2 Overall structures of human 3α-HSD3 in complex with A-dione/epi-ADT and

4-dione .............................................................................................................. 69

3.4.3 Human 3α-HSD3 in complex with A-dione/epi-ADT ...................................... 69

3.4.4 Human 3α-HSD3 in complex with 4-dione ....................................................... 70

3.4.5 3α-HSD3 expression and siRNA specific knockdown in MCF7 cells .............. 71

3.4.6 Suppression of 3α-HSD3 expression increases 5α-DHT concentration in MCF7

cells ................................................................................................................... 72

3.4.7 Suppression of 3α-HSD3 expression decreases MCF7 cell proliferation in the

presence of 5α-DHT .......................................................................................... 72

3.5 DISCUSSION ............................................................................................................. 72

3.6 FUNDING SOURCES ................................................................................................ 75

3.7 ACKNOWLEDGEMENTS ........................................................................................ 76

3.8 REFERENCES ............................................................................................................ 77

CHAPTER 4 PURIFICATION AND PRELIMINARY CRYSTALLOGENESIS OF

HUMAN ESTROGEN RECEPTOR ALPHA .......................................... 97

4.0 PREFACE ................................................................................................................... 98

4.1 EXPRESSION, PURIFICATION AND PRELIMINARY CRYSTALLOGENESIS

OF HUMAN ESTROGEN RECEPTOR ALPHA DBD-LBD ................................... 99

4.1.1 Abstract ........................................................................................................... 100

4.1.2 Introduction ..................................................................................................... 101

4.1.3 Methods ........................................................................................................... 102

4.1.3.1 Plasmids construction ......................................................................... 102

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4.1.3.2 Site-directed mutagenesis of ERα C530A .......................................... 102

4.1.3.3 Protein expression and purification .................................................... 102

4.1.3.4 Solubility test ...................................................................................... 103

4.1.3.5 Western blot analysis .......................................................................... 104

4.1.3.6 Preliminary test of crystallization ....................................................... 104

4.1.4 Results and discussion .................................................................................... 104

4.1.4.1 Construction, expression and solubility test of ERα DBD-LBD........ 104

4.1.4.2 Purification of ERα DBD-LBD .......................................................... 105

4.1.4.3 Preliminary crystallogenesis of ERα DBD-LBD ............................... 106

4.1.5 Perspective ...................................................................................................... 107

4.1.6 Acknowledgements ......................................................................................... 107

4.1.7 References ....................................................................................................... 108

4.2 A SIMPLE AND EFFICIENT PURIFICATION METHOD FOR HUMAN

ESTROGEN RECEPTOR ALPHA LBD ................................................................ 117

4.2.1 Abstract ........................................................................................................... 118

4.2.2 Introduction ..................................................................................................... 119

4.2.3 Methods .......................................................................................................... 120

4.2.3.1 Site-directed mutagenesis of ERα LBD C530A ................................. 120

4.2.3.2 Protein expression and purification .................................................... 120

4.2.3.3 Solubility test ...................................................................................... 121

4.2.4 Results and discussion .................................................................................... 121

4.2.4.1 Expression and solubility test of ERα LBD ....................................... 121

4.2.4.2 Purification of ERα LBD .................................................................... 122

4.2.5 Acknowledgements ......................................................................................... 123

4.2.6 References ....................................................................................................... 124

CHAPTER 5 CONCLUSIONS ....................................................................................... 129

5.1 STRUCTURE-FUNCTION STUDY OF HUMAN 3ΑLPHA-HSD3 SINGLE POINT

MUTATION V54L ENHANCING THE 20ΑLPHA-HSD ACTIVITY ................. 131

5.2 STRUCTURAL CLUES OF 5ΑLPHA-DHT ALTERNATIVE BINDING WITHIN

HUMAN 3ΑLPHA-HSD3 AND DOWN-REGULATION OF 3ΑLPHA-HSD3

DECREASING BREAST CANCER CELL GROWTH .......................................... 132

XV

5.3 PURIFICATION AND PRELIMINARY CRYSTALLOGENESIS OF HUMAN

ESTROGEN RECEPTOR ALPHA DBD-LBD ....................................................... 134

5.4 A SIMPLE AND EFFICIENT PURIFICATION METHOD FOR HUMAN

ESTROGEN RECEPTOR ALPHA LBD ................................................................. 135

5.5 FUTURE TOPICS .................................................................................................... 136

CHAPTER 6 REFERENCES (FOR INTRODUCTION) ............................................. 137

XVII

LIST OF PUBLICATIONS

1. Bo Zhang, Dao-Wei Zhu, Xiao-Jian Hu, Ming Zhou, Peng Shang, Sheng-Xiang Lin.

(2014). Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3): the V54L

mutation restricting the steroid alternative binding and enhancing the 20α-HSD

activity (Journal of Steroid Biochemistry and Molecular Biology 141 (2014) 135-

143).

2. Bo Zhang, Xiao-Jian Hu, Xiao-Qiang Wang, Dao-Wei Zhu, Peng Shang, Fernand

Labrie, Sheng-Xiang Lin. (2014). Human 3-alpha hydroxysteroid dehydrogenase

type 3: structural clues of 5α-DHT alternative binding and enzyme down-regulation

decreasing breast cancer cell growth (to be submitted).

3. Bo Zhang, Sheng-Xiang Lin. Expression, purification and preliminary

crystallogenesis of human estrogen receptor alpha DBD-LBD (in preparation).

4. Bo Zhang, Jean-François Thériault, Dao-Wei Zhu, Sheng-Xiang Lin. A simple and

efficient purification method for human estrogen receptor alpha LBD (in

preparation).

XIX

LIST OF FIGURES

Figure 1.1 Steroid core and its numbering system ............................................................... 2

Figure 1.2 Partial representation of the steroidogenesis ...................................................... 5

Figure 1.3 The overall structure of human 3α-HSD3 with cofactors and steroids ............ 10

Figure 1.4 The detailed interactions between 3α-HSD3 and NADP+ ................................ 12

Figure 1.5 The detailed interactions in the steroid binding site of 3α-HSD3 .................... 13

Figure 1.6 Proposed catalytic mechanism of 3α-HSDs ..................................................... 14

Figure 1.7 Structural organization of ERα ......................................................................... 17

Figure 1.8 ERα in complex with ligands and coregulators ................................................ 19

Figure 2.1 Structure of the 3α-HSD3·NADP+·progesterone complex .............................. 49

Figure 2.2 Structure of the 3α-HSD3 V54L·NADP+·progesterone complex .................... 52

Figure 2.3 Presence of the reduced form of the cofactor NADPH within the purified

protein samples of the wild type 3α-HSD3 and the V54L mutant .................. 54

Figure 2.4 Superposition of the steroid binding sites of human 3α-HSD3 related structures

......................................................................................................................... 55

Figure 3.1 The oxidoreduction of 5α-DHT catalyzed by human 3α-HSD3 in the presence

of cofactors ...................................................................................................... 85

Figure 3.2 Structure of the 3α-HSD3·NADP+·A-dione/epi-ADT complex ...................... 86

Figure 3.3 The oxidation of testosterone catalyzed by human 3α-HSD3 in the presence of

NADP+ ............................................................................................................ 89

Figure 3.4 Structure of the 3α-HSD3·NADP+·4-dione complex ....................................... 90

Figure 3.5 Human 3α-HSD3 expression and knockdown by siRNA in MCF7 cells ........ 92

Figure 3.6 Suppression of 3α-HSD3 in MCF7 cells enhanced 5α-DHT-mediated

inhibition of cell growth .................................................................................. 93

Figure 3.7 Superposition of the steroid binding sites of the related 3α-HSD3 structures . 94

XX

Figure 4.1.1 The constructions of Human ERα DBD-LBD recombinant plasmids ........ 112

Figure 4.1.2 The verification of ERα DBD-LBD expression in Rosetta2 (DE3) cells ... 113

Figure 4.1.3 Expression results of ERα DBD-LBD (179-595aa) in Rosetta2 (DE3) cells

...................................................................................................................... 114

Figure 4.1.4 Purification results of ERα DBD-LBD (179-595aa) .................................. 115

Figure 4.1.5 Preliminary crystallogenesis of ERα DBD-LBD (179-554aa) ................... 116

Figure 4.2.1 Expression result of ERα LBD ................................................................... 126

Figure 4.2.2 Solubility test of ERα LBD ......................................................................... 127

Figure 4.2.3 Purification result of ERα LBD .................................................................. 128

XXI

LIST OF TABLES

Table 2.1 Data collection and refinement statistics ........................................................... 46

Table 2.2 Steady-state kinetic parameters for 5α-DHT and progesterone reduction

catalyzed by the wild type (WT) 3α-HSD3 and the Val54Leu (V54L) mutant in

the presence of NADPH ..................................................................................... 48

Table 3.1 Data collection and refinement statistics ........................................................... 83

XXIII

LIST OF ABBREVIATIONS

3α-diol 5α-androstane-3α,17β-diol

3α-HSD 3α-hydroxysteroid dehydrogenase

3α-HSD3 3α-hydroxysteroid dehydrogenase type 3

3(α/β)-HSE 3(α/β)-hydroxysteroid dehydrogenase epimerase

3β-diol 5α-androstane-3β,17β-diol

3β-HSD 3β-hydroxysteroid dehydrogenase/Δ5-Δ

4-isomerase

3D three dimensions

4-dione 4-androstene-3,17-dione

5α-DHT 5α-dihydrotestosterone

5α-Rs 5α-reductase

17-OH-Preg 17-hydroxypregnenolone

17-OH-Prog 17-hydroxyprogesterone

17β-HSD 17β-hydroxysteroid dehydrogenase

20α-HSD 20α-hydroxysteroid dehydrogenase

20α-OHProg 20α-hydroxy-progesterone

Å angstrom

aa amino acids

A-dione 5α-androstane-3,17-dione

ADT androsterone

AF activation function region

AKR aldo-keto reductase

AR androgen receptor

β-DDM dodecyl-β-D-maltoside

β-OG β-octyl glucoside

BPH benign prostatic hyperplasia

BSA bovine serum albumin

C12E8 octaethylene glycol monododecyl ether

CHOL cholesterol

CORT cortisol

CV column volume

XXIV

DBD DNA binding domain

DD4 dihydrodiol dehydrogenase isoform 4

DES diethylstilbestrol

DHEA dehydroepiandrosterone

DHEAS dehydroepiandrosterone sulfate

DHT dihydrotestosterone

DMEM Dulbecco’s Modified Eagle’s Medium

DTT dithiothreitol

E1 estrone

E1S estrone sulfate

E2 17β-estradiol

EDTA ethylenediaminetetraacetic acid

epi-ADT epi-androsterone

ER estrogen receptor

ERα estrogen receptor alpha

ERβ estrogen receptor beta

EREs estrogen response elements

FBS fetal bovine serum

GABAA γ-aminobutyric acid type A

GC/MS gas chromatography/mass spectrometer

GR glucocorticoid receptor

GST glutathione S-transferase

HAT histone acetyltransferase

HDAC histone deacetylase

HPLC high performance liquid chromatography

IPTG isopropyl-β-D-thiogalactopyranoside

Kcat the catalytic rate constant

Km the Michaelis-Menten constant

kDa kilodalton

LB lysogeny broth

LBD ligand binding domain

XXV

M molar

MES 2-(N-morpholino)ethanesulfonic acid

mg milligram

min minute

ml milliliter

mM millimolar

MR molecular replacement

MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

NAD nicotinamide adenine dinucleotide

NADH nicotinamide adenine dinucleotide reduced form

NADP nicotinamide adenine dinucleotide phosphate

NADPH nicotinamide adenine dinucleotide phosphate reduced form

NC negative control

NcoR nuclear receptor corepressor

NDSB non detergent sulfobetaines

ng nanogram

nl nanoliter

NLS nuclear localization sequence

nM nanomolar

nm nanometer

NR nuclear receptor

Nrf2 nuclear factor erythroid-2 related factor 2

OD optical density

OHT 4-hydroxytamoxifen

P450arom aromatase

P450c17 17α-hydroxylase/17,20-lyase

P450scc cholesterol side-chain cleavage

PBS phosphate buffer saline

PCR polymerase chain reaction

PEG polyethylene glycol

pg picogram

XXVI

PMSF phenylmethylsulfonyl fluoride

PPARγ peroxisome proliferator-activated receptor gamma

PREG pregnenolone

PROG progesterone

q-RTPCR quantitative real-time PCR

RMSD root mean square deviations

RXRα retinoid X receptor alpha

SDR short-chain dehydrogenase/reductase

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

sec second

SERM selective estrogen receptor modulator

siRNA small interfering RNA

SMRT silencing mediator for retinoid and thyroid hormone receptor

SRC steroid receptor coactivator

STS steroid sulfatase

SULT sulfotransferase

Testo testosterone

TF transcription factor

TIM triose-phosphate isomerase

TLC thin layer chromatography

TR thyroid receptor

Tris tris(hydroxymethyl)aminomethane

μg microgram

μl microliter

μM micromolar

WT wild type

XXVII

Foreword

FOREWORD

This thesis includes four scientific manuscripts prepared during my graduate study. One

article has been published in the Journal of Steroid Biochemistry and Molecular Biology,

the second will be submitted soon, and the two others are in preparation.

In chapter 1 (Introduction), the general ideas on human steroidogenesis and the related

enzymes, steroid-related cancers, human 3-alpha hydroxysteroid dehydrogenase (3α-HSD)

family and 3α-HSD3, human nuclear receptors and estrogen receptor alpha are briefly

reviewed.

Chapter 2 includes one manuscript on the structure-function study of human 3α-HSD3

V54L mutation: Bo Zhang, Dao-Wei Zhu, Xiao-Jian Hu, Ming Zhou, Peng Shang, Sheng-

Xiang Lin. (2014). Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3): the

V54L mutation restricting the steroid alternative binding and enhancing the 20α-HSD

activity (Journal of Steroid Biochemistry and Molecular Biology 141 (2014) 135-143). In

this paper, I carried out all the experimental studies, solved the crystal structures and wrote

the manuscript. Dr. Sheng-Xiang Lin and I designed the study.

Chapter 3 includes one manuscript on the structure clues of 5α-DHT alternative binding

within human 3α-HSD3 and the role of the enzyme in breast cancer: Bo Zhang, Xiao-Jian

Hu, Xiao-Qiang Wang, Dao-Wei Zhu, Peng Shang, Fernand Labrie, Sheng-Xiang Lin.

(2014). Human 3-alpha hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT

alternative binding and enzyme down-regulation decreasing breast cancer cell growth (to be

submitted). In this paper, I carried out all the experimental studies except for the work

indicated below. I and Dr. Xiao-Jian Hu solved the crystal structure of the 3α-

HSD3·NADP+·A-dione/epi-ADT complex. I solved the crystal structure of the 3α-

HSD3·NADP+·4-dione complex. Mr. Xiang-Qiang Wang and I used the ELISA kit to

detect the 5α-DHT concentration. The q-RTPCR platform and the bioanalytical platform

XXVIII

(Centre Hospitalier Universitaire (CHU) de Quebec Research Center (CHUL) and Laval

University, Québec) offered the services for the q-RTPCR analysis and the GC/MS analysis.

I wrote the manuscript. Dr. Sheng-Xiang Lin and I designed the study.

Chapter 4 includes two manuscripts. (1) The first manuscript is focused on purification and

preliminary crystallogenesis of human ERα DBD-LBD: Bo Zhang, Sheng-Xiang Lin.

Expression, purification and preliminary crystallogenesis of human estrogen receptor alpha

DBD-LBD (in preparation). In this paper, I carried out all the experimental studies and

wrote the manuscript. Dr. Sheng-Xiang Lin and I designed the study. (2) The second

manuscript is focused on an efficient purification method for ERα LBD: Bo Zhang, Jean-

François Thériault, Dao-Wei Zhu, Sheng-Xiang Lin. A simple and efficient purification

method for human estrogen receptor alpha LBD (in preparation). In this paper, I carried out

the experimental studies and wrote the manuscript. Mr. Jean-François Thériault as a

summer student prepared some SDS-PAGE gels. Dr. Sheng-Xiang Lin, Dr. Dao-Wei Zhu

and I designed the study.

Chapter 5 contains the conclusions.

Chapter 6 contains the references for chapter 1.

XXIX

Acknowledgements

ACKNOWLEDGEMENTS

I express the deepest acknowledgement to Dr. Sheng-Xiang Lin, the supervisor of my Ph.D.

study at Laval University. Dr. Lin guided me into the field of the structural biology,

especially into the field of X-ray crystallography. This research field has been my pursuit

for long time. It is my honor to study in Dr. Lin’s laboratory, which has accumulated

experiences in biochemistry and X-ray crystallography for twenty years. Dr. Lin has a

broad vision in basic science and his open mind for discussions often benefits me in many

aspects. His enthusiasm for medical research and conscientious working attitude will

significantly affect me in my future career.

I also express the deepest acknowledgement to Dr. Peng Shang, the supervisor of my Ph.D.

study in China. It was Dr. Peng Shang who offered me the opportunity to transform my

research field from computational biology to experimental biology. In Dr. Peng Shang’s

laboratory, I received the basic training on molecular cloning and protein purification,

which greatly facilitated my subsequent study. His open mind and professional knowledge

affected me continuously. Without his support, I can hardly fulfill my study abroad.

I express my acknowledgement to Dr. Dao-Wei Zhu. He taught me a lot of practical

methods in protein purification and shared his valuable experiences in crystallogenesis.

Without his help, the research project could be more difficult to be carried through.

I also express my acknowledgement to Dr. Ming Zhou. She guided me in enzyme kinetic

studies and instructed me many useful skills in protein purification and crystallogenesis. I

was impressed by her capability to identify the problems and rapidly to solve them.

I thank very much Dr. Pei-min Rong, who taught me the basic skills of Western-blot. After

he retired from the research center, Dr. Rong also gave me a lot of help in daily lives.

XXX

I express my special thanks to Dr. Xiao-Jian Hu. During his visiting in our laboratory, Dr.

Hu gave me basic training in crystal data collection and X-ray crystallography. During the

attempts to obtain the crystal structures, we had many discussions together. He always

patiently answered any questions from me.

I thank very much Dr. Donald Poirier, who not only kindly offered us some chemicals that

we did not have in our laboratory, but also gave the professional answers and suggestions

for a number of questions from me.

I also express my thanks to Dr. Van Luu-The and Dr. Fernand Labrie, who provided us the

radioactive progesterone and the plasmid of human 3-alpha hydroxysteroid dehydrogenase

type 3.

I express my acknowledgements to Dr. Juliette-A. Aka, Dr. Mausumi Mazumdar, Dr.

Chen-Yan Zhang and Dr. Ying Wang. They showed me some basic knowledge in cellular

functional experiments and X-ray crystallography. I can’t forget that we had so many

meaningful discussions on research topics.

I express my thanks to Dr. Preyesh Stephen, who revised the manuscripts of chapters 1, 3

and 4 in this thesis. I express my thanks to Dr. Kathryn Leake, who revised the manuscript

of chapter 4 in this thesis. I also specially thank Mr. Jean-François Thériault for the

translation of the abstract and the summary for this thesis from English to French. Mr.

Thériault and I had numerous discussions on the research. When I met any problems, he

always helped me without hesitation.

I want to express my thanks to Dr. Feng-Fei Huang for her helpful instruction on the

method of co-immunoprecipitation. I also thank Dr. Jing-Bao Li, Dr. Sheng-Meng Di and

Dr. Wei-ning Niu for the basic training to me on molecular cloning and protein purification.

I thank Dr. Jiong Chen and Miss Wei-Qi Wang for ordering the plasmid of human estrogen

receptor alpha and several fragments of siRNA from China and sending them by post to

Canada.

XXXI

I also express my acknowledgements to Mr. Yi Han, Miss Mouna Zerradi, Mr. Xiao-Qiang

Wang, Mr. Le-Yi Lin, Mr. Jian Song, Miss Dan Xu, Miss Hui Han, Dr. Ai-Rong Qian, Mr.

Zhe Wang, Mr. Peng-Fei Yang, Miss Li-Fang Hu, Miss Chong Ding, Mr. Yong-Liang

Wang and Mr. Zhen-Kun Sun. I have received a lot of help and many useful suggestions

from them.

I want to express my acknowledgements to Ms. Nathalie Paquet, Mr. Patrick Bélanger and

Mr. Ronald Maheux for their professional services, who are from the q-RTPCR platform,

the bioanalytical platform and the image-analysis platform (Centre Hospitalier

Universitaire (CHU) de Quebec Research Center (CHUL) and Laval University, Québec),

respectively.

I express my thanks to PROTEO (The Quebec Network for Research on Protein Function,

Structure, and Engineering) and Dr. Albert Berghuis’ laboratory at McGill University,

which offered the X-ray crystallography facilities for our initial X-ray datasets collection. I

also thank LRL-CAT at the Advanced Photon Source, Argonne National Laboratory, and at

the CMCF facility, Canadian Light Source, in which X-ray datasets were collected.

I thank China Scholarship Council (CSC) for supporting my graduate study at Laval

University by providing a national scholarship. I also thank the grant (MOP 97917) to Dr.

Sheng-Xiang Lin and collaborators by CHIR (Canadian Institute of Health Research).

Finally, I express my deepest gratitude for the spiritual support from my parents and my

sisters. Their great love has always given me the courage and the strength to face the

difficulties.

1

CHAPTER 1 INTRODUCTION

2

1.1 STEROID HORMONES AND HUMAN STEROIDOGENESIS

Steroid hormones are originated from the endogenous and exogenous cholesterol and are

sharing a cyclopentanoperhydrophenanthrene nucleus (Figure 1.1), which include sex

steroids (estrogens, androgens and progestins), mineralocorticoids and glucocorticoids 1.

They have diverse physiological functions, especially involving into the regulation of

human reproduction 2; 3

. Cholesterol is characterized by 27 carbon atoms. Steroids have

been classified into several major classes based on their chemical structures, of which

cholestanes possess 27 carbons (such as cholesterol), cholanes possess 24 carbons (such as

cholic acid), pregnanes possess 21 carbons (such as progesterone), androstanes have 19

carbons (such as testosterone) and estranes have 18 carbons (such as estradiol) 4.

Steroid core Standard numbering system

Figure 1.1 Steroid core and its numbering system

Sex steroids can be synthesized in gonadal organs such as testis and ovary, which are

released into the blood circulation and transferred to distant target cells through endocrine

pathway 5. Besides, a large amount of sex steroids are synthesized in peripheral tissues

from their inactive precursors, which are dehydroepiandrosterone (DHEA) and DHEA-

sulfate (DHEA-S) from the adrenal gland 5; 6

. It is noteworthy that, in men, about 50% of

androgens are synthesized from DHEA in peripheral tissues; while in women, about 75%

or nearly 100% estrogens are synthesized before or after menopause in peripheral tissues,

3

respectively 7. Through diffusing into the target cells and binding to the nuclear hormone

receptors, sex steroids initialize the activation or repression of their target gene

transcriptions.

Different classes of enzymes are involved into the steroidogenesis and steroid metabolism 1

(Figure 1.2). In the following paragraphs, several key enzymes related to the

steroidogenesis of sex steroids will be introduced briefly.

Human steroid sulfotransferase (SULT) uses the sulfonate anion from 3’-

phosphoadenosine-5’-phosphosulfate and catalyzes the transformations of steroids to their

inactive sulfated forms 8; 9

. Particularly, estrogen sulfotransferase (gene named SULT1E1)

will sulfonate the 3-hydroxyl group of estrogen 4; 10

. Besides, steroid sulfatase (STS) is

responsible for the conversion of sulfated steroids to the active steroids 11; 12

. Steroid

sulfatase deficiency leads to the X chromosome-linked ichthyosis and the impairment of

placental estrogen synthesis 4; 13

.

The family of human 17β-hydroxysteroid dehydrogenases (17β-HSDs) catalyzes the

reduction of 17-ketosteroids or the oxidation of 17β-hydroxysteroids 10; 14; 15; 16

. To date, 15

different 17β-HSDs have been identified and they belong to the short-chain

dehydrogenase/reductase (SDR) superfamily, except for type 5 17β-HSD, which is a

member of the aldo-keto reductase (AKR) superfamily. These enzymes can be divided into

two groups that one group uses NADPH as the cofactor for reduction (such as types 1, 3, 5

and 7 of 17β-HSD), and the other group uses NAD+ as the cofactor for oxidation (such as

types 2, 4, 6 and 8 of 17β-HSD) 4. These isoenzymes differ in tissue distribution, catalytic

mechanism, substrate specificity and physiological functions 10; 17

.

Human 3β-hydroxysteroid dehydrogenases (3β-HSDs) catalyze both the conversion of

steroidal 3β-hydroxyl group to 3-keto group and the isomerisation of the double bond from

Δ5-steroid to

Δ

4-steroid

18; 19. In human, two 3β-HSDs are identified. 3β-HSD1 is expressed

in placenta and peripheral tissues. 3β-HSD2 is primarily expressed in gonads and adrenal

glands 20

.

5

Figure 1.2 Partial representation of the steroidogenesis

The name and abbreviation of steroids and enzymes are listed in the section of “LIST OF

ABBREVIATIONS” in the thesis. (This figure is from Poirier D, Expert Opin. Ther.

Patents, 2010.)

Human 5α-reductases are membrane-associated enzymes that preferentially catalyze the

transformation of testosterone (Testo) to 5α-DHT, which act as important regulators of

androgen receptor 21

. Two major isoenzymes are found in this family, and type 2 5α-

reductase is predominantly expressed in male genital skin, epididymis and prostate to

generate the most potent androgen 5α-DHT 22; 23

.

Aromatase (P450arom) is the only known enzyme in vertebrates to be responsible for the

biosynthesis of estrogens from androgens, and it catalyzes three sequential reactions of two

C19 steroids (4-dione and Testo) to produce their corresponding C18 steroids (estrone and

estradiol) with a phenolic A-ring 24; 25; 26

.

Human 3-alpha hydroxysteroid dehydrogenase family (3α-HSD) possesses different

degrees of 3-keto-, 17-keto-, and 20-ketosteroid reductase activities 27; 28

. This family

contains four isoenzymes, and belongs to the aldo-keto reductase (AKR) superfamily. In

particular, human 3α-HSD type 3 (3α-HSD3) has an essential role to inactivate the most

potent androgen 5α-dihydrotestosterone (5α-DHT), which behaves as a pre-receptor

regulator of androgen receptor (AR) in prostate cancer 29

. However, androgen 5α-DHT has

been shown to prevent breast cancer cell growth. Very few studies have been conducted to

investigate the role of 3α-HSD3 in breast cancer. Therefore, the structure-function study of

human 3α-HSD3 in breast cancer is the major research topic of this thesis and enzymes in

this family will be introduced in details in the subsequent sections.

1.2 STEROID-RELATED CANCERS

Unlike chemicals or viruses induced cancers, steroid-related cancers involved in breast,

ovary, prostate, testis, thyroid, endometrium and osteosarcoma share a distinct mechanism

6

for carcinogenesis 30; 31

. Steroids have an outstanding role to stimulate the cell division and

proliferation, and thus the random genetic errors are accumulated during these processes,

which will lead to a malignant phenotype 32

. The candidate genes of steroid-related cancers

are involved in the endocrine pathway, tumor suppressor genes and oncogenes. For

examples, human 17β-hydroxysteroid dehydrogenase type 1 gene (HSD17B1) and human

aromatase gene (CYP19) are involved in the endocrine pathway related to breast cancer;

BRCA1 and BRCA2 are responsible for repairing DNA, which are two tumor suppressor

genes and mutations in these two genes significantly increase risks for developing breast

cancer and ovarian cancer 32

.

Breast cancer and prostate cancer are two common steroid-related cancers for women and

men, respectively. According to the statistics of the American Cancer Society

(http://www.cancer.org/), about 12% women in US will develop breast cancer during the

lifetime, and about 232,340 new cases of breast cancer will be diagnosed in women for the

year of 2013 in US. Breast cancer is the second leading cause of death from cancer in

women, after lung cancer. In addition, prostate cancer is the second leading cause of death

from cancer in American men, about 17% men in US will develop prostate cancer during

the lifetime, and about 238,590 new cases of prostate cancer will be diagnosed for the year

of 2013 in US. According to the statistics of the Canadian Cancer Society

(http://www.cancer.ca), breast cancer is the second leading cause of death from cancer in

Canadian women, and it is estimated that 26% of all new cancer cases in women will be

diagnosed with breast cancer for the year of 2013; in addition, prostate cancer is the third

leading cause of death from cancer in Canadian men, and it is estimated that 25% of all new

cancer cases in men will be diagnosed with prostate cancer for the year of 2013.

Sex steroid such as 17β-estradiol has a role to stimulate cell proliferation and promote

breast cancer development 33; 34

. The rapid rate of cell proliferation can initiate mutations

due to the reduced duration of DNA repair, and increase the risk of cancer-causing

mutations 35; 36

. In particular, after menopause of female, estrogens are predominantly

produced from adrenal and ovarian androgens by aromatase in the peripheral tissues such

as skin, fat and muscle 37

. The genetic variations of aromatase are associated with an

increased risk of developing breast cancer 38

. On the other side, sex steroids such as Testo

7

and 5α-DHT play a significant role in normal prostate development and prostate cancer 39

.

The studies of Huggins and Hodges show that prostate cancer is inhibited by androgen

deprivation therapy and activated by androgen injection 40

. Particularly, the development of

normal prostate and benign prostatic hyperplasia (BPH) depends on androgen 5α-DHT. In

prostate, Testo is irreversibly catalyzed to 5α-DHT by type 2 5α-reductase, and the most

potent androgen 5α-DHT binds within androgen receptor to stimulate the cell proliferation

31; 41; 42. The genetic mutations for type 2 5α-reductase and androgen receptor are found to

suppress or block the normal prostate development and are associated with increased risk of

prostate cancer 39; 43; 44; 45

.

1.3 HUMAN 3ΑLPHA-HSD FAMILY

Human 3-alpha hydroxysteroid dehydrogenase family (3α-HSD) includes four isoenzymes

(types 1, 2, 3 and 4), which belongs to the aldo-keto reductase (AKR) superfamily. These

enzymes possess varying degrees of 3-keto-, 17-keto-, and 20-ketosteroid reductase

activities to catalyze the inactivation of androgens, estrogens, progestins, and bile acid

precursors 10; 27; 29; 46

. Human 3α-HSD types 1, 2, 3 and 4 are also named to AKR 1C4, 1C3,

1C2 and 1C1, respectively 47

. The genes of these four enzymes are clustered on

chromosome 10p14-15 and consist of nine exons 28; 48

. The protein sequences of these four

enzymes have more than 81% identity and show differential tissue distributions 27; 28; 49; 50

.

These enzymes adopt a well known triose-phosphate isomerase (TIM) barrel motif or an

(α/β)8-barrel motif and share a conserved cofactor binding site 51; 52; 53; 54

. The substrate

binding site is located at the C-terminal end of the barrel 52

. 3α-HSDs prefer NADPH over

NADH, and NADP is mostly in its reduced form in metabolically active cells 55

.

Human 3α-HSD1 (AKR1C4) is almost exclusively expressed in human liver 27

, which is

also known as hepatic dihydrodiol dehydrogenase isoform 4 (DD4) or chlordecone

reductase 56; 57

. This enzyme converts a keto group to a hydroxy group, which probably has

a significant role in the detoxification of compounds containing a keto group 50

. Besides,

working in concert with 5α/5β-reductases, 3α-HSD1 catalyzes 3-keto steroids to 3α-

hydroxy steroids, which could maintain the homeostasis of steroids and facilitate the final

elimination of steroids by sulfotransferase or glucuronosyl transferase 50; 58

.

8

Human 3α-HSD2 (also named 17β-HSD5 or AKR1C3) is expressed dominantly in prostate

and mammary glands. It plays a major role of Testo biosynthesis from 4-dione, while its

ability of 5α-DHT inactivation to 3α-diol is quite lower 59

. As known, about 50% of Testo

in men is produced by the Leydig cells of the testis, whereas the rest of Testo is produced

from the circulating 4-dione in peripheral tissues, in which 3α-HSD2 is a typical enzyme

involved in this process called intracrinology 5; 7

. In human prostate, both 3α-HSD2 and 3β-

HSD1 are highly expressed in its basal epithelial cells, and androgen receptor (AR) is

highly expressed in its luminal cells. DHEA can be converted into 4-dione by 3β-HSD1,

and then 4-dione can be catalyzed into Testo by 3α-HSD2 in basal epithelial cells. Next,

Testo is transformed into 5α-DHT by type 2 5α-reductase in both basal and luminal cells of

prostate. The most potent androgen 5α-DHT is binding to AR in luminal cells, in which

prostate cancer is developed 5; 60

. In female ovary, 3α-HSD2 is involved in the

transformation of 4-dione to Testo in the theca cells of ovary 61

. Subsequently, 4-dione and

Testo are transferred into the granulosa cells of ovary, in which they can be aromatized into

estrogens 5; 61

. In addition, 3α-HSD2 also shows 20α-HSD activity to inactivate

progesterone, which can protect the theca cells from progesterone that is produced by the

granulosa cells 61

. The expression of 3α-HSD2 can be detected in the epithelial cells of

normal and malignant breast tissue specimens from women. However, breast cancer

specimens possess significant higher expression of 3α-HSD2 than that of normal breast

tissue, and the patients with 3α-HSD2 overexpression show worse prognosis than other

patients 62; 63

. To date, a few crystal structures of human 3α-HSD2 in complex with

different substrates and inhibitors have been reported, which demonstrates the flexibility of

its substrate binding site 64; 65; 66; 67

.

Human 3α-HSD3 (AKR1C2) is one of the major research topics in this thesis, and will be

introduced in more details in the subsequent sections.

Human 3α-HSD4 (also named 20α-HSD or AKR1C1) is found in uterus, ovary and

placenta 27; 68; 69

. This enzyme is mainly responsible for the inactivation of progesterone to

yield 20α-hydroxyprogesterone (20α-OHProg) in the presence of NADPH. By controlling

progesterone levels in vivo, 3α-HSD4 is closely involved in endometrial development and

pregnancy maintenance, as well as neuronal function 70; 71

. Selective loss of 3α-HSD4 is

9

found in breast cancer samples and cell lines (T47D and MCF7), and the suppression of 3α-

HSD4 expression by specific siRNA shows an increased progesterone level in MCF10A

cells and a decreased growth of T47D cells 72

. In addition, the crystal structure of 3α-HSD4

in complex with 20α-OHProg has been reported 54

.

1.4 HUMAN 3ΑLPHA-HSD3

1.4.1 The biological functions of human 3α-HSD3

Human 3α-HSD3 (AKR1C2) is found in liver, brain, prostate, testis and adrenal 27; 50

. It

shares a high protein sequence identity of 97.8% with human 3α-HSD4 (20α-HSD) 73

. Only

seven residues are different between 3α-HSD3 and 3α-HSD4. These two enzymes show

significant difference in specificity of steroidal substrates 74

. In vitro, 3α-HSD3 can oxidize

3α-diol to 5α-DHT in the presence of NAD+. However, it acts as a reductase to degrade 5α-

DHT in prostate cells 75; 76

. As known, 5α-DHT is the most potent androgen 77

. The

inactivation of 5α-DHT by 3α-HSD3 can prevent the activation of androgen receptor 29

. A

decreased expression of 3α-HSD3 in prostate cancer was reported by Qing Ji et al., and

they also reported that 5α-DHT concentrations were remarkably higher in prostate tumor

compared with benign tissue, which could be speculated that the loss of 3α-HSD3 in

prostate cancer cells increased 5α-DHT dependent cell growth 76; 78

. However, another

research group reported that, among four types of human 3α-HSDs, 3α-HSD3 was the

major form that was overexpressed in patients with prostate cancer, and its overexpression

was associated with prostate cancer progression 79

. In breast cancer cells, the expression

levels of 3α-HSD3 and 3α-HSD4 are reduced as compared with normal tissue cells 72; 80

.

Human 3α-HSD3 is also known as the bile acid binding protein and can be inhibited by bile

acids such as ursodeoxycholate 81; 82

. Human 3α-HSD3 exhibits a remarkable activity to

reduce 5β-pregnanes and 5α-pregnanes 71

. In human brain, through the production of

neurosteroid allopregnanolone, 3α-HSD3 also has an ability to regulate the γ-aminobutyric

acid type A (GABAA) receptor, which can lead to anesthetic and anxiolytic effects 48; 83

. In

addition, 3α-HSD3 plays a role in the basal and glucocorticoid-induced 5α-DHT

inactivation in human preadipocytes 84

. In the pathogenesis of female hirsutism, a reduced

10

expression of 3α-HSD3 of hirsute women is found, which can elevate 5α-DHT level in

their tissue 85

.

Figure 1.3 The overall structure of human 3α-HSD3 with cofactors and steroids

The reported structures show that there are two monomers in one crystal asymmetric unit.

Monomer A is colored in magenta. Monomer B is colored in cyan. Atoms are colored as

follows. Green, carbon atoms of steroids (Testo) and cofactors (NADP+); blue, nitrogen;

red, oxygen; orange, phosphorus.

1.4.2 The crystal structures of human 3α-HSD3

There are three crystal structures of human 3α-HSD3 in complex with Testo/acetate,

ursodeoxycholate and citrate/acetate, respectively, are reported 52; 53; 86

. Interestingly, the

11

high resolution structure of the 3α-HSD3·NADP+·Testo/acetate complex was obtained by

co-crystallization with 4-dione. During the crystallization process, the C17 carbonyl group

of 4-dione was firstly reduced by the enzyme to produce Testo, which means that human

3α-HSD3 also possesses certain 17β-HSD activity. It is noteworthy that Testo is released

from the steroid binding site after reduction and reoriented itself with the C3 carbonyl

group pointing inside of the enzyme cavity 52

. Although 3α-HSD3 is a monomer in

solution, there are two enzyme molecules in the crystal asymmetric unit (Figure 1.3).

Moreover, an acetate molecule is located between Testo and the nicotinamide ring of the

cofactor, which is introduced from the crystallization condition. This acetate molecule is

stably binding in the catalytic site and its ketone group forms two hydrogen bonds with the

side chains of Tyr55 and His117, which mimics the C3 carbonyl group of Testo in the rat

3α-HSD3 complex 51

. Obviously, the acetate can remarkably distort the orientation of Testo

in the enzyme cavity. Thus, an adjusted crystallization condition without acetate is used to

obtain a new crystal of 3α-HSD3 in complex with 4-dione. This work is reported in chapter

3 of this thesis.

In the 3α-HSD3·NADP+·ursodeoxycholate complex, the carboxylate group of

ursodeoxycholate is binding toward the catalytic site of the enzyme, which is located at the

similar position of the acetate molecule in the Testo/acetate complex and forms hydrogen

bonds with Tyr55 and His117 53

. An overlay of these two structures shows that although

ursodeoxycholate and Testo are oriented upside down in the cavity, their C18 and C19

methyl group are pointing toward the side chains of Val54 and Tyr55 and their α-faces of

ligands are stacking with the indole ring of Trp227. Meanwhile, the side chains of Val128,

Leu306 and Leu308 in two complexes are flipping about 180°, which demonstrates that the

steroid binding site possesses a considerable flexibility.

To understand the flexibility of the steroid binding site, the 3α-

HSD3·NADP+·citrate/acetate complex was reported

86. The citrate and acetate molecules,

which were introduced from the crystallization step, were found deeply embedded in the

steroid binding pocket. The acetate molecule is located at the same position as it in the

Testo/acetate complex, whereas the volume of its steroid binding pocket is reduced

considerably compared with that of the Testo/acetate complex. In addition, the replacement

12

of two residues Arg301 and Arg304, which are adjacent to the steroid binding pocket,

remarkably influences the 3α-HSD activity of the enzyme. These observations support an

“induced-fit” mechanism that the steroid binding pocket undergoes considerable

conformational changes to create a suitable binding site for its substrates.

Figure 1.4 The detailed interactions between 3α-HSD3 and NADP+

Atoms are colored as follows. Green, cofactor (NADP+) carbon; cyan, protein carbon; blue,

nitrogen; red, oxygen; orange, phosphorus. Hydrogen bonds are shown by black dashed

lines.

1.4.3 The conserved NADP+ binding site

The cofactor is clearly defined in the crystal structures, and is maintained in an extended

conformation across the (α/β)8-barrel (Figure 1.4). Its nicotinamide ring is located at the

center of the barrel, which is stacking with the side chain of Tyr216 and forms the bottom

of the steroid binding pocket. Meanwhile, the side chains of Ser166, Asn167 and Gln190

form hydrogen bonds with the nicotinamide ring of the cofactor. The ribose group of the

13

cofactor also forms hydrogen bonds with Thr23, Tyr24 and Asp50 to stabilize itself. The

pyrophosphate portion is stabilized by a numbers of hydrogen bonds with the back-bone

nitrogen atoms of Ser217, Leu219, Ser221, His222, Lys270 and the side chain of Ser217.

In addition, the 2’-phosphate group of the adenosine moiety is stabilized by salt bridges or

hydrogen bonds with Lys270, Ser271, Tyr272 and Arg276. Human 3α-HSD3 lacks a so-

called “safety belt” to firmly catch the cofactor, which is found in aldose reductase

subfamily and is also absent in rat 3α-HSD3 and human 20α-HSD 51; 54; 87; 88

. Interestingly,

even no cofactor is introduced during the protein purification and crystallization process,

NADP+ can be found in the crystal structures of the enzyme. This suggests that cofactors

coming from the E.coli cells are used in 3α-HSD3 catalysis 52; 86

.

Figure 1.5 The detailed interactions in the steroid binding site of 3α-HSD3

(A) The steroid binding site of the 3α-HSD3·NADP+·Testo complex; (B) The steroid

binding site of the 3α-HSD3·NADP+·ursodeoxycholate (urso) complex. Atoms are colored

as follows. Green, carbon atoms of cofactors and steroids; magenta, protein carbon in

figure 1.5 (A); cyan, protein carbon in figure 1.5 (B); blue, nitrogen; red, oxygen; orange,

phosphorus. Hydrogen bonds are shown by black dashed lines.

14

Figure 1.6 Proposed catalytic mechanism of 3α-HSDs

For the reduction, Tyr55 (Y55) acting as a general acid by donating its proton to the

acceptor carbonyl of the steroid, facilitated by protonation of the tyrosyl hydroxyl group by

the imidazole ring of His117 (H117). For the oxidation, Tyr55 (Y55) acting as a general

base by using its phenolate anion to abstract a proton from steroid alcohol, facilitated by

deprotonation of the tyrosyl hydroxyl group by the ε-amino group of Lys84 (K84). (This

figure is from Schlegel B. P. et al., Biochemistry, 1998.)

1.4.4 The steroid binding site

The steroid binding site of human 3α-HSD3 is formed by loop A (residues 117-143), loop

B (residues 217-238), loop C (residues 299-323), and two short loops (residues 23-32 and

51-57), which also includes the nicotinamide ring of the cofactor that lying at the bottom of

the cavity. In particular, 12 residues mainly located on these loops clearly define the

15

boundary of the cavity, which are Tyr24, Val54, Tyr55, Trp86, His117, Val128, Ile129,

His222, Trp227, Leu306, Leu308 and Phe311 52; 53

(Figure 1.5). In the 3α-

HSD3·NADP+·ursodeoxycholate complex, there is an additional hydrogen bonds between

the 7β-hydroxyl group of ursodeoxycholate and the side chain of Tyr24, which leads to an

increase of the binding affinity for this bile acid 53

.

1.4.5 Catalytic mechanism

The catalytic mechanism of human 3α-HSD3 follows an ordered bi-bi mechanism, in which

the cofactor binds firstly and then the steroid binds to the enzyme, after the reaction, the

steroid product is released from the enzyme before the release of the cofactor 89

. Based on

the rat 3α-HSD structure, a general mechanism is suggested for the oxidoreduction

catalyzed by 3α-HSDs 51; 90; 91

(Figure 1.6). In this mechanism, the C4 atom of the

nicotinamide ring is located neighboring to four residues of Asp50, Tyr55, Lys84 and

His117. These four residues are conserved in 3α-HSDs and form a catalytic tetrad. For the

reduction, a hydride (a proton with two electrons) from the C4 position of the nicotinamide

ring is directly transferred to the carbonyl group of the steroid, in which Tyr55 acts as a

general acid to donate its proton to the carbonyl group of the steroid. For the oxidation,

Tyr55 acts as a general base to extract a proton from the hydroxyl group of the steroid,

which can assist a hydride to transfer back to the cofactor. The acid-base ability of Tyr55 is

fulfilled by forming hydrogen-bond chains with Lys84 and His117 under different pH

conditions.

1.4.6 Kinetic features of human 3α-HSD3

The previous result shows that the C17 carbonyl group of 4-dione can be reduced by human

3α-HSD3 to output Testo during the crystallization process 52

. Thus, further kinetic studies

were performed to compare the 3α-HSD activity and its 17β-HSD activity of human 3α-

HSD3. The reported results show that, for the 3α-reduction of 5α-DHT, the steady-state

kinetic parameters are Km = 1.1 μM, kcat = 1.5 min-1

and kcat/Km = 1.3 min-1

μM-1

. For the

17β-HSD reduction of 4-dione, these parameters are Km = 1.38 μM, kcat = 0.067 min-1

and

kcat/Km = 0.049 min-1

μM-1

52

. The kinetic results clearly show that the 3α-HSD activity is

more favored by the enzyme than its 17β-HSD activity. In addition, a lower kcat/Km value

16

(Km = 2.90 μM, kcat = 1.98 min-1


μM-1

) for the 3α-reduction of 5α-

DHT by the enzyme is reported from the group of Penning 92

. Besides, they also

demonstrate that four types of human 3α-HSDs convert 5α-DHT not only to 3α-diol but

also to 3β-diol, and the ratio of 3α-HSD/3β-HSD activity varies remarkably among human

3α-HSDs. In particular, human 3α-HSD3 predominantly acts as a 3α-HSD, whereas 3α-

HSD4 plays a major role of 3β-HSD 58

.

1.5 HUMAN NUCLEAR RECEPTORS

The nuclear receptors (NRs) are an evolutionarily conserved superfamily of transcription

factors (TFs) that are responsible for binding physiological hormones and other signaling

molecules 93; 94

. Activated by the binding of ligands, nuclear receptors can directly interact

with the promoter or enhancer of specific genes to initialize up-regulation or down-

regulation of transcriptions. As a consequence, nuclear receptors play important roles in

numerous physiological functions such as embryonic development, metabolic homeostasis,

salt balance and reproductive health 95; 96

. Moreover, many genes regulated by nuclear

receptors control key pathways involved in various diseases from cancers to metabolic

diseases. About 13% of U.S. FDA approved drugs use nuclear receptors as their drug

targets 97

.

1.6 HUMAN ESTROGEN RECEPTOR ALPHA

Estrogen receptor (ER) is a well-defined member of NRs superfamily. Through binding to

17β-estradiol (E2), ER controls sexual development and reproductive functions, and also

affects bone, cardiovascular, immune and central nervous systems 98

. In mammals, ERα

and ERβ are the two reported ER subtypes. ERα was firstly cloned from MCF7 human

breast cancer cell in 1980s. Human ERα gene (ESR1) is located on chromosome 6 99; 100

. In

the following ten years, human ERβ gene (ESR2) was cloned out. ESR2 is located on

chromosome 14 101; 102

. ESR1 and ESR2 both have eight exons separated by 7 introns and

other splice isoforms of both ERα and ERβ have deletion of exon3, exon4, or both 103

. ERα

and ERβ share common structural domains. In the presence of 17β-estradiol, ERα

stimulates MCF7 cell proliferation, whereas ERβ has a role to attenuate this effect 104

.

17

Figure 1.7 Structural organization of ERα

(A) ERα contains five structural regions: the N-terminal region, the DNA binding domain

(DBD), the hinge region, the ligand binding domain (LBD) and the C-terminal region. (B)

The 3D model of ERα. (Figure 1.7 (B) is from www.rcsb.org/pdb/101/motm.do?momID=45)

Similar to all the members of NRs, ERα contains five structural regions: the N-terminal

region (A/B region), the DNA binding domain (DBD or C region), the hinge region (D

region), the ligand binding domain (LBD or E region) and the C-terminal region (F region)

(Figure 1.7). The N-terminal region (residues 1-180) contains the activation function region

1 (AF-1), which modulates transcriptional activation independent of the presence of 17β-

estradiol and provides phosphorylation sites or interacts with other regulatory proteins 103

.

To be noticed, this domain is intrinsically disordered and involved in transient protein-

protein interactions, which is a common feature in NRs and certain transcription factors

http://en.wikipedia.org/wiki/N-terminus

18

such as p53 105; 106; 107

. The DNA binding domain (residues 181-263) is highly conserved in

NRs and contains two zinc-finger motifs with each zinc ion chelated with four conserved

cysteines 108; 109

. Two ERα DBDs form a homodimer directly binding to the estrogen

response elements (EREs) in specific promoters or enhancers. Typically, ERα EREs is

composed of two half-sites separated by 3 base pairs to form a palindrome segment such as

5’-AGGTCA-nnn-TGACCT-3’ 110

. The ligand binding domain (residues 303-552) is a

globular domain formed by 12 α-helices and the ligand binding pocket lies in the space

between helix3 and helix11. The ligand recognition is mainly based on a combination of

hydrophobic interactions and specific hydrogen bonding networks. ERα LBD contains a

ligand-dependent activation function region 2 (AF-2), which includes helix12 and is

responsible for the recruitment of coactivator proteins 111; 112; 113

. The hinge region (residues

264-302) between DBD and LBD contains the nuclear localization sequence (NLS) and the

structural information about this region is still not clear 103

. The C-terminal region (residues

553-595) is known to impact ligands binding and regulates LBD interaction with

coregulators 114

.

17β-estradiol is a small hydrophobic ligand, which can diffuse through the cell membrane

and bind to unliganded ERα located in cytoplasm. The liganded ERα dissociates from heat

shock proteins to form the homodimer and then translocates from the cytoplasm into

nucleus 115

. After ERα DBD binding to EREs of specific genes, the liganded ERα LBDs

interact with certain coregulators to up or down regulate genes transcription. The

coregulators including coactivators and corepressors using their Leu-X-X-Leu-Leu

(LXXLL) motif bind to the AF2 region of LBD in the presence of a ligand 116

. A well-

characterized family of coactivators is p160, which consists of three members: steroid

receptor coactivators 1 (SRC-1, also named NCoA1), SRC-2 (also named NCoA2 or GRIP-

1) and SRC-3 (also named AIB1). All three SRCs have the ability to recruit CREB-binding

protein (CBP) and its homolog p300. CBP and p-300 possess histone acetyltransferase

(HAT) activity, which promotes chromatin more accessible to form the RNA Pol II

transcription complex 103; 117; 118

. Corepressors including nuclear receptor corepressor

(NcoR) and silencing mediator for retinoid and thyroid hormone receptor (SMRT)

negatively regulate gene expression. Binding to ERα LBD in the appropriate ligand,

19

corepressors recruit proteins containing histone deacetylase (HDAC) activity, which

repress transcription by maintaining a condensed chromatin 103; 119; 120

.

Figure 1.8 ERα in complex with ligands and coregulators

ERα agonists such as 17β-estradiol cause a conformational change in the LBD, particularly

in H12 (red), which supports the formation of binding surface for the coactivator (SRC-1,

shown in purple). ERα antagonists such as raloxifene induce alternate conformations in the

LBD in which H12 is shifted away from its agonist conformation, which results in a larger

crevice that can accommodate a corepressor (SMRT, shown in blue). (Figure 1.8 is from

Huang P. et al., Annual Review of Physiology, 2010.)

ERα LBDs bind a variety of ligands with significant structural and functional diversity

(Figure 1.8). Some ligands act as agonists, while other ligands act as antagonists. The

agonists bind to LBD similar to the natural ligand 17β-estradiol such as the synthetic ligand

diethylstilbestrol (DES). Agonist binding stabilizes helix12 to form a lid on the ligand

binding pocket and thereby generates a competent AF2 region to recruit coactivator 111; 113

.

In contrast, antagonists such as Raloxifene and 4-hydroxytamoxifen (OHT) have extended

side chains to displace helix12, which prevents helix12 forming a functional AF2 region.

However, unlike coactivators, corepressors can bind to LBDs in the presence of antagonists

94; 121. In addition, SERMs (selective estrogen receptor modulators) act as agonists in

certain tissues, while they act as antagonists in other tissues. The ideal SERM should be an

20

ER antagonist in breast, and an ER agonist in bone, brain or other selected tissues.

Tamoxifen is the first well-characterized SERM and acts as antagonist in breast, but

tamoxifen results in an increase of endometrial cancer 122

. A possible explanation is that

tamoxifene is an antagonist in breast cancer cells for its recruitment of corepressors, while

tamoxifene is an agonist in endometrial cells for its recruitment of coactivators 123

.

Raloxifene is a second-generation SERM. However, it has similar function as tamoxifene

in breast cancer cells but has no negative effects on endometrial cell growth 117

. The

function of SERM suggests that the recruitment of different coregulators determines the

function of ER in different tissues.

Scientist’s interest for the structural studies of NRs has been alive for more than twenty

years. Due to difficulties in protein expression, purification and crystallization of the full-

length NRs, NRs DBDs and LBDs have been studied separately. The reported structures of

NRs DBDs and LBDs show the detailed structural-functional relationship and greatly

promote the rational drug design against these valuable targets 103; 124; 125; 126; 127; 128; 129; 130

.

In particular, the solution and crystal structures of ERα DBD with or without EREs, as well

as the crystal structures of ERα LBD in complex with agonists, antagonists and coactivator

are reported 108; 109; 110; 111; 112; 113

. However, in recent years, a growing number of studies

show that NRs DBDs and LBDs exert their functions as a whole. In 2008, Rastinejad group

reported the first full-length structure of PPARγ-RXRα heterodimer bound to DNA target,

ligand and coactivator peptide, which indicates that PPARγ LBD cooperates with both

heterodimer DBDs to enhance their binding to DNA response elements 131

. Moreover,

thyroid receptor showed higher binding affinity to its DNA response elements than its

isolated DBD alone 132

. Recently, Griffin and coworkers reported that NRs DBD binding to

DNA or its LBD binding to agonist is bi-directionally regulated by DBD and LBD 133

. In

addition, Chambon and coworkers reported that when glucocorticoid receptor (GR) bound

with “negative” DNA response elements, even GR bound with an agonist will still induce

direct transcriptional repression, which implies that DNA response elements is also a player

to regulate gene transcription 134

.

Therefore, the mechanism of bi-directional regulation between NRs DBDs bound with

DNA response elements and LBDs bound with ligands should be investigated further. This

21

is a driving force for us to purify ERα DBD-LBD and try to obtain the crystal of this

important hormone receptor in complex with its EREs.

1.7 THE TOPICS OF THE RESEARCH PROJECTS

The major topics discussed in this thesis are as following:

(1) Human 3α-HSD3 shares 97.8% protein sequence identity with 20α-HSD and only one

amino acid difference (residue 54) is located on their steroid binding pocket. Does residue

54 play a dominant role to dictate the substrate specificity for these two enzymes? How

does the structural difference caused by residue 54 play its role for the steroid

discrimination? By combining the mutagenesis, kinetics and X-ray crystallography study,

we aim to answer these questions.

(2) Androgen 5α-DHT has been shown to prevent breast cancer cells growth and human

3α-HSD3 is characterized by its major ability to inactivate 5α-DHT. So, what is the

concrete interaction between 5α-DHT and 3α-HSD3? Does 3α-HSD3 play an important

role in breast cancer cells as it does in prostate cancer cells? By combining the X-ray

crystallography and the functional studies in the cellular level, we try to give certain

answers.

Besides, two topics related to human ERα are as following:

(1) Recently, a number of studies have showed that nuclear receptor DBDs and LBDs exert

their functions as a whole. However, due to difficulties in protein expression, purification

and crystallization, the full-length ERα structure remains unfulfilled. Here, we want to

establish an expression and purification protocol of ERα DBD-LBD, and try to crystallize

ERα DBD-LBD in complex with estrogen response elements.

(2) Several methods have been developed to purify ERα LBD, such as carboxymethylation

or mutation of reactive cysteine residues. Here, we develop a simple and efficient

purification method of ERα LBD by using a combination of detergents.

23

CHAPTER 2 STRUCTURE-FUNCTION STUDY OF HUMAN 3-

ALPHA HYDROXYSTEROID DEHYDROGENASE TYPE 3 (3ΑLPHA-

HSD3) V54L MUTATION

24

2.0 PREFACE


the inactivation of 5α-dihydrotestosterone (5α-DHT), while human 20-alpha

hydroxysteroid dehydrogenase (20α-HSD) is responsible for the inactivation of

progesterone. However, there is only one amino acid difference (residue 54) between the

steroid binding pockets of 3α-HSD3 and 20α-HSD. Does residue 54 play a dominant role to

dictate the substrate specificity for these two enzymes? How does the structural difference

caused by residue 54 play its role for steroid discrimination? By combining mutagenesis,

kinetics and X-ray crystallography study, we want to find the answers for these questions.

This chapter includes one paper: Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-

HSD3): the V54L mutation restricting the steroid alternative binding and enhancing the

20α-HSD activity. In this paper, I carried out the experimental studies, solved the crystal

structures and wrote the manuscript. Dr. Dao-Wei Zhu guided the protein purification. Dr.

Xiao-Jian Hu checked the solved crystal structures. Dr. Ming Zhou guided enzyme kinetic

studies. Dr. Sheng-Xiang Lin and I designed the study.

25

Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3): the V54L mutation

restricting the steroid alternative binding and enhancing the 20α-HSD activity

Bo Zhang 1, 2

, Dao-Wei Zhu 1, Xiao-Jian Hu

3, Ming Zhou

1, Peng Shang

2, Sheng-Xiang

Lin 1, §

1 Laboratory of Molecular Endocrinology and Oncology, Centre Hospitalier Universitaire

(CHU) de Quebec Research Center (CHUL) and Laval University, Québec City, Québec,

G1V4G2, Canada

2 Key Laboratory for Space Bioscience & Biotechnology, Institute of Special

Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University,

Xi’an, P. R. China, 710072

3 School of Life Sciences, Fudan University, Shanghai, P.R. China, 200433

§ Corresponding Author: S.-X. Lin, CHUL Research Center, 2705 Boul. Laurier, Québec

City, QC, G1V4G2, Canada; Tel.: +1 418 654 2296

E-mail: [email protected]

Shortened title: Structure-function study of human 3α-HSD3 V54L mutation

mailto:[email protected]

26

2.1 ABSTRACT


the inactivation of 5α-dihydrotestosterone (5α-DHT). Notably, human 3α-HSD3 shares

97.8% sequence identity with human 20-alpha hydroxysteroid dehydrogenase (20α-HSD)

and there is only one amino acid difference (residue 54) that is located in their steroid

binding pockets. However, 20α-HSD displays a distinctive ability in transforming

progesterone to 20α-hydroxy-progesterone (20α-OHProg). In this study, to understand the

role of residue 54 in the steroid binding and discrimination, the V54L mutation in human

3α-HSD3 has been created. We have solved two crystal structures of the 3α-

HSD3·NADP+·Progesterone complex and the 3α-HSD3 V54L·NADP

+·progesterone

complex. Interestingly, progesterone adopts two different binding modes to form

complexes within the wild type enzyme, with one binding mode similar to the orientation

of a bile acid (ursodeoxycholate) in the reported ternary complex of human 3α-

HSD3·NADP+·ursodeoxycholate and the other binding mode resembling the orientation of

20α-OHProg in the ternary complex of human 20α-HSD·NADP+·20α-OHProg. However,

the V54L mutation directly restricts the steroid binding modes to a unique one, which

resembles the orientation of 20α-OHProg within human 20α-HSD. Furthermore, the kinetic

study has been carried out. The results show that the V54L mutation significantly decreases

the 3α-HSD activity for the reduction of 5α-DHT, while this mutation enhances the 20α-

HSD activity to convert progesterone.

Keywords: 3-alpha hydroxysteroid dehydrogenase, AKR1C2, 20α-HSD, mutation, kinetics,

crystal structure

Protein data bank accession codes: 4L1W and 4L1X

27

2.2 INTRODUCTION

Human 3-alpha hydroxysteroid dehydrogenases (3α-HSDs) belong to the aldo-keto

reductase (AKR) superfamily and possess 3-keto-, 17-keto-, and 20-ketosteroid reductase

activities to catalyze the inactivation of androgens, estrogens, progestins, and bile acid

precursors 1; 2; 3

. In human, four isoenzymes of 3α-HSDs share at least 81% sequence

identity and show differential tissue distributions 4; 5; 6

. These enzymes are soluble

monomeric proteins and they use NADPH as their cofactor 5. According to the

nomenclature of AKR family, human 3α-HSD types 1, 2, 3 and 4 are named AKR 1C4,

1C3, 1C2 and 1C1, respectively. Human 3α-HSDs have no significant sequence similarity

with the short-chain dehydrogenase/reductase superfamily (SDR) such as 17β-HSDs and

11β-HSD 7; 8; 9; 10

.

Human 3α-HSD3 plays a major role in the reduction of 5α-DHT to 5α-androstane-3α,17β-

diol (3α-diol), which prevents the binding and activation of androgen receptor from

overflowing androgen 6; 11

. As known, 5α-DHT is the most potent androgen and excess of

5α-DHT has been associated with the development of benign prostatic hyperplasia and

prostate cancer in human 12

. Human 3α-HSD4 also named 20α-HSD (hereafter 20α-HSD is

used to represent this enzyme) transforms progesterone into 20α-OHProg in ovary, uterus

and placenta 5; 13; 14

. Human 20α-HSD shares 97.8% sequence identity with 3α-HSD3 and

there are only seven amino acids differences between two enzymes 15

. Human 3α-HSD2

(also recognized as 17β-HSD5) exhibits much higher ability in reducing 4-androstene-3,17-

dione (4-dione) to testosterone (Testo) than its ability in transforming 5α-DHT to 3α-diol 16

.

In mammary gland, 3α-HSD2 can also catalyze estrone to 17β-estradiol and progesterone

to 20α-OHProg 5. Human 3α-HSD2 and 3α-HSD3 are found to be highly expressed in

human prostate 5; 17

. Human 3α-HSD1 is a liver specific 3α-HSD, which plays a role of

inactivation 3-keto steroids in liver 5; 6

.

The reported structure of 3α-HSD3·NADP+·Testo/acetate complex shows that the enzyme

adopts an (α/β)8-barrel motif. The steroid binding pocket, which is mainly formed by loop

A, B and C, locates at the C-terminal end of the barrel 18

. Besides, the 3α-

HSD3·NADP+·ursodeoxycholate complex and the 3α-HSD3·NADP

+·citrate·acetate

28

complex reveal that the steroid binding pocket of the enzyme possesses remarkable

flexibility to bind different substrates 19; 20

. A general catalytic mechanism shows that a

hydride is directly transferred from the C4 position of the cofactor to the carbonyl group of

substrate and the enzyme acts as the proton donor simultaneously 21

.

Particularly, there is only one amino acid difference (residue 54) between the steroid

binding pockets of human 3α-HSD3 and 20α-HSD. Based on the kinetic study of certain

steroids or substrates in human liver, the earlier research showed that the mutation of

human 20α-HSD Leu54-to-Val54 produced an enzyme that possessing the similar

characteristic as 3α-HSD3 22

. However, the detailed spatial change caused by residue 54 in

the steroid binding pockets of both enzymes awaits a comparative structural study to

acquire better comprehension on the structure-function relationship. In addition, a

comparative kinetic study for the reduction of 5α-DHT and progesterone by human 3α-

HSD or 20α-HSD, and the corresponding residue 54 mutant has not been completely

investigated.

Here, we report the crystal structures of the 3α-HSD3·NADP+·progesterone complex and

the 3α-HSD3 V54L·NADP+·progesterone complex. It can be found that progesterone in the

wild type enzyme possesses two different binding modes. However, only one binding mode

of the steroid can be observed in the mutant enzyme. Furthermore, the kinetic study shows

that the V54L mutation in human 3α-HSD3 remarkably reduces the 5α-DHT degradation

ability of the enzyme and enhances the 20α-HSD activity to reduce progesterone.

2.3 MATERIALS AND METHODS

2.3.1 Materials

DTT and β-octyl glucoside (β-OG) were purchased from Gold Biotechnology. Thrombin

was purchased from MP Biomedicals. Progesterone and 5α-DHT were purchased from

Steraloids. Diethyl ether, toluene, acetone and dichloromethane were purchased from

Fisher Scientific. Thin layer chromatography (TLC) plate was purchased from EMD

Millipore. NADPH, NADP+ and other chemicals were purchased from Sigma-Aldrich.

14C-

29

Progesterone (55 mCi/mmol) was purchased from American Radiolabeled Chemicals and

14C-5α-DHT (53.5 mCi/mmol) was purchased from PerkinElmer Life Sciences.

2.3.2 Site-directed mutagenesis

The pGEX vector encoding the wild type human 3α-HSD3 was used as a template for

conducting human 3α-HSD3 V54L site-directed mutagenesis. The mutation was performed

using QuikChange Lightning Kit (Agilent Technologies) with 5’-

CCATATTGATTCTGCACATTTATACAATAATGAGGAGC-3’ as forward primer and

verified by sequencing.

2.3.3 Protein expression and purification

The recombinant wild type human 3α-HSD3 with a glutathione S-transferase (GST) tag

was overexpressed in E. coli BL21(DE3)pLysS cells and purified by following a previously

described procedure 23

. In brief, the enzyme was firstly purified with a glutathione agarose

column in PBS buffer (pH 7.4) and the GST fusion tag was cut by thrombin overnight at

4°C. Another step of Q-sepharose column purification with a salt gradient using buffer A

(25 mM Tris-HCl (pH 7.4), 0.5 mM DTT, 0.06% β-octyl glucoside (β-OG), 1 mM EDTA,

10% Glycerol) and buffer B (buffer A with 500 mM NaCl) yields high purity enzyme. The

fractions of 3α-HSD3 were concentrated to 28-30 mg/ml in a 10 mM KH2PO4-K2HPO4

buffer (pH 7.0) containing 0.5 mM DTT, 0.06% β-OG, 1 mM EDTA, 1 mM NADP+ and

40 μM progesterone. The recombinant human 3α-HSD3 V54L was overexpressed and

purified as the wild type enzyme. Protein purity was checked by SDS-PAGE and protein

concentration was determined by Bradford assay. The concentrated 3α-

HSD3·NADP+·Progesterone or 3α-HSD3 V54L·NADP

+·progesterone was preserved at -

80°C for later crystallization.

2.3.4 Crystallization

Crystals were obtained by vapor diffusion method using both hanging drops and sitting

drops at room temperature. For both complex crystals, the reservoir solution contained 100

mM sodium cacodylate buffer (pH 6.0), 200 mM ammonium sulfate, 24-26% (w/v)

polyethylene glycol (PEG) 3350. Prior to crystallization, 40 mM progesterone stock in

30

ethanol was added into the 0.5 ml reservoir solution to reach the final 0.8 mM progesterone.

Crystals appeared in 3 days and reached to their final size in two weeks. The reservoir

solution containing 15% glycerol was used as a cryoprotectant for the flash-cooled crystals

in liquid nitrogen.

2.3.5 Data collection and structure determination

Diffraction data were collected from the Advanced Photon Source (APS) at Argonne

National Laboratory. Data images were recorded while the single crystal was rotated from 0°

to 180° by 1° steps. Data sets were indexed and integrated by iMosflm version 1.0.7 24

. The

space group for both complex crystals was P21 and the asymmetric unit for each crystal

contained two monomers. The 3α-HSD3·NADP+·progesterone and 3α-HSD3

V54L·NADP+·progesterone structures were solved by molecular replacement using Molrep

in CCP4 Program Suite 6.2.0 25; 26

. The subunit A of the 3α-HSD3·NADP+·Testo/acetate

structure omitting Testo and the solvent (Protein Data Bank code 1J96) was used as a

search model 18

. Multiple rounds of refinement and model building were performed using

the program Refmac in CCP4 27

and COOT 28

, respectively. Subsequently, NADP+ and

progesterone were fitted into the electron density map and refined. The geometry of the

final models was verified with the program PROCHECK in CCP4 29

.

2.3.6 Kinetic analysis of the wild type and the mutant 3α-HSD3

The enzyme for kinetic study was purified as described above. The only difference was that

the wild type and the mutant 3α-HSD3 were concentrated to 1 mg/ml in buffer A without

steroids and cofactors (NADPH and NADP+). The enzyme concentration was determined

by Bradford assay. Reduction reactions on 14

C-steroids were conducted. Each kinetic

reaction was performed at 37.0 ± 0.3°C in a volume of 1 ml 25 mM Tris-HCl buffer (pH

7.4) containing 10% Glycerol, 300-400 μM NADPH and 0.01 mg/ml of BSA. The reaction

was initiated by adding the enzyme to the reaction mixture. When 5α-DHT was used as

substrate, the wild type and the mutant enzyme were added to the reaction mixture to reach

to the final concentration of 0.2 μg/ml and 1.2 μg/ml, respectively. When progesterone was

used as substrate, the final concentrations of the wild type and the mutant enzyme were 1.0

μg/ml and 0.5 μg/ml, respectively. After different time intervals, the reactions were stopped

31

by the adding 3 volumes of diethyl ether. Following a typical TLC protocol previously

described 30

, steroids were separated and TLC plates were exposed and quantified by using

a Storm imaging system. Initial velocities were calculated by less than 10% steroids

consumption. At least duplicates of independent experiments were carried out for each Km

and kcat determination.

2.4 RESULTS

2.4.1 Overall structure of the wild type and the mutant 3α-HSD3

The crystal structures of two ternary complexes 3α-HSD3·NADP+·progesterone and 3α-

HSD3 V54L·NADP+·progesterone have been solved. Prior to crystallization, progesterone

was added into the 0.5 ml reservoir solution to reach the final 0.8 mM. This step can be

used to replace soaking progesterone into the crystal afterwards, which can saturate the

enzymes with excess steroids and avoid cracking the crystal during the soaking process.

Crystals of two ternary complexes 3α-HSD3·NADP+·progesterone and 3α-HSD3

V54L·NADP+·progesterone belonged to the monoclinic space group P21 and contained two

monomers (monomer A and B) in the asymmetric unit (Figure 2.1(A)). The 3α-

HSD3·NADP+·progesterone complex was refined at 2.2 Å with a crystallographic R-factor

of 14.6% and an R-free factor of 19.4%. The 3α-HSD3 V54L·NADP+·progesterone

complex was refined at 2.0 Å with an R-factor of 14.0% and an R-free factor of 18.3%.

Data collection and refinement statistics are shown in Table 2.1. The protein chains in both

refined models are highly similar to each other and to the previously reported structures of

human 3α-HSD3 in complex with Testo/acetate and ursodeoxycholate 18; 19

. The major

differences in these structures only exist in the regions around the steroid binding site.

2.4.2 Conserved NADP+ binding

The electron density map unambiguously defines the NADP+ stable binding to the enzyme,

which shows a highly similar conformation for NADP+ as described previously

18; 19. The

NADP+

phosphate group is well located between the two side chains of Lys270 and Arg276.

The π-π stacking between the NADP+ nicotinamide ring and the Tyr216 phenol group also

contributes to NADP+ stabilization. Besides, a number of hydrogen bonds and Van der

32

Waals interactions between NADP+ and the cofactor binding site have been revealed earlier

18; 19.

2.4.3 The ternary complex of human 3α-HSD3·NADP+·progesterone

The 3α-HSD3·NADP+·progesterone complex has been obtained by co-crystallization as

described in methods. The Fourier difference map clearly indicates that two progesterone

molecules are located in the steroid binding sites in monomer A and B. However,

progesterone in monomer A shows a lower average B-factors. In order to obtain

comparable B-factors, the occupancy of progesterone in monomer B is set to 0.8. As

observed in human 20α-HSD in complex with 20α-OHProg 31

, progesterone is positioned

with its O20 atom pointing toward the nicotinamide ring. In monomer A of human 3α-

HSD3, the β-face of progesterone interacts with the Trp227 side chain and its α-face

interacts mainly with Val54 and Val128. This allows the steroid C18 and C19 methyl

groups to point toward Trp227 (Figure 2.1(B)). While, in its monomer B, the two methyl

groups are pointing toward Tyr55 and the steroid α-face is in stacking with Trp227 side

chain (Figure 2.1(C)). Consequently, when monomer A and B are superimposed, two

steroids could not overlap each other and their orientations have an about 105° rotation

difference around the long axis of C10-C17 (Figure 2.1(D)). With this rotation, the distance

between the nicotinamide ring C4 atom and progesterone C20 atom is changed from 4.15 Å

in monomer A to 4.37 Å in monomer B. Meanwhile, the distance between the steroid O20

atom and His222 Nε atom is changed from 3.19 Å in monomer A to 5.46 Å in monomer B.

As known, the steroid binding site is mainly formed by loop A (residues 117-143), loop B

(residues 217-238), loop C (residues 299-323), and two short loops (residues 23-32 and 51-

57). When monomer A and B are superimposed, the RMSD value is 0.40 Å for 323 amino

acids Cα atoms. Meanwhile, the RMSD values are 1.09 Å, 0.46 Å and 0.23 Å for loop A,

loop B and loop C, respectively, which indicates that the back bone of loop A fluctuates

notably compared with loop B and loop C. Further inspection of the steroid binding site,

residues 126-141 in loop A exhibits a considerable displacement toward loop B. The

conformational changes of loop A drive the side chains of Val128, Ile129 moving closer to

the steroid, which may generate the forces to rotate progesterone in its binding site.

Moreover, the side chains of Leu54, Trp227, Leu306 and Leu308 reconcile their

33

conformations to accommodate the steroid. In addition, the crystal structure reveals that a

water molecule presents at the catalytic site and forms two hydrogen bonds with the side

chains of Tyr55 and His117, which exactly occupies the same position instead of the ketone

group from Testo, acetate or ursodeoxycholate in the reported ternary complexes 18; 19; 20; 32

.

In monomer B, this key water molecule can also participate in the stabilization of

progesterone through a hydrogen bond with the steroid O20 atom. In monomer A and B,

the orientations of the steroid C20 ketone groups as well as the distances between the C20

atoms and the nicotinamide ring C4 atoms are not suitably coordinated for the hydride

transfer, and such conformations represent the steroid binding modes before or after the

reaction.

2.4.4 The ternary complex of human 3α-HSD3 V54L·NADP+·progesterone

The 3α-HSD3 V54L·NADP+·progesterone structure shows high similarity to the ternary

complex of the wild type enzyme and the 20α-HSD ternary complex 31

. The electron

density map clearly defines that progesterone is situated in the steroid binding site, in which

the Leu54 side chain in the mutant enzyme is outstretching toward the steroid (Figure

2.2(A)). Consistent with the wild type enzyme, in the mutant enzyme, the temperature

factor for progesterone in monomer B is higher than that in monomer A. Therefore, the

occupancy of progesterone in monomer B is set to 0.8 to acquire a comparable B-factor.

However, when superimposing monomer A with B (Figure 2.2(B)), the orientations of two

steroids overlap each other quite well, which is different from the 3α-

HSD3·NADP+·progesterone structure. The β-faces of both steroids interact with the Trp227

indole group and their α-faces interact mainly with Leu54 and Val128. After superposition,

the RMSD value for 323 amino acids Cα atoms between monomer A and B is 0.35 Å. The

RMSD values for loop A, loop B and loop C are 0.85 Å, 0.34 Å and 0.18 Å, respectively.

Although loop A displays a movement to loop B and the side chains of Val128, Ile129 get

closer to the steroid at the same time, the conformation of progesterone seems be fixed in

its binding pocket and hardly any rotation can be observed. It is not difficult to understand

that the bulky side chain of Leu54 generates a steric hindrance to restrict the steroid

rotation. Even though the average B-factors for the wild type enzyme and the mutant

enzyme are quite similar (Table 2.1), the average B-factors for steroids in the mutant

34

enzyme (monomer A/monomer B) hold the relative lower B-factors (23.3/27.6 Å2) than

that in the wild type enzyme (37.7/34.4 Å2), indicating that the V54L mutation has a role in

the stabilization of progesterone. In addition, the side chains of Trp227, Leu306 display

slight movements and the side chain of Leu308 flips 90° around its Cβ. Besides, a water

molecule forms hydrogen bonds with Tyr55, His117 and situates at the catalytic site.

2.4.5 Kinetic study of the wild type and the mutant 3α-HSD3

To investigate how significantly the mutation of Val54 to Leu54 in human 3α-HSD3 alters

the enzyme activity and its steroid binding specificity, the relevant kinetic study were

carried out. Firstly, the wild type enzyme or the V54L mutant enzyme (the final

concentration of 1 μg/ml) was added into the reaction mixture to initiate the reaction, and

the reaction mixture contained 300 μM NADPH. When 5α-DHT or progesterone was used

as substrate at the concentration of 4 μM, after 10 minutes reaction at 37°C, the TLC was

performed. The result demonstrates that the V54L mutation remarkably reduces the 3α-

HSD reduction ability for 5α-DHT and enhances the 20α-HSD reduction activity for

progesterone (Figure 2.3). Furthermore, to obtain more detailed kinetic information, the

steady-state kinetic study was performed (Table 2.2). When 5α-DHT was used as substrate,

the specificity constant (kcat/Km) for the mutant enzyme was decreased to 17-fold. This

denotes that, compared with the wild type enzyme, the V54L mutation significantly reduces

the 3α-HSD3 specificity for 5α-DHT. When progesterone was used as substrate, the kcat/Km

value was increased to 4.4-fold and the mutant enzyme has gained a considerable 20α-HSD

activity.

2.5 DISCUSSION

In this study, it is interesting to show that progesterone can adopt two different binding

modes to form complexes with monomer A and B of the wild type 3α-HSD3 in a crystal

asymmetric unit, respectively. However, the V54L mutation in the enzyme directly restricts

the steroid binding modes to a unique one.

When monomer A and B in the 3α-HSD3·NADP+·progesterone complex are superimposed,

it can be clearly seen that, in monomer B, loop A has a major movement toward loop B and

35

progesterone (Figure 2.1(D)). This is mainly due to the different interacting environments

with the neighboring symmetric molecules for monomer A and B. Correspondingly, in

monomer B, the two hydrophobic residues Val128 and Ile129 in loop A are moving inside

the steroid binding pocket a little and their side chains make closer van der Waals contacts

with the A ring of the steroid. The side chain of Leu308 flips 180° around its Cβ atom and

the side chain of Leu306 owns alternative conformations. Meanwhile, progesterone

presents a 105° rotation around its C10-C17 axis. The interaction between His222 Nε atom

and the O20 atom of the steroid is within the range of hydrogen bond in monomer A. While

in monomer B, this interaction is greatly reduced and the side chain of His222 flips 180°

around its Cβ atom. The superposition shows that the steroid binding pocket of 3α-HSD3

possesses a considerable flexibility, which allows two different steroid binding modes for

progesterone. In addition, it has been proved that human 3α-HSD3 can bind with several

ligands such as citrate and ursodeoxycholate 19; 20

, which further validates the flexibility of

the steroid binding pocket.

In the 3α-HSD3 V54L·NADP+·progesterone complex, the overlay of monomer A and B

shows high similarity for the overall structure (Figure 2.2(B)). Although loop A in

monomer B also displays a shift toward loop B, the orientation of progesterone has not

been transformed. Obviously, Leu54 plays a key role to eliminate multiple orientations

which are allowed in the wild type enzyme. If progesterone in the mutant enzyme has a 105°

rotation like it does in the wild type enzyme, the C ring of the steroid will conflict with the

bulky side chain of Leu54. Therefore, despite the steroid binding pocket possessing

considerable flexibility, the V54L mutation significantly restricts the movement space of

the steroid. An overlay of both monomers A in the wild type and the mutant enzyme shows

progesterone has a minor shift toward Trp227 in the mutant enzyme and the indole ring of

Trp227 has an 11° rotation around its Cβ atom (Figure 2.4(A)). At the same time, the side

chain of Ile129 rotates about 103° around its Cγ atom and the side chain of Leu308 flips

about 180° around its Cβ atom, which represents the flexibility of this cavity. When the

V54L mutant complex and the 20α-HSD·NADP+·20α-OHProg complex are superimposed,

the plane of the steroid has a slight swing around the short axis of C13-C18. Meanwhile,

the side chains of Ile129, Trp227 and Leu308 possess subtle movements and the side chain

of Leu306 flips 180° around its Cβ atom (Figure 2.4(B)). Although six amino acids

36

difference between the 3α-HSD3 V54L mutant and 20α-HSD still remains, these residues

do not disturb the overall arrangement of the main chains in both enzymes.

In human 3α-HSD3 and 20α-HSD, the indole rings of Trp227 adopt similar orientations to

stack with the plane of the steroid and have a crucial role to stabilize the orientation of the

steroid. However, compared with their homologous human steroid 5β-reductase (AKR1D1)

33, the indole ring of Trp227 (numbering according to 3α-HSD3) rotates about 125° toward

the side chain of Val228. It can be seen that the steroid and the indole ring of Trp227 in

AKR1D1 adjust their orientations to satisfy the bulky side chains of Ile128 and Tyr129 (3α-

HSD3 numbering) in loop A stretching into the steroid binding pocket. Besides, the less

bulky side chains of Ser222 and Val306 (3α-HSD3 numbering) in loop B can accommodate

the reorientation of Trp227.

An overlay shows the orientation of ursodeoxycholate in the reported 3α-

HSD3·NADP+·ursodeoxycholate complex is quite identical to that of progesterone in

monomer B of the 3α-HSD3·NADP+·progesterone complex, in which both C18 methyl

groups are pointing toward Tyr55 side chain (Figure 2.4(C)). However, compared with the

orientation of progesterone in the 3α-HSD3 V54L·NADP+·progesterone complex, the

orientation of ursodeoxycholate needs an about 105° rotation around its C10-C17 axis to

overlap with the plane of progesterone (Figure 2.4(D)). But this rotation is not allowed for

the reason that the carboxylate group of ursodeoxycholate will collide with the imidazole

ring of His222. Besides, the overlay shows that the distance between ursodeoxycholate C18

atom and Leu54 Cγ atom in the mutant enzyme is about 2.48 Å, which implies the bulky

side chain of Leu54 will obstruct the ursodeoxycholate binding by this orientation. Hence,

the orientation of ursodeoxycholate in the wild type 3α-HSD3 should be the unique one for

the reason that the less bulky side chain of Val54 is located in the steroid binding pocket.

As reported 19; 34

, human 3α-HSD3 is the only one that can bind ursodeoxycholate among

human 3α-HSDs. All the other three types of human 3α-HSDs have Leu54 residues at this

position.

Based on the docking simulation of 5α-DHT into human 3α-HSD3 and 20α-HSD, the

swinging mechanism has been proposed that the shorter side chain of Val54 in 3α-HSD3

prefers to produce 3α-diol, while the bulky side chain of Leu54 in 20α-HSD can generate a

37

swing of 5α-DHT to produce 3β-diol 35; 36

. From our structural results, superposition of the

wild type 3α-HSD3 and the V54L mutant demonstrates that the orientation of progesterone

in their monomer A does not show such kind of swing, while the orientation of the steroid

in their monomer B displays a 105° rotation. This rotation of the steroid orientation is

similar to the flipping mechanism that the plane of the steroid can flip around its long

molecular axis to bind in its cavity 37

.

To further understand the steroid discrimination by human 3α-HSD3 and its V54L mutant,

the kinetic study has been carried out. A single V54L mutation in 3α-HSD3 significantly

decreases its 3α-reductase activity and enhances its 20α-reductase activity. For 5α-DHT

reduction, the steady-state kinetic parameters for the wild type enzyme are Km = 2.18 μM,

kcat = 3.67 min-1


μM-1

. When compared with the former results by

our group (Km = 1.1 μM, kcat = 1.5 min-1


μM-1

), the kcat/Km values are

similar to each other 18

. The relevant result from the group of Penning (Km = 2.90 μM, kcat =

1.98 min-1


μM-1

) shows a slight difference 38

. However, the kinetic

parameters for the V54L mutant (Km = 71.03 μM, kcat = 6.92 min-1


μM-1

) clearly shows its 5α-DHT reduction specificity is significantly decreased. While for

progesterone reduction, the wild type enzyme exhibits a weak activity (Km = 0.84 μM, kcat =

0.21 min-1


μM-1

), but the mutant enzyme shows a considerable

20α-reductase activity (Km = 2.13 μM, kcat = 2.35 min-1


μM-1

). The

3α-HSD3 V54L mutant imitates human 20α-HSD and its enzyme activity is higher than the

reported human 20α-HSD activity for progesterone reduction by the group of Penning (Km

= 2.65 μM, kcat = 0.29 min-1


μM-1

) and by the group of Bunce (Km =

4.20 μM, kcat = 1.77 min-1


μM-1

) 5; 39

, and is similar to the reported

result by the group of Hara (Km = 1.30 μM, kcat = 1.50 min-1


μM-1

)

40. The differences of the kinetic parameters may attribute to the different buffer systems

and temperatures used for the reactions. In addition, the kinetic study demonstrates that

17β-HSD1 also possesses certain 5α-DHT reduction activity (Km = 32.0 μM, kcat = 2.40

min-1


μM-1

) 30

, but it is far less than the specificity constant (kcat/Km)

of the wild type 3α-HSD3 and is only comparable to that of the V54L mutant enzyme.

38

Besides the V54L mutation in human 3α-HSD3, a number of mutations targeting 3α-HSDs

have been carried out to evaluate the specific roles of different residues in the enzymes. A

previous experiment showed that two human 3α-HSD3 mutants (F46Y and L172Q) possess

reduced reductase activities 41

, and these mutants have been predicted to destabilize the

cofactor binding by the method of homology modeling 42

. Furthermore, formerly, an

attempt has been done to engineer mammalian (rat) 3α-HSD to 20α-HSD 43

. Both enzyme

share 67% protein sequence identity and six amino acid residues which locate in the steroid

binding site are different. However, even when all these six distinctive residues of rat 3α-

HSD were mutated to the relevant residues of rat 20α-HSD, the 20α-HSD activity was still

not available for the mutant enzyme. Compared with the results from human 3α-HSD3

V54L mutation, one reasonable explanation is that human 3α-HSD3 and 20α-HSD share

97.8% sequence identity, which is much higher than 67% sequence identity between the

two rat enzymes. The V54L mutation in human 3α-HSD3 can generate adequate

stereospecificity for the 20α-HSD activity. While for rat 3α-HSD, until loop A, B and C in

the steroid binding site are together replaced with the equivalent loops of 20α-HSD, the

similar task can be fulfilled 43

. In addition, the important mutagenesis experiment done by

Matsuura et al. demonstrated that the replacement of human 20α-HSD Leu54 with Val54

almost produced an enzyme possessing the similar property as 3α-HSD3 22

. Our kinetic

study is complementary to their mutagenesis and kinetic work.

In summary, the alternative binding modes of progesterone have been found in the wild

type 3α-HSD3, which indicates the steroid binding pocket possessing considerable

flexibility to accommodate different orientations of a steroid. However, the bulky side

chain of Leu54 in the mutant enzyme significantly confines the spatial movement of the

steroid in its cavity and thus only one orientation presents in its ternary structure. Moreover,

the kinetic study shows that the V54L mutation is enough to transform human 3α-HSD3 to

an enzyme with a competent 20α-HSD activity, whereas its 3α-HSD activity is significantly

decreased. This study gives us a hint that the alternative binding modes may also exist for

5α-DHT in human 3α-HSD3 and be utilized by the enzyme to output different products

such as 3α-diol and 3β-diol.

39

2.6 FUNDING SOURCES

This work was supported by the Canadian Institutes of Health Research (CIHR), with a

grant to Dr. Sheng-Xiang Lin and collaborators (MOP 97917). Use of the Advanced Photon

Source, an Office of Science User Facility operated for the U.S. Department of Energy

(DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE

under Contract No.DE-AC02-06CH11357. Use of the Lilly Research Laboratories

Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon

Source was provided by Eli Lilly Company, which operates the facility.

2.7 ACKNOWLEDGEMENTS

We thank Dr. Preyesh Stephen and Dr. Zhen-Kun Sun for language checking. We thank Mr.

Ronald Maheux for the image analysis of the exposed films from the TLC plates. Bo Zhang

thanks the grant provided by CIHR (MOP 97917) and a national scholarship provided by

China Scholarship Council (CSC) for supporting his Ph.D. study. Dr. Xiao-Jian Hu thanks

the National Natural Science Foundation of China (No. 31011120381) for supporting the

international travel expenses for his visiting to Dr. Lin’s laboratory. X-ray diffraction

datasets were collected at LRL-CAT at the Advanced Photon Source, Argonne National

Laboratory.

40

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38. Jin, Y. & Penning, T. M. (2006). Multiple steps determine the overall rate of the

reduction of 5alpha-dihydrotestosterone catalyzed by human type 3 3alpha-

hydroxysteroid dehydrogenase: implications for the elimination of androgens.

Biochemistry 45, 13054-63.

39. Velica, P., Davies, N. J., Rocha, P. P., Schrewe, H., Ride, J. P. & Bunce, C. M.

(2009). Lack of functional and expression homology between human and mouse

aldo-keto reductase 1C enzymes: implications for modelling human cancers. Mol

Cancer 8, 121.

40. Higaki, Y., Usami, N., Shintani, S., Ishikura, S., El-Kabbani, O. & Hara, A. (2003).

Selective and potent inhibitors of human 20alpha-hydroxysteroid dehydrogenase

(AKR1C1) that metabolizes neurosteroids derived from progesterone. Chem Biol

Interact 143-144, 503-13.

41. Takahashi, R. H., Grigliatti, T. A., Reid, R. E. & Riggs, K. W. (2009). The effect of

allelic variation in aldo-keto reductase 1C2 on the in vitro metabolism of

dihydrotestosterone. J Pharmacol Exp Ther 329, 1032-9.

42. Arthur, J. W. & Reichardt, J. K. (2010). Modeling single nucleotide polymorphisms

in the human AKR1C1 and AKR1C2 genes: implications for functional and

genotyping analyses. PLoS One 5, e15604.

45

43. Ma, H. & Penning, T. M. (1999). Conversion of mammalian 3alpha-hydroxysteroid

dehydrogenase to 20alpha-hydroxysteroid dehydrogenase using loop chimeras:

changing specificity from androgens to progestins. Proc Natl Acad Sci U S A 96,

11161-6.

46

Table 2.1 Data collection and refinement statistics

Structure

3α-HSD3

NADP+·progesterone

3α-HSD3 V54L

NADP+·progesterone

A. Data collection

Unit cell dimensions

a, b, c (Å)

β, α=γ (degrees)

52.80, 86.82, 76.49

105.96, 90.00

53.14, 87.10, 76.87

106.31, 90.00

Space group P21 P21

Resolution range (Å) 21.50-2.20 (2.32-2.20) 25.00-2.00 (2.11-2.00)

Total reflections 123,942 (17,392) 159,275 (20,993)

Unique reflections 32,561 (4701) 44,885 (6376)

Completeness (%) 96.4 (95.9) 98.7 (96.6)

Rmerge a

0.037 (0.056) 0.037 (0.053)

I/σ(I) 26.4 (19.3) 19.2 (14.2)

B. Refinement statistics

Reflection used (R-free set) 30,919 (1640) 42,617 (2263)

R-factor b

0.146 0.140

R-free c

0.194 0.183

Root mean square deviations

Bond lengths (Å)

Bond angles (degrees)

0.017

1.902

0.019

1.941

Protein atoms d

NADP+ molecules

d

Steroid molecules d

Sulfate ion d

Water molecules d

5204

2

2

2

369

5206

2

2

2

544

Average B-factors (Å2)

All atoms

Protein atoms e

11.8

10.0/12.8

11.1

8.9/12.4

47

NADP+ e

9.8/11.4 7.9/10.4

Steroid e

Sulfate ion e

Water

37.7/34.4

13.4/24.2

14.7

23.3/27.6

14.9/26.3

17.6

Ramachandran statistics (%) f

Most favored regions

Additionally allowed regions

Generously allowed regions

Disallowed regions

93.1

6.6

0.0

0.3

93.6

6.1

0.0

0.3

a Rmerge = ΣΣi|I(h)-I(h)i|/ΣΣiI(h),where I(h) is the mean intensity after rejections.

b R-factor = Rcryst = Σ(|FO|-|FC|)/Σ|FO|, where |FO| and |FC| are the observed and calculated

structure factor amplitudes for the reflection with Miller indices h = (h, k, l), respectively.

c R-free is calculated for a “test” set of reflections, which was not included in refinement.

d Per asymmetric unit.

e Average B-factors were calculated separately for two monomers within an asymmetric

unit (monomer A/monomer B).

f Ramachandran statistics was calculated with PROCHECK in COOT. Ser221 in both

monomers of human 3α-HSD3 were found in the disallowed regions 18

.

48

Table 2.2 Steady-state kinetic parameters for 5α-DHT and progesterone reduction

catalyzed by the wild type (WT) 3α-HSD3 and the Val54Leu (V54L) mutant in the

presence of NADPH

Enzyme Substrate Km (μM) kcat (min-1

) kcat/Km (min-1

μM-1

)

3α-HSD3 WT 5α-DHT 2.18 ± 0.03 3.67 1.68

3α-HSD3 V54L 5α-DHT 71.03 ± 0.65 6.92 0.10

3α-HSD3 WT Progesterone 0.84 ± 0.01 0.21 0.25

3α-HSD3 V54L Progesterone 2.13 ± 0.13 2.35 1.10

49

Figure 2.1 Structure of the 3α-HSD3·NADP+·progesterone complex

(A) The crystal asymmetric unit contains two monomers. Monomer A is colored in

magenta. Monomer B is colored in cyan. Atoms are colored as follows. Green, carbon

atoms of steroids and cofactors; blue, nitrogen; red, oxygen; orange, phosphorus. (B) The

steroid binding site of monomer A. The 2Fo-Fc electron density map around progesterone

is contoured at 0.9 σ level. Protein carbon is colored in magenta. A water molecule is

located at the catalytic site. (C) The steroid binding site of monomer B. The 2Fo-Fc

electron density map around progesterone is contoured at 0.7 σ level. Protein carbon is

colored in cyan. Hydrogen bonds in (B) and (C) are shown by black dashed lines. (D)

Superposition of the steroid binding sites of monomer A and B. For clarity, carbon atoms of

steroids and cofactors in monomer A and B are colored in magenta and cyan, respectively.

Progesterone possesses two binding conformations in the steroid binding site of the wild

type enzyme.

52

Figure 2.2 Structure of the 3α-HSD3 V54L·NADP+·progesterone complex

(A) The steroid binding site of monomer A in the 3α-HSD3 V54L·NADP+·progesterone

complex. The 2Fo-Fc electron density map around progesterone is contoured at 1.1 σ level.

Atoms are colored as follows. Green, carbon; blue, nitrogen; red, oxygen; orange,

phosphorus. Protein chain is colored in green. A water molecule is located at the catalytic

site. Hydrogen bonds are shown by black dashed lines. (B) Superposition of the steroid

binding sites of monomer A and B in the 3α-HSD3 V54L·NADP+·progesterone complex.

Carbon and protein chain of monomer B are colored in dark gray. Progesterone possesses

only one binding conformation in the steroid binding site of the mutant enzyme.

54

Figure 2.3 Presence of the reduced form of the cofactor NADPH within the purified

protein samples of the wild type 3α-HSD3 and the V54L mutant

Lane 1, 14

C-5α-DHT marker; lane 2, the wild type 3α-HSD3 with 14

C-5α-DHT in the

reaction mixture; lane 3, the V54L mutant with 14

C-5α-DHT in the reaction mixture; lane 4,

14C-progesterone marker; lane 5, the wild type 3α-HSD3 with

14C-progesterone in the

reaction mixture; lane 6, the V54L mutant with 14

C-progesterone in the reaction mixture.

55

Figure 2.4 Superposition of the steroid binding sites of human 3α-HSD3 related

structures

(A) Superposition of the steroid binding sites of two monomer A in the 3α-

HSD3·NADP+·progesterone complex (colored as in Figure 2.1(D)) and the 3α-HSD3

V54L·NADP+·progesterone complex (colored as in Figure 2.2(A)). (B) Superposition of

the steroid binding site of monomer A in the 3α-HSD3 V54L·NADP+·progesterone

complex (colored as in Figure 2.2(A)) and that of the 20α-HSD·NADP+·20α-OHProg

complex (PDB code 1MRQ; carbon and protein chain are colored in orange). (C)

Superposition of the steroid binding site of monomer B in the 3α-

HSD3·NADP+·progesterone complex (colored as in Figure 2.1(D)) and that of the 3α-

HSD3·NADP+·ursodeoxycholate complex (PDB code 1IHI; carbon and protein chain are

colored in pink). (D) Superposition of the steroid binding site of monomer A in the 3α-

HSD3 V54L·NADP+·progesterone complex and that of the 3α-

HSD3·NADP+·ursodeoxycholate complex.

59

CHAPTER 3 STRUCTURE CLUES OF 5ΑLPHA-DHT

ALTERNATIVE BINDING WITHIN HUMAN 3ΑLPHA-HSD3 AND

THE ROLE OF THE ENZYME IN BREAST CANCER

60

3.0 PREFACE

The most potent androgen 5α-DHT has been shown to reduce breast cancer cell growth and

human 3α-HSD3 plays an essential role to inactivate 5α-DHT. However, the important

structural information about the interaction of 3α-HSD3 with its substrate 5α-DHT is still

not available. Also, the role of 3α-HSD3 in breast cancer cells is short of investigations. By

combining the X-ray crystallography and the functional studies in the cellular level, we

carry out the research on these issues.

This chapter includes one paper: Human 3-alpha hydroxysteroid dehydrogenase type 3:

structural clues of 5α-DHT alternative binding and enzyme down-regulation decreasing

breast cancer cell growth. In this paper, I carried out all the experimental studies except for

the work indicated below and wrote the manuscript. I and Dr. Xiao-Jian Hu solved the

crystal structure of the 3α-HSD3·NADP+·A-dione/epi-ADT complex. I solved the crystal

structure of the 3α-HSD3·NADP+·4-dione complex. Mr. Xiang-Qiang Wang and I used the

ELISA kit to determine the 5α-DHT concentration. Dr. Dao-Wei Zhu guided the protein

purification. The q-RTPCR was done by the q-RTPCR platform (Centre Hospitalier

Universitaire (CHU) de Quebec Research Center (CHUL) and Laval University, Québec).

The GC/MS analysis was done by the bioanalytical platform (Centre Hospitalier

Universitaire (CHU) de Quebec Research Center (CHUL) and Laval University, Québec).

Dr. Sheng-Xiang Lin and I designed the study.

61

Human 3-alpha hydroxysteroid dehydrogenase type 3: structural clues of

5α-DHT alternative binding and enzyme down-regulation decreasing

breast cancer cell growth

Bo Zhang 1, 2

, Xiao-Jian Hu 3, Xiao-Qiang Wang

1, Dao-Wei Zhu

1, Peng Shang

2, Fernand

Labrie 1, Sheng-Xiang Lin

1, §



G1V4G2, Canada




3 School of Life Sciences, Fudan University, Shanghai, P.R. China, 200433




Shortened title: Structure clues of 5α-DHT alternative binding within human 3α-HSD3 and

the role of the enzyme in breast cancer


62

3.1 ABSTRACT

Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) plays essential roles in

steroid hormone metabolism, especially in the inactivation of the most potent androgen 5α-

DHT. Besides, 5α-DHT has been shown to reduce breast cancer cell growth. However, the

important structural information about 3α-HSD3 inactivation of 5α-DHT is still not

available and the role of 3α-HSD3 in breast cancer cells is short of investigations. Here, we

reported the crystal structure of human 3α-HSD3·NADP+·A-dione/epi-ADT complex,

which was obtained by co-crystallization with 5α-DHT in the presence of NADP+.

Although 5α-DHT was introduced during the crystallization, the oxidoreduction of 5α-DHT

occurred. A-dione and epi-ADT were identified to be located in the steroid binding sites of

two monomers (monomer A and B) in a crystal asymmetric unit, respectively. An overlay

showed that A-dione and epi-ADT were oriented upside down and flipped to each other,

which provided the structural clues of 5α-DHT alternative binding within the enzyme to

yield different products. Moreover, we reported the crystal structure of the 3α-

HSD3·NADP+·4-dione complex. Using a crystallization condition without the acetate ion,

the crystal was obtained by co-crystallization with testosterone in the presence of NADP+.

The acetate ion introduced from the crystallization condition could distort the steroid

binding conformation and it should be avoided. In addition, using specific siRNA to knock

down 3α-HSD3 expression in MCF7 breast cancer cells, 5α-DHT concentration increased

while MCF7 cell growth was suppressed.

Keywords: 3α-HSD3, AKR1C2, crystal structure, 5α-DHT, alternative binding, cancer cell

growth

Protein data bank accession codes: 4NNE and 4NNF

63

3.2 INTRODUCTION

Breast cancer is a common steroid-related cancer in women 1; 2

. According to the statistics

of the American and Canadian Cancer Society, breast cancer is the second leading cause of

death from cancer in women in North America. 17β-estradiol (E2), the most potent

estrogen, stimulates the proliferation of breast cancer cells, whereas androgen 5α-

dihydrotestosterone (5α-DHT) can reduce breast cancer cell growth 3; 4; 5; 6; 7

. As known, 5α-

DHT is the most potent androgen, and is highly involved in the development of prostate

cancer 8; 9; 10; 11

. In human, 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) plays a

major role of 5α-DHT reduction to produce the inactive steroid 5α-androstan-3α,17β-diol

(3α-diol) in the presence of NADPH 12; 13; 14

, which makes 3α-HSD3 acting as a pre-

receptor regulator of androgen receptor (AR) in prostate cancer 15; 16; 17; 18

. However, the

role of 3α-HSD3 in breast cancer cells is short of investigations.

There are four isoenzymes (types 1, 2, 3 and 4) included in human 3-alpha hydroxysteroid

dehydrogenase family (3α-HSDs), which belongs to the aldo-keto reductase (AKR)

superfamily and are also named AKR 1C4, 1C3, 1C2 and 1C1, respectively 19; 20

. These

isoenzymes have varying degrees of 3-keto-, 17-keto-, and 20-ketosteroid reductase

activities to inactivate androgens, progestins, and bile acid precursors 18; 21; 22

. They are

highly homologous enzymes and show differential tissue distributions 13; 22; 23

. Human 3α-

HSD1 (AKR1C4) is a liver-specific enzyme acting on the steroid metabolism in liver 22; 23

.

Human 3α-HSD2 (AKR1C3) is highly expressed in prostate and mammary glands, and

shows a major ability to biosynthesize testosterone (Testo) from 4-androsten-3,17-dione (4-

dione) 24; 25

. Human 3α-HSD3 (AKR1C2) is expressed in several tissues including liver,

lung, brain, prostate, testis, mammary gland and adrenal 13; 22

. In vitro, 3α-HSD3 can

oxidize 3α-diol to 5α-DHT in the presence of NAD+. However, it practically acts as a

reductase to inactivate 5α-DHT depending upon the cellular environment 13; 14; 26

. Human

3α-HSD3 is also recognized as the bile acid binding protein and can be inhibited by bile

acids such as ursodeoxycholate 27; 28

. In the pathogenesis of female hirsutism, a reduced

expression of 3α-HSD3 along with an elevated 5α-DHT level is found in tissue of hirsute

women 29

. Human 3α-HSD4 (AKR1C1, also called 20-alpha hydroxysteroid

dehydrogenase (20α-HSD)) is found in mammary gland, ovary, uterus and placenta, and

64

predominantly exerts its 20-ketosteroid reductase activity to inactivate progesterone 22; 30; 31

.

It is noteworthy that 3α-HSD3 and 3α-HSD4 share 97.8% sequence identity, and only

seven residues are different between two enzymes. However, they show distinctive

specificity for steroidal substrates.

The former crystal structures of 3α-HSD3 show that this enzyme possesses an (α/β)8-barrel

motif, and its steroid binding pocket possesses considerable flexibility, which is formed by

five loops including loop A (residues 117-143), loop B (residues 217-238), loop C (residues

299-323), and two short loops (residues 23-32 and 51-57) 32; 33; 34

. Although these structures

provide the detailed information, the crucial structure of 3α-HSD3 in complex with 5α-

DHT is still not available

In this study, we reported the high-resolution crystal structure of the 3α-HSD3·NADP+·A-

dione/epi-ADT complex, which was obtained by co-crystallization with 5α-DHT.

Surprisingly, the oxidoreduction of 5α-DHT occurred during the crystallization process and

produced a mixture of steroids. A-dione and epi-ADT were identified to be located in the

enzyme cavities. Moreover, we reported the crystal structure of the 3α-HSD3·NADP+·4-

dione complex. Using a crystallization condition without the acetate ion, the steroid binding

conformation was more reasonable and less distorted. In addition, using specific siRNA to

knockdown 3α-HSD3 expression in breast cancer cells, the impacts of 3α-HSD3 expression

on the concentration of 5α-DHT and cell proliferation were further investigated.

3.3 MATERIALS AND METHODS

3.3.1 Materials

MCF7 cells and MTT cell proliferation assay kit were purchased from the American Type

Culture Collection (ATCC). Fetal bovine serum (FBS, Qualified, US origin) and

Lipofectamine 2000 transfection reagent were purchased from Life Technologies

Corporation. Dulbecco’s Modified Eagle’s Medium (DMEM) low-glucose medium was

ordered from Sigma-Aldrich. siRNA was ordered from Shanghai GenePharma. 5α-DHT

ELISA kit was ordered from Alpha Diagnostic International. 5α-DHT and Testo were

ordered from Steraloids. Diethyl ether, acetone, toluene and dichloromethane were

65

purchased from Fisher Scientific. Thin layer chromatography (TLC) plate was purchased

from EMD Millipore. 14

C-labelled 5α-DHT (53.5 mCi/mmol) was purchased from

PerkinElmer Life Sciences. 14

C-labelled Testo (55 mCi/mmol) was purchased from

American Radiolabeled Chemicals. NADPH, NADP+ and other chemicals were purchased

from Sigma-Aldrich.

3.3.2 Protein purification and crystallization

Human 3α-HSD3 was purified basically following a previously reported procedure 35

.

Briefly, the recombinant enzyme with a glutathione S-transferase (GST) tag was initially

purified with a glutathione agarose column and then the second step of purification by the

Q-sepharose column can fulfill the task. The enzyme was concentrated to about 30 mg/ml

in a 10 mM KH2PO4-K2HPO4 buffer (pH 7.0) containing 0.5 mM DTT, 0.06% β-OG, 1

mM EDTA, 1 mM NADP+, 40 μM 5α-DHT or 40 μM Testo. Crystals were obtained in the

sitting drops by using the vapor diffusion method at room temperature. Before

crystallization, 5α-DHT or Testo were added into the 0.5 ml well solution to reach the final

1 mM concentration, respectively, and the crystallization condition included 100 mM

sodium cacodylate (pH 6.0), 200 mM ammonium sulfate, 24-26% (w/v) polyethylene

glycol (PEG) 3350. Crystals appeared after three days and then reached to their full size

within two weeks. During the crystallization period, 5α-DHT can be oxidized to A-dione

and then be reduced to epi-ADT by the enzyme. The well solution containing 15% glycerol

was selected as the cryoprotective solution.

3.3.3 Data collection and structure determination

The X-ray diffraction data were collected at beamline 31-ID-D (λ= 0.979 Å) of the

Advanced Photon Source (APS) at Argonne National Laboratory. Data sets were indexed

and integrated by iMosflm 36

. Both space groups of the 3α-HSD3·NADP+·A-dione/epi-

ADT complex and the 3α-HSD3·NADP+·4-dione complex were P21. There are two

monomers (A and B) contained in an asymmetric unit for each crystal structure. In the 3α-

HSD3·NADP+·A-dione/epi-ADT complex, A-dione was found in monomer A and epi-

ADT was found in monomer B (see “RESULTS”). Using the reported 3α-HSD3 structure

(PDB code 1J96) 32

, the two crystal structures were solved by molecular replacement with

66

the program Molrep in CCP4 37; 38

. The program Refmac in CCP4 and COOT were used for

refinement and model building, respectively 39; 40

. After the amino acid residues of the

enzyme and water molecules were refined, NADP+ and steroids were fitted into the 2FO-FC

omit electron density and refined. Finally, the quality of the models was verified with

PROCHECK 41

.

3.3.4 The oxidoreduction assay of steroids and the GC/MS analysis

The reaction of 5α-DHT oxidoreduction was performed at 37.0 ± 0.3°C in a volume of 1 ml

25 mM Tris-HCl buffer (pH 7.4) containing 10% Glycerol, 0.1 mM 5α-DHT (with 0.1 μM

14C-labelled 5α-DHT), 1 mM NADP

+ and 0.01 mg/ml of BSA. The reaction was initiated

by adding the enzyme to the reaction mixture, and the final concentration of the enzyme

was 0.2 mg/ml. After overnight incubation, the reaction was terminated by adding 3 ml

diethyl ether. Duplicates of reactions were performed. Following a standard TLC protocol

42, steroids were separated. TLC plate was exposed and quantified by a Storm imaging

system. Similarly, the reaction of Testo oxidation was performed using the same

experimental condition as the reaction of 5α-DHT oxidoreduction except that 5α-DHT and

14C-labelled 5α-DHT were replaced by Testo and

14C-labelled Testo. In addition, the

reaction mixture of 5α-DHT oxidoreduction (without 14

C-labelled 5α-DHT) was analyzed

by gas chromatography and mass spectrometer (GC/MS). The GC/MS analysis was carried

out by the bioanalytical platform (Centre Hospitalier Universitaire (CHU) de Québec

Research Center (CHUL), Université Laval, Québec, Canada).

3.3.5 Cell culture

MCF7 cells were cultured in DMEM low-glucose medium in the presence of 10% FBS and

the medium was phenol red-free. Cells were maintained in a humidified incubator at 37°C

and supplied with 5% CO2. When indicated, FBS was treated with 2% (w/v) dextran-coated

charcoal overnight at 4°C to deprive the original steroids in FBS.

3.3.6 siRNA synthesis and transfection

Human 3α-HSD3 siRNA (sense: AAGCUCUAGAGGCCGUCAAAUTT; antisense:

AUUUGACGGCCUCUAGAGCUUTT) was designed according to the previous research

67

43 and the specificity of siRNA was checked by BLAST in human genome

44. Duplex 3α-

HSD3 siRNA and negative control (NC) siRNA were synthesized and purified with HPLC

by Shanghai GenePharma. siRNA was transfected by using lipofectamine 2000 and

following the manufacturer’s protocol.

3.3.7 Quantitative real-time RT-PCR

MCF7 cells were plated into a six-well plate at 4 × 105 cells per well with 2 ml medium and

cultured one bay before siRNA transfection. MCF7 cells were transfected with 100 nM 3α-

HSD3 specific siRNA or negative control siRNA for 24 hours and then cells were treated

with Trizol reagent. The treated samples were sent to the q-RTPCR platform (Centre

Hospitalier Universitaire (CHU) de Québec Research Center (CHUL), Université Laval,

Québec, Canada) for the mRNA quantification of 3α-HSD3 and 3α-HSD4. The primer

sequences of 3α-HSD3 (5’-CCTAAAAGTAAAGCTCTAGAGGCCGT-3’; 5’-CAACTCT-

GGTCGATGGGAATTGCT-3’) and 3α-HSD4 (5’-AAAGTAAAGCTTTAGAGGCC-

ACC-3’; 5’-AAATGAATAAGGTAGAGGTCAACATAA-3’) were designed based on the

previous studies 26; 45

.

3.3.8 Determination of 5α-DHT levels by Elisa assay

MCF7 cells were seeded into 24-well plate at 50,000 cells per well with 0.5 ml medium

treated with dextran-coated charcoal. After cultured for 24 hours, cells were transfected

with 100 nM 3α-HSD3 specific siRNA or negative control siRNA and grown for another

48 hours. Then, the cell culture medium was replaced with fresh charcoal treated medium

containing 0.7 nM 5α-DHT. Supernatants of the cell culture medium were collected after

48 hours. Following the protocol of its supplier, 5α-DHT levels in supernatants were

determined by using a commercial 5α-DHT ELISA kit.

3.3.9 Cell proliferation assay

Cell proliferation was determined by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide) assay. MCF7 cells were seeded into 24-well plate as the way

used in Elisa assay and cultured for 24 hours. Next, cells were transfected with 100 nM 3α-

HSD3 specific siRNA or negative control siRNA. After another 24 hours cell culture, the

68

culture medium was replaced with fresh charcoal treated medium containing different

concentrations of 5α-DHT (0.1 nM or 1.0 nM). Cell growth study was performed for 96

hours and the medium was replaced every 48 hours. MTT assay was carried out as

described previously 7. Absorbance was read at 570 nm with a reference at 650 nm.

3.4 RESULTS

3.4.1 Crystal growth of human 3α -HSD3 in complex with A-dione/epi-ADT and 4-dione

5α-DHT is the main steroid transformed by human 3α-HSD3. However, its complex

structure with the enzyme is still not available. At the beginning, crystal growth of human

3α-HSD3 in complex with 5α-DHT has been performed using the reported crystallization

condition, which includes 100 mM sodium citrate (pH 5.6), 260 mM ammonium acetate, 1

mM 5α-DHT, 26-27% (w/v) polyethylene glycol (PEG) 4000 32

. After obtaining the

crystal, the solved crystal structure showed that acetate and citrate molecules were deeply

bound in the steroid binding site, while 5α-DHT could not be observed. The whole

structure resembles the reported human 3α-HSD3·NADP+·citrate/acetate complex

34. To

solve this problem, the crystallization condition was adjusted to 100 mM sodium

cacodylate (pH 6.0), 200 mM ammonium sulfate, 1 mM 5α-DHT and 24-26% (w/v)

polyethylene glycol (PEG) 3350. By doing so, the crystal was obtained and the electron

density of the steroid was shown in the steroid binding site. Surprisingly, the solved

structure shows that the bound steroids can be fitted with A-dione and epi-ADT, but not

with 5α-DHT. The oxidoreduction of 5α-DHT happened during the crystallization and a

mixture of steroids including A-dione and epi-ADT were generated (Figure 3.1), which

phenomenon was also observed in human steroid 5β-reductase co-crystallization with 5α-

DHT 46

. In addition, in the previously reported 3α-HSD3·NADP+·Testo/acetate complex

32,

the acetate molecule with high concentration (260 mM) from the crystallization condition

saturates the active site of the enzyme and can distort the actual steroid binding

conformation. Therefore, human 3α-HSD3 has been co-crystallized with Testo by using an

adjusted crystallization condition without the acetate molecule.

69

3.4.2 Overall structures of human 3α-HSD3 in complex with A-dione/epi-ADT and 4-

dione

The crystal structures of the 3α-HSD3·NADP+·A-dione/epi-ADT complex and the 3α-

HSD3·NADP+·4-dione complex both possess the monoclinic space group of P21, and there

are two monomers (A and B) included in an asymmetric unit (Figure 3.2(A)). The 3α-

HSD3·NADP+·A-dione/epi-ADT complex was refined at 1.20 Å with a crystallographic R-

factor of 15.5% and an Rfree-factor of 17.4%. The 3α-HSD3·NADP+·4-dione complex was

refined at 1.75 Å with an R-factor of 16.4% and an Rfree-factor of 20.5%. The protein

backbones of two complexes are quite similar to each other and the root mean square

deviations (RMSD) of the backbone Cα atoms in both monomer A is 0.42 Å. However,

loop A (residues 117-143) of two complexes undergoes a considerable conformational

variation and the RMSD value for 27 Cα atoms of loop A in both monomer A is 1.32 Å. It

is noteworthy that monomer A owns a lower thermal B-factor than that of monomer B in

two complexes. The cofactor is tightly bound in the enzyme with an extended conformation

and its binding mode is almost unchanged in different complexes 32; 33; 34

. Numbers of

hydrogen bonds, salt bridges and Van der Waals interactions contribute to the stabilization

of NADP+ in the enzyme as described previously

32; 33. The Ramachandran plot shows that

above 93% residues of two complexes are located in the most favored regions. Data

collection and refinement statistics are summarized in Table 3.1.

3.4.3 Human 3α-HSD3 in complex with A-dione/epi-ADT

The human 3α-HSD3·NADP+·A-dione/epi-ADT complex was obtained by co-

crystallization in the presence of 5α-DHT. The difference Fourier map defines that two

different steroids exist in the cavity of monomer A and B, respectively. In monomer A, A-

dione well depicts the majority of electron density map with the lower average B-factors. In

monomer B, epi-ADT agrees with the electron density map with the lower average B-

factors. However, partially weak electron density can also be found to accommodate epi-

ADT in monomer A, and correspondingly, a similar phenomenon can be observed in

monomer B. Nevertheless, A-dione and epi-ADT represent the major steroid binding mode

in monomer A and B, respectively, and were determined in the structure. To be surprised,

although 5α-DHT was introduced during the crystallization, the observed electron density

70

defined that A-dione or epi-ADT bound into the cavity. Thus, reactions that simulating the

crystallization condition were performed by following the instructions in “METHODS”.

The results show that 5α-DHT is oxidized to A-dione in the presence of 1 mM NADP+, and

then 5α-DHT and A-dione are reduced to 3α-diol/3β-diol and ADT/epi-ADT, respectively

(Figure 3.1). With the overnight incubation, A-dione and epi-ADT hold the percentage of

26% and 17% among the mixture of steroids. Moreover, the GC/MS analysis further

indicates that the outputs ratio of 3α-diol/3β-diol and ADT/epi-ADT are about 2.2:1 and

2.1:1, respectively.

In monomer A, the O17 atom of A-dione is oriented toward the nicotinamide ring of

NADP+ (Figure 3.2(B)). The α-face of A-dione interacts with the side chains of Val54 and

Val128. Its β-face mainly interacts with the indole ring of Trp227, and the steroid C18 and

C19 methyl groups are pointing toward Trp227. It is worth mentioning that a water

molecule is located at the catalytic core and forms hydrogen bonds with the side chains of

Tyr55, His117 and the O17 atom of A-dione. In monomer B, the O3 atom of epi-ADT is

pointing toward the nicotinamide ring (Figure 3.2(C)). The α-face of epi-ADT is stacking

with the indole ring of Trp227, and the steroid C18 and C19 methyl groups are pointing

toward Val54 and Tyr55. Particularly, the O3 atom of epi-ADT forms a hydrogen bond

with His222 Nε atom. When monomer A and B are superimposed, the O3 and O17 atoms of

both steroids are oriented upside down to each other, and the planes of both steroids are

flipped about 145° around the long axis of C3-C17 (Figure 3.2(D)). After superposition, the

RMSD value is 0.43 Å for their backbone Cα atoms. In both monomers, loop A and a short

loop (residues 23-33) possess considerable fluctuation and the RMSD values for their Cα

atoms are 1.06 Å and 1.05 Å, respectively. The residues of Val54, Tyr55, His 117, His222

and Leu308, which belong to the steroid binding pocket, are relatively static, while the side

chains of Val128, Ile129 and Leu306 display apparent movement to consort with steroids.

In particular, the side chain of Leu306 contains two alternative conformations in both

monomers, which may correspond to the two different orientations of steroids.

3.4.4 Human 3α-HSD3 in complex with 4-dione

The human 3α-HSD3·NADP+·4-dione complex was obtained by co-crystallization with

Testo in the presence of NADP+. During the crystallization process, the oxidation of Testo

71

occured and 4-dione was generated (Figure 3.3). The difference Fourier map clearly shows

that 4-dione is located in the steroid binding site of each monomer (Figure 3.4(A)). When

monomer A and B are superimposed, the orientation and conformation of 4-dione are quite

similar to each other and only a small shift of the steroid plane toward the indole ring of

Trp227 can be observed (Figure 3.4(B)). The backbones of monomer A and B overlap each

other quite well, and the RMSD value for the backbone Cα atom is 0.36 Å. Loop A still

displays its flexibility and the RMSD value for its 27 Cα atom is 0.96 Å. The side chains of

Val54, Val128, Ile129 and Leu308 in the steroid binding pocket also exhibit subtle

adjustments in two monomers. The C3 atom and the A-ring of 4-dione is pointing toward

the nicotinamide ring. The α-face of 4-dione interacts with the side chain of Trp227, and

the C18 and C19 methyl groups of 4-dione are pointing toward the side chain of His117.

The O3 atom of 4-dione forms a hydrogen bond with His222 Nε atom.

Compared with the orientation of Testo in the reported 3α-HSD3·NADP+·Testo/acetate

complex 32

, it can be clearly seen that the plane of 4-dione in the 3α-HSD3·NADP+·4-dione

complex generates an about 45° rotation around its C3-C17 long axis (Figure 3.4(B)).

Meanwhile, the steroid goes deeper into the cavity of the enzyme, and the distance between

the C3 atoms of steroids and the C4 atom of the nicotinamide ring is changed to 4.3 Å from

the former 6.2 Å. The acetate molecule in the Testo/acetate complex, which is originated

from the crystallization condition, is situated between the steroid and the cofactor, and

occupies the catalytic site of the enzyme. Obviously, the repulsion between the O3 atom of

Testo and the ketone group of acetate remarkably twists the steroid orientation. However,

the acetate molecule is replaced by a water molecule in the 4-dione complex, and the latter

forms hydrogen bonds with the side chains of Tyr55 and His117. Moreover, the side chains

of Val54, Ile129, Trp227, Leu306, Leu308 and the nicotinamide ring have slight

adjustments to accommodate the more suitable orientation of the steroid.

3.4.5 3α-HSD3 expression and siRNA specific knockdown in MCF7 cells

The mRNA transcription of human 3α-HSD3 and 3α-HSD4 in MCF7 cells was evaluated

by quantitative RT-PCR. The results show that the mRNA level of 3α-HSD3 is about 2.5

times that of 3α-HSD4 in MCF7 cells (Figure 3.5(A)). Due to the high similarity of 3α-

HSD3 and 3α-HSD4 on the nucleotide sequences, the selective knockdown of the 3α-HSD3

72

expression, without interferencing with 3α-HSD4, was performed by a specific siRNA.

Compared with control siRNA treated cells, the quantitative RT-PCR results show that 3α-

HSD3 specific siRNA significantly suppresses 3α-HSD3 expression, while the expression

of 3α-HSD4 is affected negligibly (Figure 3.5(B)).

3.4.6 Suppression of 3α-HSD3 expression increases 5α-DHT concentration in MCF7

cells

To investigate the impact of 3α-HSD3 expression on 5α-DHT concentration in MCF7 cells,

ELISA assays were carried out using supernatants from the cell culture medium (Figure

3.6(A)). After siRNA treatment, MCF7 cells were cultured for 48 hours with the charcoal

treated medium containing 0.7 nM (203 pg/ml) 5α-DHT. Compared with the control siRNA

treated groups, the 5α-DHT concentration from the 3α-HSD3 specific siRNA treated group

shows significantly higher 5α-DHT concentration (about 154 pg/ml), which is about 2.3

times the value of the control group (about 68 pg/ml).

3.4.7 Suppression of 3α-HSD3 expression decreases MCF7 cell proliferation in the

presence of 5α-DHT

To assess the potential role of 3α-HSD3 on MCF7 cell proliferation, siRNA specific

knockdown of 3α-HSD3 expression in MCF7 cells was performed. After siRNA treatment,

MCF7 cells were cultured for 96 hours with the charcoal treated medium containing 0.1 nM

or 1.0 nM 5α-DHT. Compared with MCF7 cells transfected with control siRNA, MCF7

cells transfected with 3α-HSD3 specific siRNA exhibits significantly decreased growth

rate, with about 20% or 25% reduction of the cell growth under the cultured conditions

containing 1.0 nM or 0.1 nM 5α-DHT, respectively (Figure 3.6(B)).

3.5 DISCUSSION

In order to recognize the molecular characteristics of 5α-DHT binding to human 3α-HSD3,

a high resolution crystal was obtained by co-crystallization in the presence of 5α-DHT and

NADP+. However, during the crystallization process, 5α-DHT initiated a series of

oxidoreduction and generated a mixture of steroids. Therefore, A-dione and epi-ADT were

identified, which located in two monomers (A and B) of the enzyme in a crystal

73

asymmetric unit, respectively. In monomer A, the O17 atom of A-dione is pointing inside

into the catalytic site of the enzyme, which means 5α-DHT has been oxidized on its C17

hydroxyl group to output A-dione and then the generated A-dione resides in the cavity of

the enzyme. In monomer B, the O3 atom of epi-ADT is oriented toward the catalytic site of

the enzyme, which implies that A-dione has been reduced on its C3 carbonyl group to

produce ADT and epi-ADT, while only epi-ADT is retained in the cavity of the enzyme.

Although the assay of 5α-DHT oxidoreduction mimicking the crystallization process shows

that 5α-DHT has also been reduced to 3α-diol and 3β-diol, resembling to ADT, these two

steroids have been released from the cavity after the reactions. Nevertheless, the different

orientations of A-dione and epi-ADT provide the structural clues of 5α-DHT alternative

binding in the cavity of the enzyme to generate different steroids. In addition, both loop A

of monomer A and B possess different neighboring environments from the nearby

symmetric molecules and display an apparent flexibility. In addition, in the 3α-

HSD3·NADP+·4-dione complex, Testo was introduced before crystallization, while the

observed electron density in the cavity agreed with 4-dione. That means the C17 hydroxyl

group of Testo firstly enters into the catalytic site of 3α-HSD3, and Testo is oxidized to 4-

dione in the presence of the cofactor. Then, 4-dione is released from the catalytic site and

reenters with its C3 carbonyl group pointing inside. Interestingly, this process is reversed in

the former 3α-HSD3·NADP+·Testo/acetate complex

32. Therefore, the evidence further

supports the alternative binding modes of steroids in 3α-HSD3.

The alternative binding modes of steroids in enzymes have also been found in other

hydroxysteroid dehydrogenase such as human 17β-hydroxysteroid dehydrogenase type 1

(17β-HSD1), 3α-HSD2 (also named 17β-HSD5 or AKR1C3). In human 17β-HSD1, 5α-

DHT can be oxidized to A-dione and simultaneously be reduced to 3β-diol, which implies

the existence of the alternative binding orientations of 5α-DHT in the enzyme 42

.

Meanwhile, the structural study further verify that androgens such as Testo, A-dione and 4-

dione can bind in reverse orientation within the enzyme compared with the orientations of

estrogens such as 17β-estradiol 42; 47

. In human 3α-HSD2, the crystal structure shows that

two reverse orientations of Testo can be observed in the cavity of the enzyme 48

. Moreover,

for human steroid 5β-reductase (also named AKR1D1), the enzyme E120H mutant was co-

crystallized with 5α-DHT 46

. During the crystallization process, the oxidoreduction of 5α-

74

DHT occurred and produced a mixture of steroids. 3β-diol and epi-ADT were found to

occupy the cavity of monomer A and B in the crystal asymmetric unit, which indicated that

the alternative binding modes of steroids in the enzyme corresponds with the

multispecificity of the enzyme. Taken together, the alternative binding modes of steroids in

the enzyme originates from the pseudo self-symmetry property of steroids and the

flexibility of the steroid binding pocket.

Recently, the reported human 3α-HSD3·NADP+·progesterone complex shows that although

the D-ring of the steroid is pointing toward the cavity of the enzyme, progesterone

possesses two different orientations in the cavity. When superimposed with the 3α-

HSD3·NADP+·A-dione/epi-ADT complex, in both monomer A, the orientations of A-dione

and progesterone resemble with each other, and only the D-rings of two steroids show a

little detachment (Figure 3.7(A)). The C18 and C19 methyl groups of two steroids are

pointing toward Trp227. The side chains of Val54, His222, Leu306 and Leu308 are

flipping about 180°, which reflects the subtle flexibility of the steroid binding site. Besides,

in both monomer B, although the A, B, C and D rings of epi-ADT and progesterone are

located reversely to each other, the orientations of two steroids planes are roughly identical

to each other, and both α-face of steroids interact with the side chain of Trp227 (Figure

3.7(B)). In addition, in the reported 3α-HSD3·NADP+·ursodeoxycholate complex, an

overlay shows that the overall orientation of ursodeoxycholate is similar to that of epi-ADT

in the cavity, and there is only a little swing between two ligands around the vertical axis of

the steroid plane (Figure 3.7(C)). Therefore, two major orientations of steroids can be

found in the cavity of 3α-HSD3, which further implies the steroid binding site of 3α-HSD3

possessing considerable flexibility.

In the 3α-HSD3·NADP+·4-dione complex, the overall conformation of the enzyme is quite

identical to monomer A of the 3α-HSD3·NADP+·progesterone complex, and the RMSD

value is only 0.14 Å between the backbone Cα atoms of both monomer A. Although an

overlay shows that two steroids are oriented back to back as well as their C18 and C19

methyl groups are pointing toward the opposite directions, the orientations of two steroids

resemble with each other (Figure 3.7(D)). This indicates that the steroid binding pocket of

3α-HSD3 has sufficient space and flexibility to allow the planes of steroids flipping inside.

75

Previously, compared with benign tissue, an increased 5α-DHT concentration as well as a

decreased expression of 3α-HSD3 in prostate tumor was observed by Stolz and coworkers

16; 26. They speculated that the loss of 3α-HSD3 in prostate cancer cells increased 5α-DHT

dependent cell growth. As known, through binding to AR, excessive 5α-DHT leads to the

stimulation of prostate cancer proliferation. 3α-HSD3 eliminates 5α-DHT in prostate and

this can prevent AR activation from excessive androgens. In addition, they reported that, in

the presence of progesterone, specific knocking down of 3α-HSD4 alone or with 3α-HSD3

together in T47D cells led to decreased cell growth 43

. The former studies indicate that 5α-

DHT still plays an important role of antiproliferation in breast cancer cells, which is

mediated by AR 5; 6; 49; 50

. Human 3α-HSD3 is characterized by its major ability to

inactivate 5α-DHT, which prompts us to explore its role in breast cancer cells. In this study,

the 3α-HSD3 expression was knocked down by specific siRNA in MCF7 cells, and a

relative elevation of 5α-DHT level was observed by using ELISA assay. Meanwhile, in the

presence of 5α-DHT, suppression of 3α-HSD3 expression by specific siRNA resulted in a

decrease of MCF7 cell growth, which suggested that the suppressed expression of 3α-

HSD3 strengthened the antiproliferative effect of 5α-DHT in MCF7 cells. In addition to the

outstanding ability of estradiol (E2) production, human 17β-hydroxysteroid dehydrogenase

type 1 (17β-HSD1) also displays an ability to inactivate 5α-DHT 42

. Suppression of 17β-

HSD1 expression was shown to increase 5α-DHT level in T47D cells, which showed an

antiproliferative effect 7. However, compared with 5α-DHT inactivation ability of 17β-

HSD1 (Km = 32.0 μM, kcat = 2.40 min-1


μM-1

) 42

, 3α-HSD3

possesses a significantly higher activity to reduce 5α-DHT (Km = 1.1 μM, kcat = 1.5 min-1


μM-1

) 32

, which implies that 3α-HSD3 may play a more important

role than 17β-HSD1 to inactivate 5α-DHT.

3.6 FUNDING SOURCES

This work was supported by the Canadian Institutes of Health Research (CIHR), with a

grant to Dr. Sheng-Xiang Lin and collaborators (MOP 97917). Use of the Advanced Photon

Source, an Office of Science User Facility operated for the U.S. Department of Energy

(DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE

under Contract No.DE-AC02-06CH11357. Use of the Lilly Research Laboratories

76

Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon

Source was provided by Eli Lilly Company, which operates the facility. The Canadian

Macromolecular Crystallography Facility (CMCF) is supported by CFI, NSERC, and CIHR.

3.7 ACKNOWLEDGEMENTS

We thank Dr. Donald Poirier for offering us several steroids and the helpful discussions.

We thank Ms. Nathalie Paquet, Mr. Patrick Bélanger for the services of the q-RTPCR

analysis and the GC/MS analysis, who are from the q-RTPCR platform, the bioanalytical

platform (Centre Hospitalier Universitaire (CHU) de Quebec Research Center (CHUL) and

Laval University, Québec), respectively. We thank PROTEO (The Quebec Network for

Research on Protein Function, Structure, and Engineering) and Dr. Albert Berghuis’

laboratory at McGill University for providing the X-ray crystallography facilities for our

initial X-ray datasets collection. We thank Dr. Preyesh Stephen for the revision of the

manuscript. Bo Zhang thanks a national scholarship provided by China Scholarship

Council (CSC) and the CIHR grant (MOP 97917) to support his graduate study. X-ray

diffraction datasets were collected at LRL-CAT at the Advanced Photon Source, Argonne

National Laboratory, and at the CMCF, Canadian Light Source.

77

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83

Table 3.1 Data collection and refinement statistics

Structure 3α-HSD3

NADP+·A-dione/epi-ADT

3α-HSD3

NADP+·4-dione

A. Data collection

Unit cell dimensions

a, b, c (Å)

β, α=γ (degrees)

54.64, 86.95, 76.72

107.10, 90.00

52.90, 86.65, 76.99

106.25, 90.00

Space group P21 P21

Resolution range (Å) 17.64-1.20 (1.26-1.20) 25.00-1.75 (1.84-1.75)

Total reflections 759,961 (102,820) 238,631 (32,176)

Unique reflections 212,070 (30,491) 66,677 (9455)

Completeness (%) 99.4 (98.1) 99.3 (97.0)

Rmerge a

0.063 (0.334) 0.089 (0.260)

I/σ(I) 9.8 (3.3) 9.4 (4.2)

B. Refinement statistics

Reflection used (R-free set) 201,387 (10,646) 63,276 (3382)

R-factor b

0.155 0.164

R-free c

0.174 0.205

Root mean square deviations

Bond lengths (Å)

Bond angles (degrees)

0.028

2.590

0.023

2.404

Protein atoms d

NADP+ molecules

d

Steroid molecules d

Sulfate ion d

Water molecules d

5293

2

2

2

832

5165

2

2

2

335

Average B-factors (Å2)

All atoms

Protein atoms e

NADP+ e

12.8

10.8/11.4

9.6/10.5

13.7

12.2/14.2

10.9/13.1

Steroid e

24.6/26.0 28.4/33.4

84

Sulfate ion e

Water

8.3/8.8

23.4

19.3/25.6

19.2

Ramachandran statistics (%) f

Most favored regions

Additionally allowed regions

Generously allowed regions

Disallowed regions

93.6

6.1

0.0

0.3

94.9

4.7

0.0

0.3

a Rmerge = ΣΣi|I(h)-I(h)i|/ΣΣiI(h), where I(h) is the mean intensity after rejections.

b R-factor = Rcryst = Σ(|FO|-|FC|)/Σ|FO|, where |FO| and |FC| are the observed and calculated

structure factor amplitudes for the reflection with Miller indices h = (h, k, l), respectively.

c R-free is calculated for a “test” set of reflections, which was not included in refinement.

d Per asymmetric unit.

e Average B-factors were calculated separately for two monomers within an asymmetric

unit (monomer A/monomer B).

f Ramachandran statistics was calculated with PROCHECK in COOT. Ser221 in both

monomers of human 3α-HSD3 were found in the disallowed regions.

85

Figure 3.1 The oxidoreduction of 5α-DHT catalyzed by human 3α-HSD3 in the

presence of cofactors

(A) Reactions catalyzed by human 3α-HSD3 in the presence of cofactors. (B) An assay

mimicking the crystallization condition in the presence of NADP+ and

14C-labelled 5α-

DHT. Lane 1, the reaction without 3α-HSD3; lane 2, the reaction with 3α-HSD3.

Compared with the migration of different steroids (Anne Gangloff et al., FASEB J.,

2003,17(2):274-6), the results showed that the oxidoreduction of 5α-DHT occurred during

the crystallization and produced a mixture of products that including A-dione, ADT, epi-

ADT, 3α-diol, 3β-diol. These products were further validated by the GC/MS analysis.

86

Figure 3.2 Structure of the 3α-HSD3·NADP+·A-dione/epi-ADT complex

(A) The crystal asymmetric unit contains two monomers. Monomer A colored in magenta

is the 3α-HSD3·NADP+·A-dione complex. Monomer B colored in cyan is the 3α-

HSD3·NADP+·epi-ADT complex. Atoms are colored as follows. Green, carbon atoms of

steroids and cofactors; blue, nitrogen; red, oxygen; orange, phosphorus. (B) The steroid

binding site of monomer A. The 2Fo-Fc electron density map around A-dione is contoured

at 0.7 σ level. Protein carbon is colored in magenta. A water molecule is located at the

catalytic site. (C) The steroid binding site of monomer B. The 2Fo-Fc electron density map

around epi-ADT is contoured at 0.7 σ level. Protein carbon is colored in cyan. Hydrogen

bonds in (B) and (C) are shown by black dashed lines. (D) Superposition of the steroid

binding sites of monomer A and B. For clarity, carbon atoms of steroids and cofactors in

monomer A and B are colored in magenta and cyan, respectively. The planes of A-dione

and epi-ADT are oriented upside down and flipped.

89

Figure 3.3 The oxidation of testosterone catalyzed by human 3α-HSD3 in the

presence of NADP+

(A) Reactions catalyzed by human 3α-HSD3 in the presence of cofactors. (B) An assay

mimicking the crystallization condition in the presence of NADP+

and 14

C-labelled Testo.

Lane 1, the reaction without 3α-HSD3; lane 2, the reaction with 3α-HSD3. The result

showed that the oxidation of Testo occurred during the crystallization and 4-dione was

generated.

90

Figure 3.4 Structure of the 3α-HSD3·NADP+·4-dione complex

(A) The steroid binding site of monomer A in the 3α-HSD3·NADP+·4-dione complex. The

2Fo-Fc electron density map around 4-dione is contoured at 0.8 σ level. Atoms are colored

as follows. Green, carbon; blue, nitrogen; red, oxygen; orange, phosphorus. Protein chain is

colored in green. A water molecule is located at the catalytic site. Hydrogen bonds are

shown by black dashed lines. (B) Superposition of the steroid binding sites of monomer A

and B in the 3α-HSD3·NADP+·4-dione complex, and that of monomer A in the 3α-

HSD3·NADP+·Testo/acetate complex (PDB code 1J96; carbon and protein chain are

colored in orange; an acetate molecule is located at the catalytic site and distorts the steroid

binding conformation). Carbon and protein chain of monomer B in the 3α-

HSD3·NADP+·4-dione complex are colored in dark gray.

92

Figure 3.5 Human 3α-HSD3 expression and knockdown by siRNA in MCF7 cells

(A) The relative mRNA levels of human 3α-HSD3 and 3α-HSD4 in MCF7 cells were

evaluated by quantitative RT-PCR. (B) Compared with the control siRNA treated cells, the

quantitative RT-PCR results showed that 3α-HSD3 specific siRNA substantially

suppressed 3α-HSD3 expression but not 3α-HSD4. Experiments were repeated three times.

93

Figure 3.6 Suppression of 3α-HSD3 in MCF7 cells enhanced 5α-DHT-mediated

inhibition of cell growth

(A) After siRNA transfection, MCF7 cells were cultured for 48 hours with the charcoal-

treated medium containing 0.7 nM (203 pg/ml) 5α-DHT. Steroid concentrations were

determined by ELISA assay using supernatants from the cell culture medium. Compared

with the control siRNA treated groups, the 5α-DHT concentration from the 3α-HSD3

specific siRNA treated group showed significantly increased 5α-DHT concentration.

Experiments were repeated three times in triplicate. (B) After siRNA transfection, MCF7

cells were cultured for 96 hours with the charcoal-treated medium containing 0.1 nM or 1.0

nM 5α-DHT. Cell growth was evaluated by MTT assay. Experiments were repeated three

times in triplicate.

94

Figure 3.7 Superposition of the steroid binding sites of the related 3α-HSD3

structures

(A) Superposition of the steroid binding sites of two monomer A in the 3α-

HSD3·NADP+·A-dione/epi-ADT complex (carbon and protein chain are colored in

magenta) and the 3α-HSD3·NADP+·progesterone complex (carbon and protein chain are

colored in dark gray). The other atoms are colored as follows. Blue, nitrogen; red, oxygen;

orange, phosphorus. (B) Superposition of the steroid binding sites of two monomer B in the

3α-HSD3·NADP+·A-dione/epi-ADT complex (carbon and protein chain are colored in

cyan) and the 3α-HSD3·NADP+·progesterone complex (carbon and protein chain are

colored in orange). (C) Superposition of the steroid binding sites of two monomer B in the

3α-HSD3·NADP+·A-dione/epi-ADT complex and the 3α-HSD3·NADP

+·ursodeoxycholate

complex (carbon and protein chain are colored in pink). (D) Superposition of the steroid

binding sites of two monomer A in the 3α-HSD3·NADP+·4-dione complex (carbon and

protein chain are colored in green) and the 3α-HSD3·NADP+·progesterone complex.

97

CHAPTER 4 PURIFICATION AND PRELIMINARY

CRYSTALLOGENESIS OF HUMAN ESTROGEN RECEPTOR

ALPHA

98

4.0 PREFACE

Although there are more than twenty years of the structural studies on estrogen receptor

alpha (ERα), due to the difficulties in protein expression, purification and crystallization,

the full-length ERα structure remains an unfulfilled task. Recently, more and more studies

showed that the DNA binding domain (DBD) and the ligand binding domain (LBD) of

nuclear receptors (NRs), which including ERα DBD and LBD, exerted their functions as a

whole. The mechanism of bi-directional regulation between ERα DBD and LBD should be

investigated further. Therefore, we determined to establish an expression and purification

protocol of the ERα fragment containing its DBD-LBD domain, and to crystallize ERα

DBD-LBD in complex with estrogen response elements (EREs).

In addition, several methods have been developed previously to purify ERα LBD, such as

carboxymethylations or mutations of reactive cysteine residues of ERα LBD. During the

purification, the target protein was found mainly existing in the precipitate. Therefore, we

developed a simple and efficient purification method for ERα LBD, which used a

combination of detergents to purify ERα LBD from the precipitate.

This chapter includes two papers: (1) Expression, purification and preliminary

crystallogenesis of human estrogen receptor alpha DBD-LBD. In this paper, I carried out

the experimental studies and wrote the manuscript. Dr. Sheng-Xiang Lin and I designed the

study. (2) A simple and efficient purification method for human estrogen receptor alpha

LBD. In this paper, I carried out the experimental studies and wrote the manuscript. Mr.

Jean-François Thériault, as a summer student, prepared some SDS-PAGE gels. Dr. Sheng-

Xiang Lin, Dr. Dao-Wei Zhu and I designed the study.

99

4.1 EXPRESSION, PURIFICATION AND PRELIMINARY

CRYSTALLOGENESIS OF HUMAN ESTROGEN RECEPTOR ALPHA

DBD-LBD

Expression, purification and preliminary crystallogenesis of human

estrogen receptor alpha DBD-LBD

Bo Zhang 1, 2

, Sheng-Xiang Lin 1, §



G1V4G2, Canada







Shortened title: purification and preliminary crystallogenesis of human ERα DBD-LBD


100

4.1.1 Abstract

Through binding to 17β-estradiol, estrogen receptor alpha (ERα) forms a homodimer and

translocates from the cytoplasm into nucleus to initialize the transcription of specific genes.

ERα plays important physiological functions in human such as controlling sexual

development and reproductive functions. Due to difficulties in protein expression,

purification and crystallization, the full-length ERα structure remains an unfulfilled task. In

this study, we established an expression and purification protocol of the ERα fragment

containing the DNA binding domain and the ligand binding domain (DBD-LBD) of ERα.

Initially, ERα DBD-LBD with the C530A single point mutation was expressed in E.coli

Rosetta2 (DE3) cells. The expression temperature for the target protein was optimized at

25 °C for 12 hours, and 0.2 mM IPTG was used to induce the target protein for expression

in the supernatant with an acceptable quantity. During the purification, the target protein

was prone to aggregation. High salt concentration was found significantly preventing the

protein aggregation and increasing the solubility of ERα DBD-LBD. Through two steps of

purification by the nickel column and the gel filtration column, ERα DBD-LDB was

concentrated to 6.0 mg/ml with the purity above 95%. Preliminary crystallogenesis of ERα

DBD-LBD in complex with estrogen response elements (EREs) was performed. This study

provided basic knowledge to facilitate the acquisition of the fine crystals for ERα DBD-

LBD in complex with EREs.

Keywords: estrogen receptor alpha (ERα), the DNA binding domain (DBD), the ligand

binding domain (LBD), detergent, purification, preliminary crystallogenesis.

101

4.1.2 Introduction

Estrogen receptor alpha (ERα) is a well defined member of Nuclear Receptor superfamily

(NRs). Through binding to 17β-estradiol (E2), ERα controls sexual development and

reproductive functions, and also affects bone, cardiovascular, immune and central nervous

systems 1. Similar to all the members of NRs, ERα contains five structural regions: the N-

terminal region (A/B region: 1-180 amino acids (aa)), the DNA binding domain (DBD or C

region: 181-263aa), the hinge region (D region: 264-302aa), the ligand binding domain

(LBD or E region: 303-552aa), and the C-terminal region (F region: 553-595aa).

The structural studies on NRs LBDs and DBDs have been carried out for more than twenty

years. The solution and crystal structures of ERα DBD without or with estrogen response

elements (EREs) were firstly reported by Schwabe et al 2; 3; 4

. Several years later, the crystal

structures of ERα LBD bound to agonists, antagonists and coactivator were released 5; 6; 7

.

Together with other reported NRs DBDs and LBDs, these structures provided remarkable

insights into the structural and functional basis of NRs and significantly improved the

knowledge of rational drug design against these targets 8; 9; 10; 11; 12; 13; 14; 15

. However, the

purification processes of NRs DBDs and LBDs are full of challenges. NRs DBDs are prone

to aggregation and their solubility is relatively low in solutions 16

. NRs LBDs are unstable

in the absence of their ligands. In particularly, ERα LBD is notorious for aggregation due to

three reactive cysteine residues (Cys381, Cys417 and Cys530). Several methods have been

developed to purify ERα LBD such as carboxymethylation or mutation of cysteine residues

5; 6; 17, denaturation and renaturation

7, or application of reducing agents or fusion protein

18;

19.

Due to the difficulties in protein expression, purification and crystallization, the full-length

NRs structure remains unfulfilled 20

. In 2008, Rastinejad’s group reported the first full-

length structures of intact NRs (PPARγ-RXRα heterodimer) bound to their DNA target 21

.

They found that PPARγ LBD directly impacted the binding of PPARγ DBD to its DNA

elements, which challenged the long-held notion that the NRs DBDs and LBDs maintained

their autonomous and self-reliant functions. Recently, Figueira et al. also reported that

thyroid receptor (TR) DBD-LBD had a higher binding affinity to its DNA elements than

the isolated DBD 22

. Furthermore, the hormone response elements, possessing specific

102

DNA sequences, can regulate ligands binding and receptor activation 15; 23; 24

. Thus, the

mechanism of bidirectional regulation between NRs DBDs and LBDs should be exploited

in depth. It is obvious that only the PPARγ-RXRα heterodimer structure is not adequate to

provide a model that how NRs homodimers are assembled to exert their functions properly.

More structural studies are required to fully understand the functional relationship between

NRs DBDs and LBDs. It has been well established that the N-terminal region of ERα is

intrinsically disordered and this part does not have a stable conformation. Therefore,

expression, purification and preliminary crystallogenesis of human ERα DBD-LBD are the

objectives of this study.

4.1.3 Methods

4.1.3.1 Plasmids construction

The human full-length ERα plasmid was purchased from Genecopoeia (Shanghai, China).

Two nucleotide sequences encoding ERα 179-554aa (amino acids) and 179-595aa were

amplified by PCR and were inserted into the pGEM-T vector, respectively, which included

ERα DBD-LBD. The unique forward primer contained the BamHI site: 5’-

CGCGGATCCGCCAAGGAGACTCGCTAC-3’ and each of the two reverse primers

contained the XhoI site: 5’-CCGCTCGAGTTAGCTAGTGGGCGCATGT-3’, 5’-

CCGCTCGAGTCAGACCGTGGCAGGG-3’. After validation by sequencing, the

recombinant pGEM-T vectors were digested with BamHI and XhoI, and two nucleotide

segments encoding ERα DBD-LBD were cloned into the corresponding sites of the pET-

28a(+) vectors.

4.1.3.2 Site-directed mutagenesis of ERα C530A

The recombinant pET-28a(+) vectors encoding human ERα 179-554aa and 179-595aa were

used as the templates for conducting the site-directed mutagenesis of C530A. The

mutations were carried out by using QuikChange Lightning Kit (Agilent Technologies)

with 5’-CTGTACAGCATGAAGGCCAAGAACGTGGTGCC-3’ as forward primer and

verified by sequencing.

4.1.3.3 Protein expression and purification

http://en.wikipedia.org/wiki/N-terminus

103

The pET-28a(+) constructs encoding ERα 179-554aa and 179-595aa with the C530A single

point mutations were expressed in E.coli Rosetta2 (DE3) cells. LB medium was

supplemented with 50 μg/ml kanamycin and 35 μg/ml chloramphenicol. The cells were

cultivated at 37 °C for 3 hours until OD600 reached 0.7, and protein expression was induced

with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for an additional 12 hours at

25 °C in the presence of 20 μM estradiol. The cells were harvested by centrifugation and

the pellets were stored at -80 °C. The frozen cells were thawed in buffer A (50 mM Tris-

HCl (pH 7.4), 10 mM imidazole, 500 mM NaCl, 10% glycerol, 20 mM β-mercaptoethanol,

1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μM estradiol, 0.1% β-OG (β-Octyl

glucoside)) with the addition of 1 mg/ml lysozyme and 1 μg/ml each of protease inhibitors

(leupeptin, chymostatin, antipain, aprotinin, pepstatin A and benzamidin). After incubation

on ice for 20 minutes, the cells were lysed by sonication. The supernatant was obtained via

centrifugation at 181,000 g for 50 minutes and loaded onto a 3 ml Ni-NTA agarose column

pre-equilibrated with buffer A, followed by incubation at 4 °C for 3 hours. Afterwards, the

nickel column was washed with buffer A containing an imidazole gradient from 30 mM to

200 mM. Peak fractions were pooled and concentrated using an ultra centrifugal filter with

a 30 kDa molecular-weight cutoff (Millipore). Further separation was achieved by running

the gel filtration on a Sephacryl S-200 column previously equilibrated in buffer A without

imidazole. The eluted fractions were concentrated and the buffer was exchanged to 25 mM

Tris-HCl (pH 7.4), 250 mM NaCl, 80 mM MgCl2, 5 μM zinc chloride, 10% glycerol, 2.5

mM DTT, 20 μM estradiol and 0.1% β-OG, by three rounds of dilution and concentration.

The purity of the concentrated ERα 179-554aa and 179-595aa was estimated by SDS-

PAGE. The protein concentration was determined by the Bradford method.

4.1.3.4 Solubility test

The E.coli cells containing the ERα DBD-LBD recombinant plasmids were grown at 37 °C

in 200 ml LB media for 2.5 hours until OD600 reached 0.6. Next, the expression of the

target protein was induced with 0.2 mM, 0.5 mM or 1.0 mM IPTG at 37 °C for 4 hours or

at 18 °C overnight in the presence of 20 μM estradiol. The cell pellets were harvested and

resuspended in 10 ml buffer A, followed by sonication and centrifugation. The supernatant

was discarded. The precipitate was resuspended again in 10 ml buffer A without 0.1% β-

104

OG and each solubility test used 0.5 ml resuspended sample. 0.4% (w/v) each of Triton X-

100, C12E8 (Octaethylene glycol monododecyl ether), β-DDM (dodecyl-β-D-maltoside), β-

OG, Emulgen913 or 0.5 M NDSB-201 (Non detergent sulfobetaines) was added to the 0.5

ml sample, respectively. Each sample was slowly agitated overnight at 4 °C and the results

were verified by SDS-PAGE.

4.1.3.5 Western blot analysis

Western blot was performed using primary rabbit anti-ERα polyclonal antibody (ab70170)

(Abcam) with a 1:10,000 dilution. Horseradish peroxidase-conjugated secondary goat anti-

rabbit antibody (Santa Cruz Biotechnology) was utilized by a 1:7,500 dilution. Protein

signal was detected with ECL reagents (PerkinElmer) and exposed to chemiluminescence

film (GE Healthcare).

4.1.3.6 Preliminary test of crystallization

To obtain the crystal of ERα DBD-LBD in complex with EREs, PAGE purified single-

stranded EREs were purchased from IDT Company and resuspended in 50 mM Tris-HCl

buffer (pH 7.4), 100 mM NaCl. Next, the complementary EREs strands were mixed

together at a 1:1 molar ratio, heated to 95 °C and then annealed to 5 °C over 4 hours. The

purified ERα 179-554aa and 179-595aa mixed with the double-stranded EREs at a 2:1.2

molar ratio. The crystallization was performed using the vapor diffusion method in a

hanging drop at room temperature and 4 °C. The Nextal screen (Qiagen), the Natrix screen

(Hampton Research) and several homemade screen conditions were used for the

preliminary crystallogenesis.

4.1.4 Results and discussion

4.1.4.1 Construction, expression and solubility test of ERα DBD-LBD

Following the procedures in Methods, two DNA fragments encoding human ERα DBD-

LBD (179-554aa and 179-595aa) were cloned into pET-28a(+) vector, respectively (Figure

4.1.1). The recombinant vectors with the mutations of C530A were validated by sequencing.

105

After transforming the E.coli BL21 (DE3) competent cells with the recombinant vectors,

the expression of ERα DBD-LBD was induced by 0.5 mM IPTG at 37 °C for 4 hours. Due

to a lack of the target protein expression, the E. coli Rosetta2 (DE3) competent cells were

tested as an alternative. The E. coli Rosetta2 strains contained seven tRNAs rare codons

and the expression of the target protein was appeared (Figure 4.1.2). Next, the cells were

cultivated at 37 °C until OD600 reached 0.6, and then the expression of target protein was

induced with different concentrations of IPTG at 0.2 mM, 0.5 mM, 1.0 mM and cultured

for 4 hours at 37 °C. However, after sonication and centrifugation, the target protein was

present mostly in the precipitate, not in the supernatant. Therefore, several commonly used

detergents were used for the protein solubility test. The equivalent precipitate was

resuspended in buffer A, each containing 0.4% of Triton X-100, C12E8, β-DDM, β-OG,

Emulgen913 or 0.5M NDSB-201, respectively, and slowly agitated at 4 °C overnight. After

centrifugation, the target protein was still present in the sediment. So, in this case, these

detergents had little efficacy for improving the solubility of ERα DBD-LBD (data not

shown here). Furthermore, the cells were grown at 37 °C for 3 hours until OD600 reached

0.7 and the expression of ERα 179-595aa was induced with 0.2 mM IPTG at 18 °C for 20

hours. The lower temperature facilitated the expression of the target protein in the

supernatant, but the quantity was relatively low (Figure 4.1.3(A)). Meanwhile, the

solubility tests for the precipitate treated with different detergents were also carried out and

the results showed that detergents had little effect. Finally, 0.2mM IPTG as well as a cell

culture for 12 hours at 25 °C were used to induce the expression of ERα DBD-LBD to an

acceptable quantity in the supernatant (Figure 4.1.3(B)).

4.1.4.2 Purification of ERα DBD-LBD

The frozen pellets from 2 liters of the E.coli cells expressing ERα DBD-LBD were thawed

in buffer B (50 mM Tris-HCl (pH 8.0), 10 mM imidazole, 200 mM NaCl, 10% glycerol, 20

mM β-mercaptoethanol, 1 mM PMSF and 20 μM estradiol) with the addition of lysozyme

and protease inhibitors. After sonication and centrifugation, the supernatant was loaded

onto the nickel column and incubated at 4 °C to assist binding for 3 hours. The target

protein was eluted with buffer B containing 200 mM imidazole and 0.1% β-OG. However,

immediately following the elution, the target protein began to aggregate. To address this

106

problem, the solubility test was performed. 0.4% (w/v) each of Triton X-100, C12E8, β-

DDM, β-OG, Emulgen913 or 0.5 M NDSB-201 was employed to improve the solubility of

the target protein. Only NDSB-201 was found to be mildly efficacious. Interestingly, high

salt concentrations (above 400 mM NaCl) were found to significantly increase the

solubility of ERα DBD-LBD. Thus, buffer A containing 500 mM NaCl was ultimately used

to purify the target protein instead of buffer B. Following the procedures as mentioned

above, the supernatant sample was loaded onto the nickel column and the column was

washed with buffer A containing a gradient of imidazole concentration from 30 mM to 80

mM. Then, the target protein was eluted with buffer A containing 200 mM imidazole. After

the nickel column purification, further step of purification was achieved by using a

Sephacryl S-200 gel filtration column (Figure 4.1.4). ERα DBD-LBD was concentrated to

0.5ml of 6.0 mg/ml in a buffer containing 25 mM Tris-HCl (pH 7.4), 250 mM NaCl, 80

mM MgCl2, 5 μM zinc chloride, 10% glycerol, 2.5 mM DTT, 20 μM estradiol and 0.1% β-

OG. The purity of the protein was estimated by SDS-PAGE and above 95%. This protocol

was applied to the purification of both ERα DBD-LBD fragments (179-554aa and 179-

595aa). In addition, a Western blot was performed to further verify the target band

representing ERα DBD-LBD.

4.1.4.3 Preliminary crystallogenesis of ERα DBD-LBD

Before crystallization, single stands of EREs were annealed to form a palindrome DNA

segment and then mixed with ERα DBD-LBD (179-554aa) at a 1.2:2 molar ratio. 96

conditions of the Nextal screen were initially used to crystallize ERα DBD-LBD (179-

554aa) in complex with EREs at room temperature and at 4 °C, yet no protein crystal was

obtained. Later, the Natrix screen was used to grow the crystal and was still not successful.

Then, by adjusting some crystallization conditions from the related studies 3; 21; 25; 26; 27; 28

,

several uniquely designed crystallization conditions were tested to grow the crystal. A

number of tiny needle crystals appeared at 4 °C after two weeks in one optimized

crystallization condition, which contained 7.5% (w/v) PEG3350 in 0.1 M MES buffer (pH

7.0) with 0.1 M MgCl2 and 0.2 M NaCl (Figure 4.1.5). The needle crystals were too tiny to

be scooped out from the hanging drop for SDS-PAGE verification, but they were unlikely

to be salt crystals. A hanging drops that mimicking the crystallization condition without the

107

target protein did not yield any salt crystal. Besides, salts MgCl2 and NaCl possessed high

solubility even at 4 °C and the precipitant concentration (7.5% (w/v) PEG3350) was rather

low.

4.1.5 Perspective

In this work, a purification protocol for ERα DBD-LBD was established. The preliminary

crystallogenesis of ERα DBD-LBD in complex with EREs was performed and only tiny

needle crystals were obtained. In the future, more crystallization conditions will be used to

grow high-quality crystals. If the crystal structure of ERα DBD-LBD in complex with

EREs is obtained, how ERα DBD and LBD interact with each other and function as a

whole will be exploited in depth. Moreover, the functional experiments based on the crystal

structure will be designed, such as single point mutations of certain amino acids which may

have significant roles in DBD-LDB dimerization, EREs recognition and coregulator

binding. The co-crystallization of ERα DBD-LBD with its partner proteins will be carried

out in the future studies. The partner proteins of ERα involved in the important functions in

breast cancer will be chosen as the candidates, such as FoxA1 or nm23-h1 29; 30; 31; 32; 33; 34

.

4.1.6 Acknowledgements

We thank Dr. Jiong Chen for purchasing the human full-length ERα plasmid from

Genecopoeia (Shanghai, China). We thank Dr. Kathryn Leake and Dr. Preyesh Stephen for

the revision of the manuscript. Bo Zhang thanks China Scholarship Council (CSC) for

supporting his graduate study with a national scholarship.

108

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17. Gangloff, M., Ruff, M., Eiler, S., Duclaud, S., Wurtz, J. M. & Moras, D. (2001).

Crystal structure of a mutant hERalpha ligand-binding domain reveals key

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18. Eiler, S., Gangloff, M., Duclaud, S., Moras, D. & Ruff, M. (2001). Overexpression,

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19. Cura, V., Gangloff, M., Eiler, S., Moras, D. & Ruff, M. (2008). Cleaved thioredoxin

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112

Figure 4.1.1 The constructions of Human ERα DBD-LBD recombinant plasmids

In (A) and (B), Lanes 1 and 2 are PCR results of two different clones of the pET-28a(+)

recombinant plasmids encoding ERα 179-554aa and 179-595aa, respectively; lanes 3

and 4 are the corresponding recombinant plasmids digested by two restriction enzymes

(BamHI and XhoI).

113

Figure 4.1.2 The verification of ERα DBD-LBD expression in Rosetta2 (DE3)

cells

(A) PCR results of two different Rosetta2 (DE3) clones containing ERα 179-554aa and

179-595aa encoding plasmids, respectively, lanes 1 and 2 encoding ERα 179-554aa and

lanes 3 and 4 encoding ERα 179-595aa; (B) ERα 179-554aa and 179-595aa expression

induced by IPTG in Rosetta2 (DE3) cells, lanes 1 and 2 are none-induced and induced

results of ERα 179-554aa, lanes 3 and 4 are none-induced and induced results of ERα

179-595aa.

114

Figure 4.1.3 Expression results of ERα DBD-LBD (179-595aa) in Rosetta2 (DE3)

cells

(A) and (B) are ERα 179-595aa expression in 18 °C and 25 °C, respectively. Lane 1 is the

total protein in the cells, lane 2 is protein in the supernatant and lane 3 is protein in the

precipitate.

115

Figure 4.1.4 Purification results of ERα DBD-LBD (179-595aa)

(A) Lanes 1 and 2 are the peak fractions of ERα 179-595aa eluted from the nickel column,

Lanes 3 and 4 are the peak fractions of ERα 179-595aa eluted from the gel filtration

column and the purity is above 95%; (B) two purified samples of ERα 179-595aa are

verified by Western blot.

116

Figure 4.1.5 Preliminary crystallogenesis of ERα DBD-LBD (179-554aa)

The needle crystals were obtained at 4 °C in a crystallization condition containing 7.5%

(w/v) PEG3350, 0.1 M MES buffer (pH 7.0), 0.1 M MgCl2 and 0.2 M NaCl.

117

4.2 A SIMPLE AND EFFICIENT PURIFICATION METHOD FOR

HUMAN ESTROGEN RECEPTOR ALPHA LBD

A simple and efficient purification method for human estrogen receptor

alpha LBD

Bo Zhang 1, 2

, Jean-François Thériault 1, Dao-Wei Zhu

1, Sheng-Xiang Lin

1, §



G1V4G2, Canada







Shortened title: an efficient purification method for human ERα LBD


118

4.2.1 Abstract

Human estrogen receptor alpha (ERα), activated by the binding of 17β-estradiol (E2),

exerts its essential effects on sexual development and reproductive functions. It has been

well established that the ligand binding domain (LBD) of ERα is sensitive to oxidation and

aggregation. Several methods have been developed to purify ERα LBD, such as

carboxymethylations or mutations of reactive cysteine residues, denaturation and

renaturation, introducing high concentration of reducing agent or fusion protein. Here, a

novel simple and efficient purification method for ERα LBD with a mutation of C530A

was reported. The target protein was expressed in E. coli BL21(DE3) cells. After the cell

pellets were lysed and centrifuged, the precipitate was used for purification. Several

detergents such as Triton X-100 and β-DDM (dodecyl-β-D-maltoside) were selected to

dissolve the precipitate and above 70% purity of the target protein was reached by the

treatment with detergents. Moreover, using a combination of detergents (Triton X-100 and

β-OG (β-Octyl glucoside)), one step of the purification by the nickel column could yield the

homogeneous and highly purified protein.

Keywords: estrogen receptor alpha (ERα), the ligand binding domain (LBD), detergent,

precipitate, purification.

119

4.2.2 Introduction

Nuclear receptors (NRs) act as transcription factors through binding a variety of

physiological hormones to regulate the transcription of specific genes, which play critical

roles in numerous physiological functions such as reproductive health and embryonic

development 1; 2

. Defined as a member of the NRs superfamily, estrogen receptor alpha

(ERα) is activated by the binding of 17β-estradiol (E2) to the ligand binding domain (LBD)

of ERα. The liganded ERα targets specific promoters or enhancers of genes through its

DNA binding domain (DBD) binding to estrogen response elements (EREs) and exerts its

effects on sexual development and reproductive functions 3; 4

. ERα LBD can bind a variety

of ligands, and part of its ligands act as agonists while others function as antagonists.

Through binding to agonists, the conformation of ERα LBD helix12 is rearranged, which

makes ERα LBD qualified to recruit coactivators such as steroid receptor coactivator 1

(SRC-1) 5; 6

. In contrast, through binding to antagonists, ERα LBD possesses the ability to

recruit corepressors such as nuclear receptor corepressor (NcoR) 1; 7

.

Although the crystal structures of ERα LBD in complex with agonists and antagonists have

been reported 5; 6; 8

, the purification processes of ERα LBD are full of challenges. There are

four cysteine residues in ERα LBD, and among them three residues (Cys381, Cys417 and

Cys530) are exposed to solvent and are sensitive to oxidation 9. Moreover, ERα LBD is

recognized by its hydrophobic character and is unstable without binding its ligands, which

makes it prone to aggregation. To date, several methods have been established to solve the

difficulties in purification. Initially, carboxymethylation of three reactive cysteine residues

in ERα LBD was successfully used to purify and crystallize this protein 5; 6; 9

. In addition,

denaturation and renaturation of ERα LBD was used to accomplish purification 8. The triple

cysteine to serine mutagenesis of ERα LBD was also introduced to reduce the difficulties of

purification 10

. Eiler and coworkers obtained the purified native form ERα LBD by using

high concentration of reducing agent to avoid the oxidation of cysteine residues during the

purification process 11

. In addition, Cura and coworkers developed a cleavable fusion

protein to increase the solubility of the target protein, and successfully purified ERα LBD 12

.

In this study, the residue Cys530 in ERα LBD was initially mutated to alanine. ERα LBD

Cys530 was the most reactive residue and could form the intermolecular disulfide bond

120

without high concentration of reducing agent 8. During the purification process of ERα

LBD, even under the optimized expression conditions in E. coli BL21(DE3) cells, the

major part of the target protein was predominantly kept in the precipitate. The soluble

portion in the supernatant was rather poor. This impelled us to develop a simple and

efficient purification method to obtain ERα LBD from the precipitate by using a

combination of different detergents.

4.2.3 Methods

4.2.3.1 Site-directed mutagenesis of ERα LBD C530A

The DNA fragment encoding human ERα LBD (297-554aa (amino acids)) was cloned into

pET-28a(+) vector. Using the recombinant vector as the template, the single point mutation

of ERα LBD C530A was performed by following the QuikChange Lightning Kit (Agilent

Technologies) protocol with 5’-CTGTACAGCATGAAGGCCAAGAACGTGGTGCC-3’

as forward primer and the result was verified by sequencing.

4.2.3.2 Protein expression and purification

Human ERα LBD with a mutation of C530A was expressed in E. coli BL21(DE3) cells.

The cells were grown at 37°C for 3 hours until OD600 reached 0.6, and then cells were

induced with 0.2 mM IPTG at 18°C in the presence of 10 μM 17β-estradiol overnight,

harvested and lysed in buffer A (50 mM Tris-HCl (pH 8.0), 10 mM imidazole, 200 mM

NaCl, 10% glycerol, 20 mM β-mercaptoethanol, 1 mM PMSF and 10 μM 17β-estradiol)

with the addition of 1 mg/ml lysozyme and 1 μg/ml each of protease inhibitors (leupeptin,

chymostatin, antipain, aprotinin, pepstatin A and benzamidin). After sonication and

centrifugation, the supernatant was discarded. The precipitate was resuspended in buffer A

with 0.4% Triton X-100 and protease inhibitors, and was slowly agitated via a magnetic

stirrer at 4°C overnight. After centrifugation for the second time, the supernatant was

collected and loaded onto the nickel column pre-equilibrated in buffer A with 0.2% Triton

X-100 and 0.1% β-OG. After incubation at 4°C for 3 hours, the nickel column was washed

with 15 column volumes (CV) buffer A containing 0.2% Triton X-100 and 0.1% β-OG, and

then washed with 15 CV buffer A containing 30 mM imidazole, 0.1% Triton X-100, 0.1%

121

β-OG and another step of 15 CV buffer A containing 30 mM imidazole and 0.1% β-OG.

Next, the target protein was eluted with 10 CV buffer A containing 200 mM imidazole and

0.1% β-OG. The eluted fractions were concentrated by a centrifugal filter with a 10 kDa

molecular weight cutoff and the buffer was exchanged to 50 mM Tris-HCl (pH 8.0), 100

mM NaCl, 5% glycerol, 5 mM DTT, 0.5 M NDSB-201 (non detergent sulfobetaines), 10

μM 17β-estradiol and 0.1% β-OG by three rounds of dilution and concentration. The purity

of the concentrated ERα LBD was estimated by SDS-PAGE and above 98%. The protein

concentration was determined as approximately 9.1 mg/ml using the Bradford method.

4.2.3.3 Solubility test

The E.coli cells containing the ERα LBD C530A recombinant plasmids were grown in 200

ml LB (Lysogeny broth) medium at 37°C for 2.5 hours until OD600 reached 0.6. Next, the

E.coli cells were induced by 0.2 mM IPTG at 18°C for 14-16 hours in the presence of 10

μM 17β-estradiol. The cell pellets were harvested, resuspended in 10 ml buffer A, sonicated

and centrifuged. Supernatant was discarded and precipitate was resuspended in 10 ml

buffer A. Each solubility test required 0.5 ml resuspended sample. 0.4% (w/v) each of

Triton X-100, C12E8 (octaethylene glycol monododecyl ether), β-DDM (dodecyl-β-D-

maltoside), β-OG (β-octyl glucoside) and Emulgen913 was added to the 0.5 ml sample,

respectively, and samples were slowly agitated overnight at 4°C. After samples were

centrifuged, the supernatant and the precipitate were checked by SDS-PAGE.

4.2.4 Results and discussion

4.2.4.1 Expression and solubility test of ERα LBD

The expression of the ERα LBD C530A mutant was induced with 0.2 mM IPTG at 18°C

for 14-16 hours in the presence of 10 μM 17β-estradiol in E.coli BL21 (DE3) cells. The cell

pellets were harvested and lysed in buffer A with 1 mg/ml lysozyme and 1 μg/ml each of

protease inhibitors (leupeptin, chymostatin, antipain, aprotinin, pepstatin A and

benzamidin). After sonication and centrifugation, the target protein predominantly existed

in the precipitate (Figure 4.2.1). Then, 0.4% (w/v) each of Triton X-100, Tween-20, C12E8,

β-DDM, β-OG and Emulgen913 was applied to the solubility test. The results showed that

122

Triton X-100, C12E8, β-DDM and Emulgen913 considerably improved the target protein to

be present in the supernatant. Particularly, above 70% purity was achieved by the treatment

with Triton X-100, C12E8 and Emulgen913 (Figure 4.2.2).

4.2.4.2 Purification of ERα LBD

Hereafter, the precipitate was used for the purification of ERα LBD. The precipitate was

resuspended again in buffer A containing 0.4% Triton X-100 and was then slowly agitated

at 4°C overnight. After centrifugation, the supernatant was obtained, filtered and loaded

onto the nickel column pre-equilibrated in buffer A with 0.2% Triton X-100 and 0.045% β-

DDM. After incubation at 4°C for 3 hours, the target protein was eluted with buffer A

containing 200 mM imidazole and 0.045% β-DDM. However, the target protein began to

aggregate soon after the elution from the nickel column. Therefore, 0.03% C12E8, 0.035%

Emulgen913 and 0.1% β-OG were tested to replace 0.045% β-DDM, respectively. 0.1% β-

OG was found to significantly relieve aggregation. Additionally, 0.5 M NDSB-201 was

also added to buffer A to improve the solubility of the target protein. Following the

procedures mentioned in Methods, the eluted fractions in buffer A containing 200 mM

imidazole, 0.1% β-OG and 0.5 M NDSB-201 were concentrated and then the buffer was

exchanged to 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol, 5 mM DTT, 0.1% β-

OG, 0.5 M NDSB-201 and 10 μM 17β-estradiol. This protocol used one step of the nickel

column to complete the purification. The protein concentration reached 9.1 mg/ml and the

purity was estimated to be above 98% by SDS-PAGE (Figure 4.2.3). Although further

purification by a Sephacryl S-200 gel-filtration column could improve the result, this step

was not necessary. A unique absorption peak during the gel filtration step indicated that the

target protein purified by the nickel column was rather homogeneous and the oligomeric

potential of ERα LBD was not observed. In addition, following this protocol, 25-30 mg of

ERα LBD could be obtained from a liter of the E.coli cells. This protocol was also

appropriate for the purification of ERα LBD in its native form (without the C530A

mutation). However, high concentration of the reducing agent was required for this task 11

.

123

4.2.5 Acknowledgements

The authors wish to thank Dr. Jiong Chen for purchasing the human full-length ERα

plasmid from Genecopoeia (Shanghai, China). The authors thank Dr. Kathryn Leake and

Dr. Preyesh Stephen for the revision of the manuscript. Bo Zhang appreciates China

Scholarship Council (CSC) for providing him a national scholarship.

124

4.2.6 References



72, 247-72.

2. Olefsky, J. M. (2001). Nuclear receptor minireview series. J Biol Chem 276, 36863-

4.

3. Korach, K. S. (1994). Insights from the study of animals lacking functional estrogen

receptor. Science 266, 1524-7.




905-31.







7. Bain, D. L., Heneghan, A. F., Connaghan-Jones, K. D. & Miura, M. T. (2007).

Nuclear receptor structure: implications for function. Annu Rev Physiol 69, 201-20.




9. Hegy, G. B., Shackleton, C. H., Carlquist, M., Bonn, T., Engstrom, O., Sjoholm, P.

& Witkowska, H. E. (1996). Carboxymethylation of the human estrogen receptor

ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping shows

that cysteine 447 does not react with iodoacetic acid. Steroids 61, 367-73.

10. Gangloff, M., Ruff, M., Eiler, S., Duclaud, S., Wurtz, J. M. & Moras, D. (2001).

Crystal structure of a mutant hERalpha ligand-binding domain reveals key

structural features for the mechanism of partial agonism. J Biol Chem 276, 15059-

65.

125

11. Eiler, S., Gangloff, M., Duclaud, S., Moras, D. & Ruff, M. (2001). Overexpression,

purification, and crystal structure of native ER alpha LBD. Protein Expr Purif 22,

165-73.

12. Cura, V., Gangloff, M., Eiler, S., Moras, D. & Ruff, M. (2008). Cleaved thioredoxin

fusion protein enables the crystallization of poorly soluble ERalpha in complex with

synthetic ligands. Acta Crystallogr Sect F Struct Biol Cryst Commun 64, 54-7.

126

Figure 4.2.1 Expression result of ERα LBD

The E. coli cell pellets were lysed in buffer, sonicated and centrifuged. The total protein,

the supernatant and the precipitate are shown in lanes 1, 2 and 3, respectively.

127

Figure 4.2.2 Solubility test of ERα LBD

The E.coli cell pellets were lysed in buffer, sonicated and centrifuged. The supernatant

was discarded and the precipitate was preserved. The precipitate was resuspended and

treated with 0.4% (w/v) each of Triton X-100, C12E8, β-DDM, Emulgen913,

respectively; lanes 1, 3, 5 and 7 are proteins remained in the precipitate and lanes 2, 4, 6

and 8 are proteins dissolved from the precipitate.

128

Figure 4.2.3 Purification result of ERα LBD

Lanes 1 and 2 are the peak fractions of ERα LBD eluted from the nickel column, the purity

of ERα LBD is above 98%.

129

CHAPTER 5 CONCLUSIONS

130

Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) has an essential role to

inactivate the most potent androgen 5α-dihydrotestosterone (5α-DHT). Meanwhile, 5α-

DHT has been shown to reduce breast cancer cells growth and the role of 3α-HSD3 in

breast cancer is short of investigations. The goal of my study is to investigate the structure-

function relationship of 3α-HSD3 in complex with various steroids, and to further

investigate the potential role of 3α-HSD3 in breast cancer cells. In addition, estrogen

receptor alpha (ERα) controls sexual development and reproductive functions. Another goal

of my study is to purify the ERα fragment including its DNA binding domain and ligand

binding domain (DBD-LBD), and try to obtain the crystal structure of ERα DBD-LBD in

complex with estrogen response elements (EREs). The main accomplishments are as

following:

(1) To understand the role of residue 54 in the steroid binding and discrimination, the

Val54-to-Leu54 (V54L) mutation in human 3α-HSD3 has been created. We report two

crystal structures of the 3α-HSD3·NADP+·Progesterone complex and the 3α-HSD3

V54L·NADP+·progesterone complex. These crystal structures show that progesterone in

the wild type enzyme possesses two different binding modes, which indicates the steroid

binding pocket possessing considerable flexibility to accommodate different orientations of

a steroid. However, in the mutant enzyme, the bulky side chain of Leu54 significantly

confines the spatial movement of the steroid and thus only one orientation presents in the

steroid binding pocket. Moreover, we report that the V54L mutation in 3α-HSD3

significantly decreases its 5α-DHT inactivation ability and this mutation enhances the 20-

alpha hydroxysteroid dehydrogenase (20α-HSD) activity to catalyze progesterone.

(2) We report the crystal structure of the 3α-HSD3·NADP+·A-dione/epi-ADT complex,

which is obtained by co-crystallization with 5α-DHT. Although 5α-DHT was introduced

during the crystallization, the oxidoreduction of 5α-DHT occurred. A-dione and epi-ADT

were identified to be located in the steroid binding sites of two monomers (monomer A and

B) in a crystal asymmetric unit, respectively. An overlay of monomer A and B shows that

A-dione and epi-ADT are oriented upside down and flipped to each other, which provides

the structural clues of 5α-DHT alternative binding within the enzyme to yield different

products. Moreover, using a crystallization condition without acetate ion, we report the

131

crystal structure of the 3α-HSD3·NADP+·4-dione complex. In addition, using specific

siRNA to knock down 3α-HSD3 expression in MCF7 breast cancer cells, 5α-DHT

concentration increased while the cell growth was suppressed.

(3) We set up an expression and purification protocol of ERα DBD-LBD. Preliminary

crystallogenesis of ERα DBD-LBD in complex with estrogen response elements (EREs) is

carried out. This study provides the basic knowledge to facilitate the acquisition of the fine

crystals for ERα DBD-LBD in complex with EREs.

(4) We report a simple and efficient purification method for human ERα LBD.

5.1 STRUCTURE-FUNCTION STUDY OF HUMAN 3ΑLPHA-HSD3

SINGLE POINT MUTATION V54L ENHANCING THE 20ΑLPHA-HSD

ACTIVITY

Human 3α-HSD3 plays an essential role to inactivate androgen 5α-DHT, while human 20α-

HSD is responsible for inactivation of progesterone. However, there is only one amino acid

difference (residue 54) between the steroid binding pockets of 3α-HSD3 and 20α-HSD. By

combining mutagenesis, X-ray crystallography and enzyme kinetics study, we explore the

role of residue 54 in the steroid binding and discrimination.

We report the crystal structure of the 3α-HSD3·NADP+·Progesterone complex. There are

two monomers (monomer A and B) in a crystal asymmetric unit. In monomer A, the α-face

of progesterone interacts with Val54 and Val128 and the steroid C18 and C19 methyl

groups pointing toward Trp227. However, in monomer B, the steroid α-face is in stacking

with Trp227 and the two methyl groups of the steroid are pointing toward Tyr55. When

two monomers are superimposed, it shows that loop A fluctuates notably compared with

loop B and loop C, and two steroids in the cavities could not overlap each other and their

orientations have a difference of ~105° rotation around the long axis of C10-C17. The side

chains of His222 and Leu308 flip 180° and the side chain of Leu306 owns alternative

conformations. These results demonstrate that the steroid binding pocket of 3α-HSD3

possesses considerable flexibility to accommodate different orientations of a steroid.

132

We also report the crystal structure of the 3α-HSD3 V54L·NADP+·progesterone complex.

An overlay of monomer A and B in the crystal asymmetric unit shows high similarity for

their overall structures, and the orientations of two steroids overlap each other quite well.

The α-faces of both steroids interact with Leu54 and Val128, and their β-faces interact with

the Trp227 indole group. The bulky side chain of Leu54 in the mutant enzyme is

outstretching toward the steroid and obviously generates a steric hindrance to restrict the

steroid rotation. Moreover, steroids in the mutant enzyme possess relatively lower B-factors

in monomer A and B (23.3/27.6 Å2) than that in the wild type enzyme (37.7/34.4 Å

2),

which implies the V54L mutation has a role of stabilization for progesterone. Therefore, the

V54L mutation significantly restricts the spatial movement of the steroid and eliminates

multiple orientations which are allowed in the wild type enzyme.

To further understand the steroid discrimination by human 3α-HSD3 and its V54L mutant,

the kinetic study has been carried out. The V54L mutation in 3α-HSD3 significantly

decreases its 3α-reductase activity and enhances its 20α-reductase activity. For 5α-DHT

reduction, the kinetic parameters for the wild type enzyme (Km = 2.18 μM, kcat = 3.67 min-1


μM-1

) and the mutant enzyme (Km = 71.03 μM, kcat = 6.92 min-1

and

kcat/Km = 0.10 min-1

μM-1

) indicate that the 5α-DHT reduction ability is significantly

decreased in the mutant enzyme. While for progesterone reduction, the kinetic parameters

for the wild type enzyme (Km = 0.84 μM, kcat = 0.21 min-1


μM-1

)

and the mutant enzyme (Km = 2.13 μM, kcat = 2.35 min-1


μM-1

)

indicate that the wild type enzyme possesses a weak activity and the mutant enzyme shows

a considerable 20α-reduction activity. The results imply that, for those homologous

enzymes, only one residue alteration at the substrate binding pocket is enough to transform

the substrate specificity for the enzyme.

5.2 STRUCTURAL CLUES OF 5ΑLPHA-DHT ALTERNATIVE BINDING

WITHIN HUMAN 3ΑLPHA-HSD3 AND DOWN-REGULATION OF

3ΑLPHA-HSD3 DECREASING BREAST CANCER CELL GROWTH

Androgen 5α-DHT has been shown to prevent breast cancer cells growth. In human, 3α-

HSD3 functions as the 3-ketosteroid reductase to inactivate 5α-DHT in cellular

133

environment. However, the role of 3α-HSD3 in breast cancer cells is short of investigations.

In this study, we try to obtain the crystal structure of 3α-HSD3 in complex with 5α-DHT. In

addition, using specific siRNA to knockdown 3α-HSD3 expression in breast cancer cells

(MCF7), the impacts of 3α-HSD3 expression on 5α-DHT concentration and cell

proliferation are investigated.

A high-resolution crystal structure of human 3α-HSD3·NADP+·A-dione/epi-ADT complex

is obtained by co-crystallization with 5α-DHT in presence of NADP+. Although 5α-DHT is

introduced during the crystallization, the oxidoreduction of 5α-DHT occurs and generates a

mixture of steroids. A-dione and epi-ADT are identified to be located in the cavities of two

monomers (monomer A and B) in a crystal asymmetric unit, respectively. In monomer A,

the D-ring of A-dione is oriented toward the nicotinamide ring. The α-face of A-dione

interacts with the side chain of Val54, Trp86 and Val128. Its β-face mainly interacts with

the indole ring of Trp227, and the steroid C18 and C19 methyl groups are pointing toward

Trp227. In monomer B, the A-ring of epi-ADT is pointing toward the nicotinamide ring.

The α-face of epi-ADT is stacking with the indole ring of Trp227, and the steroid C18 and

C19 methyl groups are pointing toward Val54 and Tyr55. When monomer A and B are

superimposed, two steroids are oriented upside down to each other, and the planes of two

steroids are flipped about 145° around the long axis of C3-C17. In both monomers,

fluctuation in loop A is prominent. The side chains of Val128, Ile129 and Leu306 display

apparent movement to consort with steroids. The different orientations of A-dione and epi-

ADT provide the structural clues of 5α-DHT having the alternative binding modes in the

cavity of the enzyme to generate different products. The alternative binding modes of

steroids in the enzyme originate from the pseudo self-symmetry property of androgens and

the flexibility of the steroid binding pocket.

The crystal structure of human 3α-HSD3·NADP+·4-dione complex is obtained by co-

crystallization with Testo in the presence of NADP+. During the crystallization process, the

oxidation of Testo occurs and 4-dione is identified to be located in the steroid binding

pockets of monomer A and B in a crystal asymmetric unit. When superimposing monomer

A and B, the orientation and conformation of 4-dione are quite similar to each other. Loop

A still displays its flexibility. The A-ring of 4-dione is pointing toward the nicotinamide

134

ring. The α-face of 4-dione interacts with the side chain of Trp227, and the C18 and C19

methyl groups of 4-dione are pointing toward the side chain of His117. Compared with the

orientation of Testo in the reported 3α-HSD3·NADP+·Testo/acetate complex, the

orientation of 4-dione in the 3α-HSD3·NADP+·4-dione complex generates ~45° rotation

around its C3-C17 long axis. Meanwhile, the steroid goes deeper into the cavity of the

enzyme. The acetate molecule in the Testo/acetate complex, which is originated from the

crystallization condition, occupies the active site of the enzyme and distorts the actual

steroid binding conformation. However, using a crystallization condition without the

acetate molecule, the steroid binding conformation of 4-dione was more reasonable and less

distorted.

In addition, the 3α-HSD3 expression is knocked down by specific siRNA in MCF7 cells,

and a relative elevation of 5α-DHT level is detected by using ELISA assay. Meanwhile, in

the presence of 5α-DHT, suppression of 3α-HSD3 expression by specific siRNA leads to a

decrease of MCF7 cell growth, which can be speculated that the suppressed expression of

3α-HSD3 strengthens the antiproliferative effect of 5α-DHT in MCF7 cells. It is

noteworthy that suppression of 17β-HSD1 expression is reported to increase 5α-DHT level

in T47D cells, which shows an antiproliferative effect. However, compared with 5α-DHT

inactivation ability of 17β-HSD1, 3α-HSD3 possesses a significant higher activity to

convert 5α-DHT, which implies that 3α-HSD3 may undertake a more important role than

17β-HSD1 to inactivate 5α-DHT in breast cancer cells.

5.3 PURIFICATION AND PRELIMINARY CRYSTALLOGENESIS OF

HUMAN ESTROGEN RECEPTOR ALPHA DBD-LBD

ERα plays important physiological functions in human such as controlling sexual

development and reproductive functions. However, due to the difficulties in protein

purification and crystallization, the full-length ERα structure remains an unfulfilled task. In

this study, we set up an expression and purification protocol of ERα fragment containing its

DNA binding domain and ligand binding domain (DBD-LBD).

At the beginning, the recombinant plasmid encoding ERα DBD-LBD with the C530A

single point mutation was transformed into E.coli Rosetta2 (DE3) cells. The expressions of

135

the target protein were induced with different concentrations of IPTG at 37°C. However,

after sonication and centrifugation, the target protein existed mostly in the precipitant, not

in the supernatant. Several commonly used detergents were used to improve the protein

solubility, whereas these detergents had little efficacy for improving the solubility of ERα

DBD-LBD. After optimization of the expressing conditions, 25°C for 12 hours and 0.2 mM

IPTG were determined to induce the target protein to be expressed in the supernatant with

an acceptable quantity. During the purification, the target protein was prone to aggregate.

However, high salt concentration (above 400 mM NaCl) was found significantly preventing

the protein aggregation and increasing the solubility of ERα DBD-LBD. Through two steps

of purification by the nickel column and the gel filtration column, ERα DBD-LDB was

concentrated to 6.0 mg/ml and the purity was above 95 %. In addition, Western blot further

proved the target band representing ERα DBD-LBD. Preliminary crystallogenesis of ERα

DBD-LBD in complex with estrogen response elements (EREs) was carried out. This study

provided the basic knowledge to facilitate the acquisition of the fine crystals for ERα DBD-

LBD in complex with EREs.

5.4 A SIMPLE AND EFFICIENT PURIFICATION METHOD FOR

HUMAN ESTROGEN RECEPTOR ALPHA LBD

ERα ligand binding domain (LBD) is sensitive to oxidation and aggregation. Previously,

several methods were developed to purify ERα LBD, such as carboxymethylation or

mutation of reactive cysteine residues, denaturation and renaturation, introducing high

concentration of reducing agents or fusion protein. Here, we reported a novel simple and

efficient purification method for human ERα LBD.

ERα LBD was induced to express in E.coli BL21 (DE3) cells with 0.2 mM IPTG at 18°C.

The cell pellets were harvested and lysed. After sonication and centrifugation, compared

with the supernatant, the target protein was found mainly existing in the precipitant. Several

detergents were selected to dissolve the precipitant. It was found that Triton X-100, C12E8,

β-DDM, Emulgen913 considerably improved the target protein to exist in the supernatant,

and above 70 % purity of the target protein has been reached by treatment with 0.4 % of

Triton X-100, C12E8 and Emulgen913. Thus, 0.4 % Triton X-100 was chosen to extract the

136

target protein from the precipitant. Furthermore, using a combination of Triton X-100 and

β-OG, the purification of ERα LBD could be fulfilled by only using the nickel column. The

protein concentration reached to 9.1 mg/ml and the purity was above 98 %. Further step of

purification by the gel filtration column could polish the purification result, but not

necessary.

5.5 FUTURE TOPICS

Although some progress has been made, more structural and functional studies need to be

carried out to further clarify the role of 3α-HSD3 in breast cancer cells. For example:

(1) Our study shows the structural clues of 5α-DHT and progesterone alternative binding

within 3α-HSD3. However, different from the productive binding orientation of the steroid

in rat 3α-HSD, steroids display the binding orientations before or after the reactions within

3α-HSD3. Therefore, the molecular dynamics simulation can be carried out to provide the

dynamic information of 3α-HSD3 utilizing the alternative binding modes of steroids to

output different products.

(2) Due to the role of the inactivation of 5α-DHT by 3α-HSD3, certain leading compounds

can be designed to inhibit 3α-HSD3. This may have an effect on increasing the 5α-DHT

concentration in breast cancer cells and decreasing the growth of breast cancer cells.

(3) It has been shown that the transcription of 3α-HSD3 is regulated by transcription factor

Nrf2 (nuclear factor erythroid-2 related factor 2). The role of Nrf2 in breast cancer cells

should be further studied. Moreover, the impact of 3α-HSD3 down-regulation by siRNA on

the gene and protein profile of breast cancer cells should be investigated. The differentially

expressed candidates that may get involved in breast cancer should be further investigated.

In addition, for human ERα DBD-LBD, more crystallization conditions and the target

protein in complex with different estrogen response elements should be attempted to obtain

high-quality crystals. The co-crystallization of ERα DBD-LBD with its partner proteins

such as FoxA1 and nm23-h1, which are closely related to breast cancer, will be another

direction of the research.

137

CHAPTER 6 REFERENCES (FOR INTRODUCTION)

138

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