crystal structure of the catalytic and ubiquitin

Crystal structure of the catalytic and ubiquitin-associated domains of

the protein kinase MARK2 / PAR-1 from Rattus norvegicus (Berkenhout, 1769)

Thesis submitted to the Department of Biology, Faculty of Mathematics, Informatics and Natural Sciences of the University of Hamburg in partial

fulfillment of the requirements for the degree of Ph.D.

By

Saravanan Panneerselvam from India

Hamburg 2006

Table of Contents i

1 Introduction 1 1.1 The protein kinase superfamily 1 1.2 Classification of the protein kinase superfamily 1

1.3 Identification of MAP/microtubule affinity regulating kinase (MARK) 3

1.3.1 Homologues of MARK 6

1.3.2 Activation of MARK 9

1.3.3 Inhibition of MARK 9

1.3.4 Proteins interacting with MARK 10

1.4 Ubiquitin binding domains 11

1.4.1 The ubiquitin-associated domain (UBA) 11

1.4.2 Structure of the UBA domain 12

1.4.3 The UBA domain of the MARK 13

1.5 Aim of the work 14

2 Materials and methods 15

2.1 Materials 2.1.1 Chemicals 15

2.1.2 Enzymes 15

2.1.3 Bacterial strains 16

2.1.4 Cloning vectors 17

2.1.5 Expression vectors 17

2.1.6 Media 17

2.1.7 Crystallization

2.1.7.1 Crystallization supplies and tools 18

2.1.7.2 Crystallization solutions 19

2.1.8 Equipment and accessories 19

2.2 Molecular biology and microbiological methods

2.2.1 Culture and storage of E. coli strains 20

2.2.2 Transformation of E. coli strains 20

2.2.3 Isolation of plasmid DNA 21

2.2.4 Determination of DNA concentration and purity 21

Table of Contents ii

2.2.5 Ligation reaction 21

2.2.6 Restriction analysis of DNA 22

2.2.7 DNA sequencing 22

2.2.8 Mutagenesis of DNA 23

2.2.9 Gene cloning using the Invitrogen Gateway technology 24

2.3 Protein methods

2.3.1 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 29

2.3.2 Protein expression and purification 30

2.3.3 Cell lysis and solubility test 30

2.3.4 Chromatography 31

2.3.4.1 Ni-NTA affinity chromatography 31

2.3.4.2 GST affinity chromatography 31

2.3.4.3 Anion and cation exchange chromatography 32

2.3.4.4 Gel filtration chromatography 32

2.3.5 Determination of concentration of proteins 33

2.3.6 Concentrating the protein solution 33

2.3.7 Protein kinase assay 33

2.3.8 Limited proteolysis 34

2.3.9 N-terminal sequencing 34

2.3.10 Mass spectrometry 34

2.3.11 Detailed protocols on purification of MARK2 wild type and mutant proteins 35

2.3.11.1 Expression 35

2.3.11.2 Purification 35

2.3.11.3 Selenomethionine labelling 36

2.4 Crystallographic methods

2.4.1 Crystallization techniques 36

2.4.2 Cryoprotection of crystals 38

2.4.3 Data collection 38

2.4.4 Methods for phase determination 39

Table of Contents iii

3 Results 41 3.1 Cloning of different constructs of MARK2 41

3.2 Identification of structurally folded part of MARK2 42


3.2.2 N-terminal sequencing and mass spectrometry analysis 43

3.3 Cloning of the stable fragment of MARK2 44

3.4 Expression and purification 45

3.5 Kinase activities of the purified protein 47

3.6 Preparation of selenomethionine labelled protein 48

3.7 Crystallization 48

3.8 Data collection and structure determination 50

3.8.1 Molecular replacement 53

3.8.2 Heavy atom derivatives of MARK2 crystals 53

3.8.3 Model building and refinement 54

3.8.4 Crystals of selenomethionine labelled MARK2 55

3.9 Overall structure of the MARK2 catalytic and UBA domains 56

3.10 Structure of the catalytic domain 56

3.10.1 Conformation of the activation loop 58

3.10.2 Intermolecular disulfide bridge 59

3.10.3 Dimerization 60

3.11 Structure of the UBA domain 61

3.12 UBA linker and common docking domain for kinase activators 66

4 Discussion 68

4.1 Activation segment 70

4.2 Activation loop of MARK2 interferes with substrate binding 70

4.3 Catalytic cleft and nucleotide binding site 73

4.4 Conformation of the catalytic loop 74

4.5 Dimerization 75

4.6 C-terminal extension of the kinase core 77

4.7 UBA domain and regulation of MARK2 78

Table of Contents iv

5 Summary 81

6 References 83

7 Appendix 96 7.1 Abbreviations 96

7.2 List of figures 98

7.3 List of tables 100

7.4 Oligonucleotides 100

7.5 Purification buffers 102

7.6 List of Coordinates 103

8 Acknowledgements 104

9 Curriculum vitae 105

10 Declaration 107

Introduction 1

1 Introduction 1.1 The protein kinase superfamily Protein kinases comprise one of the largest families of proteins in eukaryotic organisms.

They catalyze the phosphorylation of cellular proteins on serine, threonine or tyrosine

residues in order to alter their functional properties. They are hence central to cellular

signaling networks that co-ordinate various activities like metabolism, stress response,

transcription, translation, DNA replication and cell cycle control, development of

organs, neuronal signaling and apoptosis. Improper functioning of these enzymes is

often manifested in various human diseases and has been implicated in several types of

cancers.

There are at least 518 protein kinases identified in the human genome, which is nearly

1.7% of all human genes (Manning et al., 2002). All the protein kinases have a strong

sequence similarity in their catalytic domains. Despite their sequence similarity, protein

kinases have different substrate specificities, mechanisms of regulation, modes of

action, etc. The phylogenetic tree (Fig. 1.1) depicts the relationships between members

of the superfamily of human protein kinases.

1.2 Classification of the protein kinase superfamily

Protein kinases are classified on the basis of aminoacid sequence similarity in the

catalytic domain. Generally, the protein kinase superfamily is divided into 9 major

groups, 90 families and 145 subfamilies (Hanks and Hunter, 1995, Manning et al.,

2002).

The major groups are as follows:

AGC group - includes the cyclic-nucleotide-dependent protein kinase

families, βARK and ribosomal S6 kinase families

CaMK group - includes the families of protein kinases regulated by

Ca2+/calmodulin, the Snf1/AMPK families and other

close relatives

Introduction 2

Fig. 1.1: Phylogenetic tree of the complete superfamily of human protein kinases. Most protein kinases belong to a single superfamily of enzymes whose catalytic domains are related in sequence and structure. The main diagram illustrates the similarity between the protein sequences of these catalytic domains. The inset diagram shows trees for seven atypical protein kinase families (Manning et al., 2002). The MARK subfamily is highlighted with a red circle.

Introduction 3

PTK group - conventional protein tyrosine kinases

CMGC group - includes the CDK, MAPK, GSK3 and CLK protein

kinase families

STE group - includes homologues of Ste7/MAP2K, Ste11/MAP3K

and Ste20/MAP4K protein kinases

CK1 group - includes the CK1, TTBK and VRK protein kinase

families

TKL group - Tyrosine kinases like kinases, includes kinase families

that resemble both tyrosine and serine/threonine kinases

like MLK, LISK, IRAK and STRK families.

RGC group - kinases that are similar in domain sequences to tyrosine

kinases

OPK group - other protein kinases that are not falling into major

groups

In addition to these major groups of kinases, there were some other proteins reported to

have protein kinase activity but lack sequence similarity with other protein kinases.

These proteins have been classified as atypical protein kinases.

1.3 Identification of MAP/microtubule affinity regulating kinase (MARK)

Microtubules (MTs) serve as tracks for cellular transport, and regulate cell shape and

polarity. Rapid transitions between stable and dynamic forms of MTs are central to

these processes. This dynamic instability is regulated by a number of cellular factors,

including the structural MT-associated proteins (MAPs), which in turn are regulated by

phosphorylation. Tau is a microtubule-associated protein prominent in the brain,

particularly in the axonal compartment of neurons, where it helps to stabilize

microtubules. Microtubules in turn serve as tracks for the intracellular transport of

vesicles and organelles, for providing stability of axons, and for growth cone advance.

The tau-microtubule interaction is regulated by phosphorylation, especially at the

KXGS motifs in the repeat domain of tau which represents the core of the microtubule-

binding domain (Biernat et al., 1993). The same domain also forms the core of the

abnormal tau aggregates ("paired helical filaments", PHF) in Alzheimer's disease. Both

functions, microtubule binding and PHF assembly, are efficiently suppressed when tau

Introduction 4

is phosphorylated by MAP/microtubule affinity regulating kinases. Thus, excess

activation of MARK in cells leads to microtubule breakdown because they are not

properly stabilized. A selection of protein kinases that are phosphorylating tau at

different sites are shown in Fig. 1.2.

Fig. 1.2: Bar diagram of human tau and the sites phosphorylated by different protein kinases. Tau is a microtubule-associated protein, and htau40 is the longest isoform of the splice variants which contains additional N-terminal inserts (I1 and I2). It has an N-terminal projection domain and a C-terminal microtubule binding domain in which the KXGS repeats are located. Tau protein can be phosphorylated by many different kinases. The SP/TP motifs are the main targets for proline directed kinases such as GSK3β, CDK5 and MAP kinase. The main targets of PKA are S214 and to a lesser extent KXGS repeats. The main targets of MARKs are KXGS repeats; the phosphorylation on S262 particularly by MARKs detaches tau from microtubules which in turn causes increased microtubule dynamics and tau aggregation (Biernat et al., 2001; Drewes et al., 1997).

A second function of tau is its interference with motor proteins moving along

microtubules; this function is also fine-tuned in axons by MARK (Mandelkow et al.,

2004). Using the phosphorylation of tau as a readout, MARK was purified and cloned

from brain tissue (Drewes et al., 1997).

PKAMAPKGSK3ßCDK5

C3 4441

I1N P1 P2 1 2I21 TP

153

181TP

175TP

SP205

SP199

SP202

212TP

217TP

231TP

235SP

SP396

SP422

SP404

S214

S262S293

S324

S356

KXGS

MARK

SP46

TP69

TP50

TP111

Projection domain

Microtuble binding domainRepeat region

PKAMAPKGSK3ßCDK5

C3 4441441

I1N P1 P2 1 2I211 TP

153

181TP

175TP

SP205

SP199

SP202

SP205

SP199

SP202

212TP

217TP

231TP

235SP

SP396

SP422

SP404

SP396

SP422

SP404

S214

S262S293

S324

S356

KXGS

MARK

SP46

TP69

TP50

TP111

SP46

TP69

TP50

TP111

Projection domain

Microtuble binding domainRepeat region

Introduction 5

Compared to other kinases, MARK is a relatively large protein (~720-790 aa) which

contains several domains: an N-terminal leader sequence, a typical kinase catalytic

domain, an ubiquitin-associated domain (UBA), a spacer and a tail domain containing

the KA1 (kinase-associated) motif characteristic for the family of kinases ending with

the ELKL motif (Fig. 1.3). Four isoforms of MARK (1-4) were found in mammals,

encoded by different genes, with additional splicing variants (Drewes, 2004; Drewes et

al., 1997).

Fig. 1.3: Domain organization of human MARK2. Residue numbers refer to the longest isoform of human MARK2 (Swiss-Prot entry Q7KZI7). CD, common docking domain; UBA - ubiquitin associated domain; KA1, kinase associated domain 1 and the tail domain ends with ELKL motif, typical for this family of kinases. T208 phosphorylation by MARKK or LKB1 is necessary for MARK activation (Timm et al., 2003; Lizcano et al., 2004) and T596 phosphorylation by aPKC leads to binding of 14-3-3 proteins and negatively regulates the MARK kinase activity (Hurov et al., 2004).

MARKK LKB1 aPKC

MARKK LKB1 aPKC

Introduction 6

1.3.1 Homologues of MARK

MARK kinases are conserved from yeast to human and share a similar primary

structural organization. A sequence alignment of MARKs and their homologous

proteins is shown in Fig. 1.4. In table 1.1, genes encoding the MARK/PAR-1/KIN1

subfamily in animal and yeast species are summarized.

Organism Genes

Mammals MARK1 MARK2 MARK3/C-TAK1/p78 MARK4/MARKL1 pEG3/MELK/MPK38

D. melanogaster PAR-1 C. elegans PAR-1 S. cerevisiae KIN1

KIN2 S. pombe KIN1

Table 1.1: Genes encoding MARK/PAR-1/KIN1 kinases in animal and yeast species (Tassan et al., 2004)

PAR-1: PAR-1 kinase shows a very high homology to MARK. PAR-1 kinase was

initially identified in C. elegans and later in D. melanogaster. In both organisms this

kinase plays a major role in the anterior-posterior (A/P) axis formation and cell polarity.

Many of the identified PAR-1 substrates are involved in cell polarization and oogenesis

(Kemphues, 2000; Pellettieri and Seydoux, 2002).

KIN1: The yeast kinases KIN1 and KIN2 also share a striking homology to MARK

kinases. These kinases belong to the SNF1 kinase family of the Ca2+/calmodulin-

dependent kinase II (CaMK II) group. In S. pombe it has been shown that KIN1 kinases

are involved in the formation of cell morphology (Levin and Bishop, 1990)

Introduction 7

Members of the MARK/PAR-1 family occur in most organisms examined so far.

Analysis of the human genome showed that there are four members of the human

MARK family which belong to the class of CaMK II kinases (Fig. 1.1; Hanks and

Hunter, 1995; Manning et al., 2002). They occur in a variety of cell types and

presumably serve a variety of functions, depending on isoform or localization. Table 1.2

summarizes the different isoforms of mammalian MARKs and their different substrates.

Table 1.2: Isoforms, localization and substrates of mammalian MARK kinases (Drewes et al., 2004; Riechmann et al., 2004)

Tau, MAP2, MAP4High in brain, glioma, testis

MARKL1, hPAR-1d

MARK4

PTPH1,Cdc25C, KSR1, Plakophilin2, Dishevelled

Highest in brain and pancreas

EMK2; KP78; hPAR-1a; C-TAK1

MARK3

Tau, MAP2, MAP4Dcx, Oskar, Raf, Exuperantia

Similar to MARK1EMK1; hPAR-1b

MARK2

Tau, MAP2, MAP4High in brain, spleen, skeletal, muscle, pancreas, kidney and heart

EMK3; hPAR-1c

MARK1

SubstratesExpressionPattern

SynonymsIsoform

Tau, MAP2, MAP4High in brain, glioma, testis

MARKL1, hPAR-1d

MARK4

PTPH1,Cdc25C, KSR1, Plakophilin2, Dishevelled

Highest in brain and pancreas

EMK2; KP78; hPAR-1a; C-TAK1

MARK3

Tau, MAP2, MAP4Dcx, Oskar, Raf, Exuperantia

Similar to MARK1EMK1; hPAR-1b

MARK2

Tau, MAP2, MAP4High in brain, spleen, skeletal, muscle, pancreas, kidney and heart

EMK3; hPAR-1c

MARK1

SubstratesExpressionPattern

SynonymsIsoform

Introduction 8

Fig. 1.4: Multiple sequence alignment of MARK/PAR-1 kinase family members. The sequence numbering is that of human MARK2 kinase. Colour coding: Invariant residues white on red background; conservatively substituted residues red. The sequences are highly similar in the catalytic domain. Sequences used here were from: human (Q7KZI7), rat (O08679) for MARK, C. elegans for PAR-1 (Q17346) and S. cerevisiae for KIN1 (P13185). Swiss-Prot data base accession numbers are given in parentheses. This figure was prepared using ESPript (Gouet et al., 1999).

Introduction 9

1.3.2 Activation of MARK

Protein kinases are normally regulated by phosphorylation in their "activation loop"

which controls the access of the substrate to the catalytic centre (Huse and Kuriyan,

2002; Johnson et al., 1996). Like other kinases, MARK family members can be

activated by phosphorylation in the activation loop. This can be achieved by the protein

kinase MARKK (MARK-kinase) which phosphorylates T208 in MARK2 (Timm et al.,

2003). This kinase was also found in the context of activation of MEKs and named

TAO-1 (Hutchison et al., 1998). MARK can also be phosphorylated at T208 and

activated by LKB1 which plays a role in tumor suppression (Lizcano et al., 2004).

A further level of regulation lies in the association with other proteins and domains. By

immunofluorescence, MARK2 has a vesicular distribution, but the target proteins (such

as tau) are often cytosolic. A change in localization may be achieved via the non-

catalytic domains of MARK, and indeed the deletion of the spacer domain causes a

mislocalization in D. melanogaster (Vaccari et al., 2005).

1.3.3 Inhibition of MARK

A notable feature of MARK isolated from brain tissue is its double phosphorylation in

the activation loop (at T208 and S212 in MARK2, (Drewes et al., 1997)). While

phosphorylation of T208, the target site of MARKK or LKB1, activates the kinase,

phosphorylation of S212 is probably inhibitory, but the responsible kinase is unknown

at present. Mutation of this serine to glutamic acid or alanine abolishes the kinase

activity (Timm et al., 2003). By comparing the MARK sequence with the closely

related kinase, CHK1, it appears that this residue is involved in aligning the catalytic

residue in the proper position during the phosphotransfer reaction (Chen et al., 2000).

Moreover the oncogenic serine/threonine kinase Pim-1 interacts with MARK3 (C-

TAK1) and phosphorylates the kinase domain of MARK which inhibits the MARK

activity. The residue phosphorylated by Pim-1 is yet to be identified (Bachmann et al.,

2004).

Introduction 10

A recent study by Matenia et al. reveals that the MARK kinase activity can be inhibited

also by some other mechanism. The PAK5 kinase, a member of the mammalian p21

activated kinases family inhibits MARK activity towards tau protein. This inhibition is

mainly based on the protein-protein interaction between the MARK and PAK5 catalytic

domains, rather than by phosphorylation (Matenia et al., 2005).

1.3.4 Proteins interacting with MARK

MARK interacts with the cytosolic scaffold protein 14-3-3 (alias Par-5) during D.

melanogaster cell polarity and development (Benton et al., 2002; Macara, 2004a;

Macara, 2004b) and 14-3-3 is also involved in many cellular functions and localization

of various proteins. 14-3-3 frequently interacts with phosphorylated proteins and

recruits them to different cellular compartments. Remarkably, binding between MARK

and 14-3-3 occurs without the usual requirement of a phosphorylated peptide. Indeed,

MARK does not bind to the canonical binding groove of 14-3-3 but to its C-terminus

(Benton et al., 2002). It appears also that MARK phosphorylates other partners which

then bind to 14-3-3, such as Cdc25C, KSR1, plakophilin, or Raf-1 (Benton et al., 2002;

Muller et al., 2003). This suggests that MARK regulates 14-3-3 activity towards its

binding partners.

On the other hand MARK2 is phosphorylated by aPKC at T596, a conserved residue in

all MARK isoforms. This phosphorylation increases its 14-3-3 binding activity and

negatively regulates the kinase activity and its plasma membrane localisation (Hurov et

al., 2004). Moreover, proteomic analysis of MARK shows its interaction with different

proteins involved in cytoskeleton organization (Brajenovic et al., 2004).

Recently, it has been shown that the yeast homologues of MARK, the KIN1 and KIN2

kinases, interact with t-SNARE, Sec9 and the Lgl homologue Sro7, proteins which are

involved in the final stage of exocytosis. It has also been shown that the conserved 42

amino acids at the carboxy terminal KA1 domain (Kinase associated domain 1) interact

with the kinase catalytic domain and/or N-terminus and leads to kinase auto-inhibition

(Elbert et al., 2005).

Introduction 11

1.4 Ubiquitin binding domains

Ubiquitin is a regulatory protein which takes part in numerous biological processes,

including targeted protein degradation, endocytic sorting, transcriptional control,

intracellular localization and retroviral virion budding. This small 76 aminoacid protein

is present in all eukaryotes and is highly conserved from yeast to humans. Nine

ubiquitin binding domains are identified so far in different proteins (Hicke et al., 2005):

CUE - coupling ubiquitin to endoplasmic reticulum degradation

UIM - ubiquitin interacting motif

NZF - Npl4 zinc finger motif

UBA - ubiquitin associated domain

UEV - ubiquitin conjugating Enzyme Variant

GAT - Gga and Tom1 domain

GLUE - GRAM-like ubiquitin-binding in Eap45

PAZ (ZnF-UBP) - polyubiquitin-associated zinc finger

VHS - Vps27, HRS, STAM

All of these domains are found in various proteins and tend to have a role in ubiquitin

dependent pathways. Among these nine ubiquitin binding domains, the UBA domain is

one of the best characterized domains from different proteins.

1.4.1 The ubiquitin associated domain

The UBA domain is a commonly occurring sequence motif of ~45 amino acids which

was initially identified in proteins involved in ubiquitin/proteasome pathways, and later

found in diverse proteins involved in the DNA excision-repair and cell signaling via

protein kinases (Mueller and Feigon, 2002). The UBA domain was the first ubiquitin

binding motif to be described. This domain was identified through sequence database

searches as a moderately conserved ~45 residue sequence found in a variety of proteins

(Hofmann and Bucher, 1996). So far, 127 UBA domains from 107 human proteins have

been identified, most of them are implicated in the ubiquitin-proteasome degradation

machinery (Chen and Madura, 2002).

Introduction 12

UBA domains have been shown to bind mono-, di-, tri-, and tetra-ubiquitin in vitro but

appear to bind to polyubiquitin with a higher affinity. It is thought that

polyubiquitinated proteins represent the true in vivo binding substrates. Some UBA

domains appear to homo- and heterodimerize and to bind other proteins like Vpr protein

of human immunodeficiency virus type 1 (Dieckmann et al., 1998).

As well as having different affinities towards mono- and polyubiquitins, UBA domains

are specific for the linkage of polyubiquitins. According to the ubiquitin linkage

specificity, the UBA domains are divided into four classes (Raasi et al., 2005):

Class 1 UBA domains selective for lysine-48 linked polyubiquitin chains

Class 2 UBA domains selective for lysine-63 linked polyubiquitin chains

Class 3 UBA domains not binding to polyubiquitin chains

Class 4 UBA domains binding to any polyubiquitin chains.

p62 is a novel cellular protein which was initially identified in humans as a phospho

tyrosine independent ligand of the src homology 2 (SH2) domain of p56lck, a member

of the c-src family of cytoplasmic tyrosine kinases. In addition to the SH2 domain, p62

possesses several structural motifs, including a ubiquitin associated (UBA) domain that

is capable of binding ubiquitin noncovalently. It has been shown that the UBA domain

of p62 binds to various proteins that are involved in neurodegenerative disorders such as

Alzheimer’s disease (Pridgeon et al., 2003). The important interacting proteins include,

myelin basic protein, 14-3-3 zeta isoform, syntaxin binding protein, FK506 binding

protein 14, homeobox protein Meis2, transketolase, heat shock cognate hsp70, reelin

isoform b, CaMKII, Unc51 like kinase II and nuclear receptor co-repressor 1.

1.4.2 Structure of the UBA domain

There are several UBA domain structures from various proteins solved mainly by the

NMR method. Interestingly, all these proteins share very low sequence similarity in

their UBA domains, but form a very similar three helical fold structure (Fig. 1.5). Most

of these structures have a conserved large hydrophobic surface patch which has been

predicted to play a role in protein-protein interactions (Mueller et al., 2002).

Introduction 13

Fig. 1.5: Structure of the UBA domain from HHR23A. (PDB code: 1IFY; Mueller et al., 2002) a: Ribbon representation of UBA domain structure, coloured according to the secondary structure elements. b: Surface representation of the UBA domain using the following colour coding: red, acidic residues Glu and Asp; blue, basic residues Arg and Lys; orange, polar residues Asn, Gln, His, Ser and Thr; white, hydrophobic residues Ala, Gly, Phe, Ile, Pro, Met, Leu, Tyr and Val. The major accessible residues on the hydrophobic surface, Met173, Gly174, Tyr175, Leu199 and Ile202, are marked. The surface area of the hydrophobic surface patch is about 470 Å2, which corresponds to ~17% of the total surface area of about 2830 Å2. This Figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

1.4.3 The UBA domain of MARK

Interestingly, MARK kinases are one of the sub-families of protein kinases containing a

UBA domain downstream to their catalytic domain. The function of the UBA domain in

the MARK kinases remains unclear. The presence of a UBA domain adjacent to the

catalytic domain suggests potential interactions with diverse proteins involved in the

ubiquitin proteasome pathway, DNA repair, or cell signaling (Brajenovic et al., 2004;

Hofmann and Bucher, 1996).

Y175

M173

G174

I202

L199

C

Na b

α3

α2

α1Y175

M173

G174

I202

L199

C

Na b

Y175

M173

G174

I202

L199

C

N

Y175

M173

G174

I202

L199Y175

M173

G174

I202

L199

C

N

C

Na b

α3

α2

α1

Introduction 14

1.5 Aim of the work

MAP/microtubule affinity regulating kinases (MARK) are a family of protein

serine/threonine kinases which have been identified by their ability to phosphorylate the

microtubule-associated proteins tau, MAP2 and MAP4. Phosphorylation of the neuronal

MAP tau on serine262 dramatically reduces its MT binding capacity and leads to the

formation of neurofibrillary tangles, which is a hallmark of Alzheimer’s disease. The

homologues of MARK kinases in C. elegans and D. melanogaster play important roles

in embryonic polarity and cell cytoskeleton regulations. All these functions make

MARK an important drug target for Alzheimer’s disease. It has been proven that

regulating a protein kinase activity is a helpful approach to treat many diseases like

cancer etc. In order to develop an inhibitor specific to MARK, there is a need for a high

resolution structure of MARK.

The aim of this study was therefore to elucidate the X-ray crystal structure of MARK

kinase. Generally, protein kinases can attain different conformations (active, inactive

and semi-active) depending upon their phosphorylation status, cellular localization and

availability of partner molecules. This makes the analysis of the regulation of protein

kinases a complex task. To understand this, the crystal structures in different

conformations have to be determined. All protein kinase structures determined so far

show a similar fold in their catalytic domains, but they are highly specific regarding

their substrates. While the biochemical studies reveal many different substrates for

MARK kinase, the reason for substrate specificity remains to be answered. The MARK

kinases have different interacting partners, but for most of the interacting proteins the

mode of interaction is yet to be identified. The goal of this study was to express, purify,

crystallize and to solve the structure of MARK kinase in various conformations.

Materials and Methods

15

2 Materials and methods

2.1 Materials

2.1.1 Chemicals

All chemicals used were of the highest purity available (ACS grade) and were

purchased from the following companies:

Amersham Pharmacia Biotech

AppliChem

Acros Organics

Fluka

Kodak

Merck

New England Biolabs

Qiagen

Sigma

In addition, the chemicals for crystallization were purchased from the following

companies as pre-formulated screens or separate reagents:

Hampton Research

Jena Biosciences

Molecular Dimensions

2.1.2 Enzymes

All restriction enzymes used for DNA engineering and the T4 DNA ligases were

purchased from the companies New England Biolabs, United States Biochemical and

Stratagene. The BP-clonase and LR-clonase enzyme mixtures were purchased from

Invitrogen.


16

2.1.3 Bacterial strains

Strain Genotype Features XL2-Blue recA1 endA1 gyrA96 thi-1

hsdR17 supE44 ReIA1 lac[F’ proAB laclqZΔM15 Tn10 (Tetr) Amy Camr]α

Host for cloning and plasmid propagation (Stratagene).

DH5α Library Efficiency

F-φ80lacZ ΔM15 Δ(lacZYA-argF)U169 recA1endA1 hsdR17 (rkmk

+)phoA supE44 thi-1gyr A96 relA1 λ

Host for cloning and propagation of Gateway vectors (Invitrogen).

Library Efficiency DB3.1

F- gyrA462 endA1 (sr1-recA) mcrB mrr hsdS20(rB-, mB-) supE44 ara-14 galK2 lacY1 proA2 rpsL20(SmR) xyl-5 - leu mtl1

The DB3.1 E. coli strain is resistant to ccdB effects and can support the propagation of plasmids containing the ccdB gene (Invitrogen).

BL21-AI F- ompT hsdS(rΒ -, mB- ) dcm

araB:T7RNAP-tetA

The T7 RNA polymerase gene is contained in the araB locus of the araBAD operon, allowing the regulation of the expression of the T7 RNA polymerase by the sugars L-arabinose and glucose. Glucose represses basal expression. The strain is suitable for high yield expression from T7 based expression vector (Invitrogen).

B834 F- ompT hsdS6(rB- mB-) gal dcm met

Methionine auxotropic cell strain, used for selenomethionine labelling (Novagen).

Table 2.1: Cell strains and feature list. The first column lists the names of the E. coli strains used for cloning, vector propagation and protein expression; the second column contains the genotype and the third some remarks about the purpose of use and their features.

Table 2.1 shows the bacterial strains used in this study: XL2-Blue (Bullock, 1987) and

library efficiency DH5α (Hanahan, 1983) were used as cloning hosts, BL21 (DE3)

(Studier et al., 1990), library efficiency DB3.1 (Bernard and Couturier, 1992), BL21

AI (Studier, 1986, Lee et al., 1987) and B834 (DE3) (Wood, 1966) were used for

protein expression.


17

2.1.4 Cloning Vectors

Most inserts used in this study were amplified by PCR and cloned into the pDONR201

vector of the recombination-based Gateway cloning system (2.2.9). All vectors used in

this study are shown in Table 2.2.

2.1.5 Expression Vectors

The expression vectors used in this study for the high-yield expression of recombinant

proteins carry cloned inserts under the control of the T7 promoter. Thus, only E. coli

strains engineered to express the T7 RNA polymerase upon IPTG induction can be

used for expression, e.g. BL21 (DE3). The Gateway vectors used in this study are of 2

types: the donor vector pDONR201 and the destination vectors pDEST15/17 (Table

2.2). The BL21 AI strain that was used for expression of Gateway expression vectors is

arabinose inducible (araBAD promoter).

Table 2.2: Summary of the vectors used in this study.

2.1.6 Media

Antibiotics: All antibiotic solutions were dissolved either in water or ethanol

depending on the solubility of the corresponding antibiotic. The water soluble

antibiotics were sterilized by filtering through a 0.22 µM sterile filter. The stock

solutions were prepared with the following concentrations: Ampicillin-15 mg/ml in

ddH2O, Kanamycin-25 mg/ml in ddH2O, Chloramphenicol-34 mg/ml in ethanol and

Carbenicillin-15 mg/ml in ddH2O.

Vector Expression System Features

pDONR201 Used for generation of entry clones

KanR

pDEST15 E. coli AmpR, N-terminal GST tag

pDEST17 E. coli AmpR, N-terminal His6 tag

pET16b E. coli AmpR, N-terminal His10 tag

pET28a E. coli KanR, N-terminal His10 tag


18

Luria-Bertani (LB) medium: 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast

extract, 1% (w/v) NaCl; sterilized by autoclaving. Purchased from Life Technologies

as dried powder.

LB agar plates: LB medium and 1.5 % (w/v) bacteriological agar. Sterilized by

autoclaving. Plates were poured when the temperature reached ~50°C.

SOB medium: 2% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract, 0.5 % (w/v)

NaCl. A solution of KCl was added to a final concentration of 25 mM. The pH was

adjusted to 7 with NaOH and the solution sterilized by autoclaving. Before use a sterile

solution of MgCl2 was added to a final concentration of 0.1 M.

SOC medium: SOB medium supplemented with 1.8 % glucose.

M9 Minimal Medium: one litre of 5X stock of M9 medium was prepared by

dissolving 30g Na2HPO4, 15g KH2PO4, 5g NH4Cl and 2.5g NaCl in distilled water and

sterilized by autoclaving. To make one liter of M9 medium, 200 ml of 5X M9 salts, 1

ml of 1 M MgSO4, and 10 ml of 40% glucose were mixed and diluted up to one litre

with sterile water.


2.1.7.1 Crystallization supplies and tools

Crystallization supplies and tools including crystallization plates, siliconised cover

slides (round and square slides of different thickness), sealing tape, forceps and tools

for crystal manipulation were purchased from Hampton Research.


19

2.1.7.2 Crystallization solutions

Crystallization screens purchased from Hampton Research:

Crystal screen 1 Additive screen 1

Crystal screen 2 Additive screen 2

PEG/Ion screen Additive screen 3

Grid screen Na-Malonate Crystal screen Index

Grid screen PEG6000 Grid screen Ammonium sulfate

SaltRx screen Crystal screen Lite

Crystallization screens purchased from Jena Biosciences:

JB High throughput Screen I and II

2.1.8 Equipment and accessories

Centrifuges:

Cold centrifuge J2-21 M/E Beckman

Ultracentrifuge Beckman

Table centrifuge 5402 Eppendorf

Table centrifuge 5415C Eppendorf

Rotors:

JA-10, JA-20 Beckman

45Ti Beckman

Äkta purification system and corresponding accessories:

Mono-S column HR10/10 Pharmacia

HiLoadTM 16/60 SuperdexTM 200 Pharmacia

Ni-NTA Superflow Qiagen

GST-sepharose beads Amersham Biosciences

Sample loops 1ml, 2ml, 50ml Pharmacia


20

Other equipments and accessories:

French press cell Aminco

Gel dryer model 583 Bio-Rad

Incubator shaker model Innova 4330 New Brunswick Scientific

UV/visible spectrophotometer Ultraspec1000 Pharmacia

Dynamic light scattering Firma Dierks and Partner

Scintillation counter Tricarb 1900 CA, Packard


2.2.1 Culture and storage of E. coli

Bacteria cells were grown on LB agar plates or in liquid LB medium at 37°C unless

stated otherwise. For positive selection, media and plates were supplemented with the

appropriate antibiotics in the following concentrations: 50 μg/ml ampicillin, 50 μg/ml

carbenicillin, 25 μg/ml kanamycin. All media and manipulation tools were sterilized by

autoclaving, or if heat-labile, by filtration through a 0.22 μm filter. For permanent

storage at –80oC, cell strains were flash-frozen in liquid nitrogen after mixing with

glycerol for cryoprotection. BL21 and XL2 blue strains were preserved in 30% (v/v)

glycerol in LB; DH5α strain in 50% (v/v) glycerol in LB.

2.2.2 Transformation of E. coli strains

E. coli cells competent for transformation were either purchased from the companies as

listed in table 2.1 or prepared in the laboratory. XL2-Blue, DH5α library efficiency and

BL21 AI cells were transformed by the heat-shock method: 20-100 ng of DNA was

added to an aliquot of competent cells (~20-50 μl) previously thawed on ice for

approximately 10 minutes. The mixture of DNA and competent cells was incubated on

ice for 30 minutes. After the heat-shock at 42oC for 30 seconds, 200-400 μl of SOC

medium was added and the cells were incubated at 37°C for 1 hour with shaking.

Finally, 100-200 μl cells were plated on a selective medium plate and incubated

overnight at 37oC.


21

2.2.3 Isolation of plasmid DNA

All plasmid mini-preparations were carried out with the Invisorb Spin Plasmid Mini

Kit (Invitek) following the user manual. All midi-preparations were carried out with

the Nucleobond AX Kit (Macherey-Nagel) according to the manual.

2.2.4 Determination of DNA concentration and purity

The concentration and the degree of purity of double stranded plasmid DNA was

determined based on the Beer-Lambert Law by measuring the absorbance at 260 nm

and 280 nm:

A260=ε260 c l and A260 x 50= μg/ml (when l= 1 cm) A260 is the absorbance at 260 nm, ε260 is the molar absorbtion coefficient, c is the molar

concentration and l is the optical path. For a protein-free and RNA-free solution of

DNA the ratio of A260/ A280 should be close to 2. Protein contaminants would decrease

this ratio, whereas RNA contamination would increase it. To further estimate the

concentration and purity of DNA preparations agarose gel electrophoresis was carried

out.

2.2.5 Ligation reaction

The components below were mixed in a 500µl Eppendorf tube. The mix was incubated

at 16°C overnight. The molar ratio between the digested vector and the digested insert

was around 1:5.

10x Buffer 1µl

Ligase (5U/µl) 1µl

Digested vector 200 ng

Digested insert 200 ng

H2O x µl

Total volume 10 µl


22

2.2.6 Restriction digestion analysis of DNA

DNA samples were analyzed with the use of restriction digestion enzymes (New

England Biolabs). In each case the sample was mixed with the desired restriction

enzyme/s and the proper reaction buffer (New England Biolabs) in a final volume of

20 μl. The mixture was incubated for 60 min at 37° C. The digested DNA was loaded

immediately onto an agarose gel to check the result of the restriction digestion

analysis.

2.2.7 DNA sequencing

DNA sequencing reactions were performed to confirm the sequence of a construct and

the existence of mutations, especially after PCR amplification steps. The reactions

were performed using fluorescent dye labelling and the Sanger Method (Sanger et al.,

1977) in a Robocycler Gradient 96 PCR machine. The protocol for the temperature

cycle reaction was:

Terminator ready reaction mix 8 μl

dsDNA 500 ng

Primer (10pmol/ μl) 1 μl

ddH2O to a final volume of 20 μl

The PCR program for sequencing was:

1. Denaturation 96°C 10 sec

2. Annealing 45°C 5 sec X 30 cycles

3. Elongation 60°C 4 min

After the reaction, the DNA was precipitated by using the Pellet Paint NF Co-

Precipitant (Novagen). To the 20 μl reaction mixture, 1 μl Pellet Paint NF Co-

Precipitant and 80 μl of 75% ethanol were added. The contents were mixed by

vortexing, and centrifuged at 13 krpm for 10 min at RT. Then the DNA pellet was

washed with 250 µl of 70% ethanol to remove any trace of salts in the DNA pellet and

centrifuged at 13krpm, for 10 min at RT. The pellet was then air dried and resuspended

in 30 μl of HPLC-grade dd H2O.


23

The ABI PRISM 310 Genetic Analyser (PE Applied Biosystems) was used to sequence

the DNA. The sequencing results were analyzed with the VectorNTI software

(Informax).

2.2.8 Mutagenesis of DNA

All mutations described in this thesis were created by site-directed mutagenesis, which

was performed using the Quick Change Site-Directed Mutagenesis Kit (Stratagene).

The method utilizes the Pfu Ultra High Fidelity polymerase to replicate the parental

plasmid by using two synthetic oligonucleotide primers containing the desired

mutation.

The primers, each complementary to opposite strands of the vector, are extended

during temperature cycling by the DNA polymerase. Temperature cycling generates

copies of the plasmid by linear amplification, incorporating the mutation of interest.

The temperature cycling reaction was performed as follows:

10x Pfu Ultra High Fidelity buffer 2 μl

ds DNA template (25ng/ μl) 5 μl

dNTPs (2.5 mM) 2 μl

Primer 1 (0.5 pmoles/μl) 1 μl

Primer 2 (0.5 pmoles/μl) 1 μl

Pfu polymerase (2.5 U/μl) 0,5 μl

ddH2O to a final volume of 20 μl

The temperature cycling program used was:

Step Time Temperature Cycles

Initial

denaturation

30 seconds 95°C 1

Denaturation 30 seconds 95°C

Annealing 1 minute 55 – 58°C

Extension 12 minutes 68°C

16


24

The primer annealing temperature was calculated according to the melting temperature

(Tm) of the primers and the extension time was calculated according to the length of

the plasmid. Next, a treatment with DpnI endonuclease was carried out to digest the

parental methylated DNA template, allowing the selection of the newly synthesized

DNA containing the mutation. 2-4 μl of this reaction mixture was used to transform

XL2-Blue cells. Alternatively, some of the mutants were created by cutting and

ligating the mutated DNA fragment from the full length mutant clones.

2.2.9 Gene cloning using the Invitrogen Gateway cloning technology

The Gateway cloning technology is a method that enables rapid cloning of a gene in

various expression systems (Invitrogen). Gateway uses a well-characterized lambda

phage site-specific recombination system, thus restriction enzymes and ligases are not

required in any step. Two reactions, ‘BP-reaction’ and ‘LR-reaction’, constitute the

Gateway cloning technology.

Reaction Reaction sites Product Product Structure

BP reaction attB x attP Entry clone attL1-gene-attL2

LR reaction attL x attR Expression clone attB1-gene-attB2

Table 2.3: Summary of reactions and nomenclature of the Gateway cloning technology

First, an entry clone is generated from a PCR product that spans the attB recombination

sequences (BP reaction, Fig.2.1). Once a positive clone is verified and sequenced the

second step is to transfer the gene of interest to a variety of expression vectors (LR

reaction; Fig.2.1), featuring different tags and/or different expression systems (e.g. E.

coli, insect cells). The ccdB gene interferes with the E. coli DNA gyrase. Thus, every

cell that takes up an unreacted vector that still carries the ccdB gene or a by-product,

will fail to grow.


25

Generation of MARK2 constructs with the Gateway technology

The first step to enter the system was to obtain the proper PCR product with the

required attB recombination sequences. In this study, this was achieved in two steps,

with a set of primers specific for each construct and a set of primers for the completion

of the attB sites. The resulting DNA sequence would be attB1 – TEV cleavage site –

fragment of interest – attB2.

Fig. 2.1: Overview of the Gateway cloning technology.

The encoding sequence of the TEV cleavage site is incorporated in order to be able to

remove the tag after purification of the relevant protein for crystallization purposes.

The primers were designed as follows:

Primers to amplify the ORF:

attB1-Tev-f (forward primer)

5’-AAAAAGCAGGCTTC GAAAACCTGTATTTTCAGGGC-

coding sequence-3’

attB1 (12 bases) - 2 bases for frame - TEV cleavage site

attB2-stop-r (reverse primer)

5’-AGAAAGCTGGGTC TTA – coding sequence-3’

AttB2 (13 bases) - stop codon

BP reaction to generate an entry clone

LR reaction to generate an expression clone

BP reaction to generate an entry cloneBP reaction to generate an entry clone

LR reaction to generate an expression cloneLR reaction to generate an expression clone


26

Adapter primers to generate complete attB sites:

AttB1f (forward primer) 5’-GGGGACAACTTTGTACAAAAAAGCAGGCT-3’ AttB2r (reverse primer) 5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’ (The underlined sequences show the parts that overlap in both gene specific and

adaptor primers)

PCR reactions

The first PCR step was done with primers that do not have very long non-specific

overhangs in order to obtain a high success rate in amplification. The attB sites that are

needed, were generated with a second PCR step using the adapter primers.

1st PCR

Template DNA 200 ng

10x reaction buffer 5 μl

Forward primer 10 pmoles

Reverse primer 10 pmoles

dNTPs Mix 5 mM

H2O up to 50 µl

The PCR program used was as follows:


Initial

denaturation

1 minute 95 °C 1x

Denaturation 15 seconds 95 °C

Annealing 30 seconds Depends on each

primer

Extension 30 seconds 68 °C

10x

2nd PCR


27

First PCR product 10µl

10x reaction buffer 5 μl

Forward primer (attB1f) 40 pmoles

Reverse primer (attB2r) 40 pmoles

dNTPs Mix 5 mM

H2O up to 50 µl

The cycling parameters for the second PCR were:


Initial

denaturation

1 minute 95 °C 1x


Annealing 30 seconds 45 °C


5x


Annealing 30 seconds 55 °C


20x

The PCR products were treated with DpnI in order to digest the template plasmid.

PCR product 50 µl

10x DpnI buffer 5 µl

DpnI 10 units

30 minutes at 37oC (digestion reaction)

15 minutes 65 oC (DpnI heat inactivation)

The PCR products were then analyzed by agarose gel electrophoresis and purified by

using the pellet paint protocol (refer to section 2.2.7).


28

BP reaction:

Purified PCR product 200 ng

pDONR201 (150 ng/μl) 2.5 μl

5x BP reaction buffer 5 μl

BP-clonase enzyme mix 2 μl

ddH2O up to 25 μl

The mixture was incubated at 25oC overnight and then 2 μl of proteinase-K was added

to stop the reaction. Next, 1-3 μl were used to transform DH5α library efficiency E.

coli cells. The clones were analyzed by setting up a double digestion with specific

enzymes. Positive clones were then sequenced (section 2.2.7) using the primers attL1-f

(proximal to attL1) and attL2-r (proximal to attL2) and gene specific primers

(Appendix). When a positive clone was confirmed for correct sequence, it was used for

setting up an LR reaction in order to generate an expression clone.

LR reaction:

LR buffer 5x 4 μl

Destination vector (150 ng/ μl) 3 μl

Entry clone (150 ng/ μl) 1 μl

Topoisomerase I 0.5 μl

LR clonase mix 2 μl

ddH2O up to 20 μl

The topoisomerase was added to relax the entry clone plasmid as a supercoiled state of

entry vectors was often observed in agarose gel electrophoresis. The supercoiled state

of DNA lowers the efficiency of LR clonase mix.

The mixture was incubated at 25oC overnight and then 2 μl of proteinase K was added

to stop the reaction. 1-2 μl of this DNA product was transformed into either DH5α

library efficiency competent cells for plasmid propagation, or BL21 AI cells for

expression of the recombinant protein.


29

2.3 Protein methods

2.3.1 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed in the lab following a modified protocol (Matsudaira and

Burgess, 1978; Laemmli, 1970). The stacking gel was 4% acrylamide and the

separating gel was 10% or 17% (Table 2.4). Protein samples were diluted 1:1 with 2x

SDS-PAGE loading buffer (Laemmli, 1970), and heated for 2 min at 95°C.

Electrophoresis was carried out at 150V and maximal 35mA in SDS-PAGE running

buffer (25 mM Tris-HCI, 190 mM Glycine, 0.1% (w/v) SDS). The gels were then

stained in a 0.1% (w/v) solution of Coomassie brilliant blue R-250, 45% (v/v)

methanol and 9% (v/v) acetic acid for 20 min on a shaking platform. Next, the gels

were destained in an intensive destaining solution (50 % (v/v) methanol, 10% (v/v)

acetic acid) for 20 min and for a minimum of 1 hour in a normal destaining solution

(5% (v/v) methanol, 7.5% (v/v) acetic acid).

Molecular weight marker proteins (Biofermentas) were:

Protein name Molecular Weight (in kDa)

ß-Galactosidase 116

Bovine serum albumin 66.2

Ovalbumin 45.0

Lactate-dehydrogenase 35.0

Restriction endonuclease Bsp98I 25.0

Lactoglobulin 18.0

Lysozyme 14.4


30

Table 2.4: Solutions for preparing SDS-PAGE (volumes are in ml)

2.3.2 Protein expression and purification

The following strategy was applied for every new expression construct: First, 5 ml LB

supplemented with the appropriate antibiotics was inoculated with the desired

expression strain and grown at 37°C overnight. This pre-culture was used to inoculate

a new 100 ml LB culture, which was left to grow at 37°C, shaking at 280 rpm until the

optical density was reached to 0.6. A sample of 1 ml was centrifuged and kept as a un-

induced control and the rest was induced with IPTG to final concentration of 0.5 mM

or with arabinose to a final concentration of 0.2 %, depending on the E. coli cell strain

used for expression. The cultures were left to grow at lower temperature 25-30°C for

overnight. Cells were then harvested by centrifugation at 8 krpm for 5 min (Eppendorf

5810R) and resuspended in lysis buffer.

2.3.3 Cell lysis and solubility test

Cells were lysed using a French press (Aminco). First, cells were thoroughly

resuspended in lysis buffer (2-4 ml of lysis buffer/100ml culture) and then they were

transferred to a French press cell. Application of 20,000 PSI in two rounds ensured the

lysis. Lysates were kept on ice and centrifuged at 14 krpm for 20 min at 4°C. A sample

Separating gel

Stacking gel

Components 10% 17% 4%

40 % Acrylamide / Bis-acrylamide (37.5:1)

15.0 25.6 5.4

Tris HCl (1M pH 8.8)

22.0 22.0 -

Tris HCl (0.25 M pH 6.8)

- - 27.0

10%SDS 0.6 0.6 0.54

TEMED 0.12 0.12 0.108

10% APS 0.065 0.065 0.15

H2O

22.0 11.4 20.9


31

of the supernatant in which all soluble proteins were present and the pellet resuspended

in lysis buffer were loaded onto an SDS gel together with samples before and after

induction. In this way expression and solubility of the desired protein was tested in a

first approach. For large-scale production of proteins, cultures of 2-6 litres were grown

as described above.

2.3.4 Chromatography

Purification of proteins was performed by fast performance liquid chromatography

(FPLC) using Äkta purifier and Äkta explorer machines (Pharmacia).

2.3.4.1 Ni-NTA affinity chromatography

Immobilised metal affinity chromatography (IMAC) makes use of the binding

properties of metals towards proteins for purification purposes; nickel-nitriloacetic (Ni-

NTA) resin (QIAGEN) contains chelated nickel, which is able to specifically bind to

stretches of polyhistidine in proteins. Most expression systems include a tag of six

histidines either at the N- or at the C-terminus or on both.

The resin was cast on a Pharmacia self-packed XK26 column or a batch protocol was

performed with the use of Biorad columns. The material was rinsed with ddH2O to

remove the 20% (v/v) ethanol preservative and equilibrated with the appropriate

loading buffer. The buffers should not contain DTT or other reducing agents in high

concentrations, as this might strip the nickel from the resin. Once the column was

equilibrated, the sample was passed twice over the column. After loading, the column

was washed with loading buffer (~10-20 column volumes) and finally, the protein was

eluted with 3 volumes of elution buffer. All buffers are listed in the Appendix.

2.3.4.2 GST affinity chromatography

Glutathione Sepharose (Pharmacia) is an agarose material coupled with glutathione,

which is frequently used for purification of GST tagged proteins. Glutathione

Sepharose resin was self-packed in a column (Pharmacia) or prepacked 5 ml GST Hi-

Trap columns (Pharmacia) were used. The column was rinsed with 10 volumes of


32

ddH20 and 10 volumes of loading buffer. The sample was loaded onto the column with

a low flow rate of 0.2 ml/min and passed twice over the column. Next, the column was

washed with 7-10 volumes of loading buffer. Elution was achieved with 4 volumes of

GST elution buffer. Eluate fractions were pooled together and kept at 4oC for further

processing.

2.3.4.3 Anion and cation exchange chromatography

Protein separation by ion exchange chromatography depends on the reversible

adsorption of charged molecules to an immobilised ion exchange group of opposite

charge. Varying conditions such as ionic strength and pH can control these

interactions. To ensure electrostatic binding, the total ionic strength needs to be low.

Generally, 100mM NaCl was included into the anion exchange buffers. The columns

for anion exchange chromatography (AIEX) and cation exchange chromatography

(CIEX) were MonoQ HR 10/10 and MonoS HR 10/10 (Pharmacia) respectively. After

equilibration with 5 column volumes of the AIEX/CIEX buffer A, the sample was

loaded and the washing step followed with 5-7 column volumes of AIEX/CIEX buffer

A. The elution was performed with a linear gradient of AIEX/CIEX buffer B in two

steps: first from 0 to 60% in 5-8 column volumes and then to 100% in 1-2 column

volumes. Eluted fractions containing the protein of interest were pooled together.

2.3.4.4 Gel filtration chromatography

On a gel filtration (or size exclusion) column the molecules are separated according to

differences in their sizes. The concentrated protein solution is injected into a 1 ml loop

with a injection needle (Pharmacia). Small molecules which can diffuse into the pores

of the gel beads are delayed in their passage through the column in contrast to the

larger molecules, which cannot diffuse into the gel beads. The larger molecules thus

leave the column first, followed by the smaller ones in order of their sizes. Gel

filtration was performed with a Superdex G-200 HR 16/60 column (Pharmacia).


33

2.3.5 Determination of the protein concentration

Protein concentrations were estimated by using the Bradford method (Bradford, 1976).

The assay is based on the observation that the absorbance maximum for an acidic

solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when

binding to the protein occurs. Bovine serum albumin solution is used as a standard.

1µg to 5 µg of BSA solution in 10µl were used to calculate a standard curve. The test

proteins were measured in two different dilutions. To these samples 200µl of Bradford

reagent (Bio Rad) were added and mixed. The absorbance at 595 nm was measured in

a Microtitre plate reader (BioLynx 2.2) and the protein concentrations were calculated

from the standard curve.

2.3.6 Concentrating the protein solution

The protein solutions were concentrated using the Amicon (Millipore) device. In this

device, a membrane with a molecular weight cut-off smaller than the protein of interest

is placed at the bottom of a cell, which is filled with the protein solution. The cell is

then placed in a 50 ml Falcon tube and centrifuged at 3000g. In this way, the flow

through contains only lower molecular weight components, while the protein is

concentrated in the chamber.

2.3.7 Protein kinase assay

Kinase activities were assayed in 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM

EGTA, 0.5 mM PMSF, 0.5 mM DTT and 0.5 mM benzamidine for 30 minutes at

30°C. Final concentration of [32P] ATP (3.7*107 MBq/mol; Amersham Biosciences)

and substrate peptide were 100 μM. The substrate peptide derived from the first repeat

of tau protein containing S262 in the KXGS motif (TR1-peptide NVKSKIGSTENLK,

Drewes et al. 1997). Reactions were stopped by addition of half the volume of 30

%(w/v) TCA. After centrifugation, the supernatant was applied to phosphocellulose-

paperdiscs, washed with phosphoric acid (0.1 M), dried by air and radioactivity was

measured in a scintillation counter (Tricarb 1900 CA, Packard).


34

2.3.8 Limited proteolysis

Limited proteolysis is one of the novel techniques to identify the structurally folded

part of the protein (Fontana et al., 2004). In a number of studies it has been

demonstrated that the sites of limited proteolysis along the polypeptide chain of a

protein are characterized by enhanced backbone flexibility, implying that proteolytic

probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis

was used to analyze the partly folded (molten globule) states of several proteins, such

as apomyoglobin, lactalbumin, calcium-binding lysozymes, cytochrome C and human

growth hormone.

The N-terminal GST tagged MARK2 construct (GST-MARK2 [1-364]) was subjected to

limited proteolysis with different proteases like trypsin, chymotrypsin, GluC, AspN,

and thermolysin in a ratio of 200 to 1. 100 µg of purified MARK protein was mixed

with 0.5 µg of corresponding proteases in the protease reaction buffer (Appendix). This

reaction mixture was incubated at 37ºC and samples were taken at different time points

(10, 30, 60, 120 min). The reactions were quenched by adding 1mM PMSF and boiling

the samples with 2x SDS loading buffer. As a control, a sample was incubated at 37ºC

for 120 minutes to check for the heat stability of the protein. The results were analyzed

by SDS-PAGE and further with N-terminal amino acid sequencing and mass

spectrometry.

2.3.9 N-terminal aminoacid sequencing

N-terminal sequencing for MARK2 stable fragment was performed with Procise-cLC

(ABI-Perkin Elmer) protein Sequencer. The MARK2 protein (GST-MARK2 [1-364])

was digested with trypsin in a ratio of 200 to 1 and the stable fragment was purified by

gel filtration chromatography. This purified protein was applied on to a PVDF

membrane and subjected to 5 cycles of sequencing reactions.

2.3.10 Mass spectrometry

Mass spectrometry analysis was performed with a SELDI Mass-Spectrometer PBS-I

(Ciphergen, USA). 2µl of the protein sample were mixed with equal amount of matrix


35

(Sinapinic Acid in 50% acetonitrile and 0.5% trifluoric acid). From this mixture, 3µl

were loaded on to a H4 Protein Chip (Ciphergen, USA) in two times and each time the

chip was dried for ~15 minutes in the hot air oven. Mass spectrometry analysis was

used to find the molecular weights of stable fragment of MARK2 and

selenomethionine labelled proteins.

2.3.11 Detailed protocols on purification and crystallization of MARK2 wild type

and mutant proteins

2.3.11.1 Expression

Proteins (wild type and mutant proteins) were expressed in E. coli strain BL21 AI

(Invitrogen). Cells were induced overnight at 24°C by adding arabinose to a final

concentration of 0.2% at OD600 ≈ 0.6. Cells from a litre culture was harvested by

centrifugation and resuspended in 40 ml lysis buffer (50mM Hepes pH 7.2, 300 mM

NaCl, 5% glycerol) and supplemented with one tablet of EDTA-free complete protease

inhibitor cocktail (Roche). Resuspended cells were lysed by passing two times through

a French press cell (Aminco).

2.3.11.2 Purification

The expressed proteins were purified in four steps: Ni-NTA affinity chromatography,

TEV protease cleavage to remove the His-tag, ion exchange chromatography and gel

filtration chromatography. Clarified lysate were applied to a self-packed Ni-NTA

affinity column of 5ml Ni-NTA beads (Qiagen). The protein was eluted with a 0 to

1000 mM gradient of imidazole in buffer A (50 mM Hepes pH 7.2, 300 mM NaCl and

5% glycerol). Pure protein fractions were pooled and mixed with purified TEV

protease in a ratio of 1:20 to cleave off the His-tag. The protein mixture was dialyzed

overnight against buffer B (50 mM Hepes pH 7.2, 200 mM NaCl, 5% glycerol, 1 mM

EGTA, 1 mM DTT). Around 1 ml Ni-NTA beads were added to the dialyzed protein to

remove the His-tagged TEV protease and the uncleaved MARK2 protein. After one

hour, the Ni-NTA beads were removed and the salt concentration was reduced to

100 mM by dilution with salt free buffer (50 mM Hepes pH 7.2 and 5% glycerol). The


36

proteins were further purified by MonoS cation exchange chromatography (MonoS HR

10/10 column, Amersham Biosciences) using a 100 to 1000 mM NaCl gradient .

Pooled fractions were concentrated using Ultrafree-30 concentrators and applied to a

gel filtration column (Hiload 16/60 Superdex G200, Amersham Biosciences)

equilibrated with 50 mM Bis-Tris pH 6.5 (H2SO4), 250 mM NaCl, 5% glycerol). The

pure protein fractions were pooled and concentrated to ~20 mg/ml. The concentrated

protein solution was then aliquoted in 0.2 ml PCR tubes and shock frozen in liquid

nitrogen.

2.3.11.3 Selenomethionine labelling

Selenomethionine labelled protein of the double mutant MARK2 was prepared by

expression in methionine auxotrophic E. coli strain B834 (Invitrogen) (Table 2.1) using

M9 minimal medium, supplemented with all amino acids except methionine that was

substituted by selenomethionine (Acros Organics) (40 mg per litre of medium). The

purification procedure was essentially the same as for the unlabelled proteins. The

percentage of incorporation of selenomethionine was estimated by mass spectrometry.


2.4.1 Crystallization techniques

A variety of methods exist to crystallize biological macromolecules. All of them aim to

bring the protein solution into a supersaturated state. Among them, vapour diffusion is

the most widely used method that also has been used in this study. A droplet

containing the protein solution to be crystallized, buffer, crystallizing agent and

additives is equilibrated against a reservoir containing a solution of crystallizing agent

at a higher concentration than in the droplet. Equilibration proceeds by diffusion of the

volatile species (water or organic solvent) until the vapour pressure in the droplet is

equal to the one in the reservoir. If equilibration occurs by water exchange (from the

drop to the reservoir), it leads to a decreasing volume of the droplet. This method was

used in this study in two variations, either as hanging or as sitting drops (Fig. 2.2).


37

Crystallization solutions and supplies are described in the material section 2.1.7. Prior

to each crystallization experiment, the highly concentrated protein solution (10-20

mg/ml) was centrifuged in an Eppendorf tabletop centrifuge at 13 krpm for 10 minutes

at 4oC, in order to separate precipitates. First screens were set up in Crystal Quick 96

well sitting drop plates (Hampton Research) by mixing 1 µl of protein with 1µl of

reservoir solution. During the optimization trials, the hanging drop method was

preferred.

Fig. 2.2: Set up of crystallization trials using: a) the sitting or b) the hanging drop vapour diffusion technique.

For each trial 2 μl of protein solution was mixed with 2 μl of reservoir. The plates were

sealed with tape in the case of sitting drop and with a siliconised cover slip in the case

of hanging drop trials and kept at 20°C and/or at 4°C. The plates were examined each

day during the first week and 2 times a week during the first 2 months. The initial

screens were performed by the use of the commercial screens described in section

2.1.7.2.

Occasionally, the technique of seeding crystals was performed in an attempt to

improve the size and quality of micro crystals, irregular-shaped crystals or to avoid

excessive nucleation. First, a drop with existing micro crystals was transferred to an

Eppendorf tube, diluted with the proper buffer and a small plastic ball was added.

Vortexing this solution resulted in breaking the nuclei/crystals and made it possible to

transfer a small portion of them by a horse tail hair to new drops of lower

concentration of protein and/or precipitant agent. This technique can lead to a slower

growth thus yielding bigger crystals.

a ba b


38

2.4.2 Cryoprotection of crystals

The high intensity of synchrotron radiation can lead to radiation damage of the protein

crystals. The interaction between the beam and the crystal generates thermal disorder

and may even break bonds within the protein. A common method to prevent this

radiation damage is freezing the crystals in a stream of cold nitrogen and collect the

diffraction data at low temperature (Garman and Schneider, 1997). But the high

solvent content (~50%) of protein crystals can lead to ice crystal formation during

freezing and these ice crystals will destroy the crystal integrity and disturb the protein

diffraction. This problem can be overcome by soaking the crystal in a cryoprotectant

solution which maintains the crystal quality and prevents the formation of ice crystals

(Garman and Schneider, 1997). MARK2 crystals were soaked in different

cryoprotecting agents like glycerol, MPD and ethylene glycol and tested for the

diffraction quality.

2.4.3 Data collection

X-ray diffraction data were collected using synchrotron radiation at the beamline of the

X13 Consortium for Protein Crystallography at HASYLAB (DESY, Hamburg). X13 is

a monochromatic, fixed-wavelength beamline, set at 0.802 Å and other characteristic

features of this beam line are given below:

Beamline X13

Institute DESY, EMBL and University of Hamburg, Hamburg

Wavelength 0.802Å

Optics Triangular cut Si (111) monochromator crystal

Mirror continuous bent Rh-coated focusing mirror

Detector MAR300 CCD

Cooling device Oxford-Cryosystem


39

2.4.4 Methods of Phase determination

The determination of the three-dimensional structure of macromolecules using X-ray

crystal diffraction techniques requires the measurement of amplitudes and the

calculation of phases for each diffraction point. Amplitudes |F (h,k,l)| can be directly

measured from diffracting crystals, phases α (h,k,l) have to be determined indirectly.

Thus, methods were developed to calculate phases. These include molecular

replacement (MR), multiple isomorphous replacement (MIR) and multi wavelength

anomalous dispersion (MAD) method.

Molecular Replacement (MR)

The molecular replacement method makes use of a known three-dimensional structure

of a homologous protein as an appropriate starting model to provide initial phases for

the unknown structure. Protein kinases normally have a similar fold in their catalytic

domain. Therefore, the crystal structures of other protein kinases can be used as a

search model for solving the MARK2 crystal structure.

Multiple Isomorphous Replacement (MIR)

Isomorphous replacement requires the introduction of high atomic number elements

(heavy atoms), such as mercury, platinum, uranium, and so forth, into the

macromolecule without disrupting its structure of packing in the crystal. Thus, a

perfect isomorphous derivative is one in which the only change between it and the

native crystal is the incorporation of one or more heavy atoms. This is commonly done

by soaking crystals of native molecules in a solution containing the desired heavy

atom. 1 mM stock solutions of various heavy atom salts were prepared with the

cryoprotectant solution, and the MARK2 crystals were transferred to these solutions,

soaked for few hours, and tested for heavy atom incorporation.

Multiple-wavelength anomalous dispersion (MAD)

Multiple-wavelength anomalous dispersion method utilizes the property of heavy

atoms to absorb X-rays of specific wavelength which causes the anomalous scattering

or anomalous dispersion. One common way to use MAD is to introduce

selenomethionine (SeMet) in place of methionine residues in a protein. The selenium


40

atoms (which replace the sulfur atoms) have a strong anomalous signal at wavelengths

that can be obtained from synchrotron X-ray sources. Selenomethionine labelled

protein of the MARK2 was prepared by expression in methionine auxotrophic E. coli

strain B834 (Table 2.1) and crystals were grown by using the wild type protein

crystallization conditions.

Results 41

3. RESULTS

3.1 Cloning of MARK2 constructs

MARK is a multi domain protein which contains an N-terminal leader sequence, a

catalytic domain, an ubiquitin-associated domain (UBA), a spacer and a tail domain

containing the KA1 (kinase-associated) motif characteristic for the family of kinases

ending with the ELKL motif (Fig. 1.3). In order to solve the crystal structure of the

MARK2 kinase domain, different constructs (Fig. 3.1) were made by using

recombination based Gateway cloning techniques (2.2.9). All constructs were designed

on the basis of the domain features found by using the SMART protein analysis server

(http://smart.embl-heidelberg.de/).

Fig. 3.1: Summary of different constructs of rnMARK2 and their solubility upon prokaryotic expression. N, N-terminal leader sequence; C, kinase catalytic domain; U, ubiquitin-associated domain. The constructs NC and C alone were nearly insoluble when expressed in E. coli. Constructs CU was found to be partially soluble and NCU has a high solubility. All constructs were cloned either with N-terminal 6X His-tag or N-terminal GST-tag for affinity purification.

3041

CN - - -NC

30453

C - - -C

362

YK364

53

C U +CU

1 362

YK364CN U + + +NCU

Solublitylevel

Short Name

3041

CN - - -NC

3041

CN

30430411

CCNN - - -NC

30453

C - - -C

30453

C

30453 3043045353

CC - - -C

362

YK364

53

C U +CU

362

YK364

53

C U

362

YK364

53 362362

YK364

5353

CC UU +CU

1 362

YK364CN U + + +NCU

1 362

YK364CN U

1 362

YK364

11 362362

YK364CCNN UU + + +NCU

Solublitylevel

Short Name

Solubility level

Short Name

3043041

CN - - -NC

30453

C - - -C

1

CN - - -NC

30453

C - - -C

362

YK364

53

C U +CU

362

YK364

53

C U +CU

1 362

YK364CN U + + +NCU

Solublitylevel

Short Name

304

1 362

YK364CN U + + +NCU

Solublitylevel

Short Name

3041

CN - - -NC

3041

CN - - -NC

3041

CN

3043041

CN

30430411

CCNN - - -NC

304

11

CCNN - - -NC

30453

C - - -C

30453

C

30453

C - - -C

30453

C

30453 3043045353

CC - - -C

53 3043045353

CC - - -C

362

YK364

53

C U +CU

362

YK364

53

C U +CU

362

YK364

53

C U

362

YK364

53

C U

362

YK364

53 362

YK364

53 362362

YK364

5353

CC UU +CU

362362

YK364

5353

CC UU +CU

1 362

YK364CN U + + +NCU

1 362

YK364CN U + + +NCU

1 362

YK364CN U

1 362

YK364CN U

1 362

YK364

1 362

YK364

11 362362

YK364CCNN UU + + +NCU

Solublitylevel

Short Name

Solubility level

Short Name

Results 42

These protein constructs were transformed and expressed using BL21 AI (Table 2.1)

cells. While all these constructs were expressed at high level in E. coli cells, the

solubility remained a problem for most of them. Only the protein constructs containing

the N, C and U domains (residues 1 to 364) (NCU), were found to be highly soluble

and able to be purified to homogeneity. This purified protein was used to screen for

crystallization conditions. However these initial crystallization trails were

unsuccessful.

Based on the above results, it was assumed that some parts of the MARK2 protein

remained unfolded during prokaryotic expression thus preventing the formation of

crystals.

3.2 Identification of the structurally folded part of MARK2 kinase

3.2.1 Limited proteolysis

The structurally folded part of the MARK2 protein was identified by limited

proteolysis. The highly soluble N-terminal GST tagged MARK2 construct (GST-

MARK2 [1-364]) was subjected to limited proteolysis with proteases including trypsin

and AspN in a ratio of 200 to 1. The reaction mixture was incubated at 37ºC and

samples were taken at different time points (10, 30, 60 and 120 minutes). The reactions

were quenched by adding 1mM PMSF and boiling the samples with SDS loading

buffer (Laemmli, 1970). As a control, a sample without proteases was incubated at 37º

C for 120 minutes to check for the heat stability of the protein. The results were

analyzed by SDS-PAGE (Fig. 3.2).

Results 43

Fig. 3.2: Limited proteolysis of MARK2. GST-MARK2[1-364] protein was subjected to limited proteolysis with trypsin and AspN proteases and samples were taken at different time points (in minutes); M, molecular weight markers; C, control sample to measure the heat stability of the protein. Limited proteolysis of MARK2 revealed a stable fragment of molecular weight >35

kDa which suggested that the stable fragment comprises more than the catalytic

domain.

3.2.2 N-terminal sequencing and mass spectrometry analysis

From the limited proteolysis experiments, a fragment of approximately 37 kDa in

molecular weight was found as the stable folded part of MARK2. To identify the exact

aminoacid sequence of this fragment, N-terminal sequencing analysis and mass

spectrometry analysis were done. The N-terminal sequencing analysis revealed

aminoacid sequence N’- NSAT - C’ as the beginning of the fragment, which

corresponds from the 39th residue of the rnMARK2 full length protein.

Further analysis of the stable fragment by mass spectrometry for molecular weight

estimation, revealed a molecular weight of 37563 Da. By combining these results and

the molecular weight predictions according to the aminoacid sequence, it was

confirmed that GST-MARK2 [1-364] was cleaved by trypsin before asparagine 39 (N39)

at the N-terminus while the C-terminus (K364) of the protein remained intact.

11666.2

45.0

35.0

25.0

18.014.4

M C 10 60 12030 10 60 12030

Trypsin AspN

MARK2 fragment

GST

11666.2

45.0

35.0

25.0

18.014.4

M C 10 60 12030 10 60 12030

Trypsin AspN

MARK2 fragment

GST

Results 44

3.3 Cloning of the stable fragment of MARK2

The identified stable fragment of MARK2 (N39 - K364) was cloned by using the

Gateway technology (section 2.2.9). In short, the stable fragment of rnMARK2 (N39 –

K364) (GeneBank No. CAB06295; Drewes et al., 1997) was cloned into the gateway

expression vector pDEST17, which has an N-terminal 6 x histidine tag for affinity

purification. A TEV protease recognition site (ENLYFQG) (Phan et al., 2002) was

incorporated at the beginning of the MARK2 protein sequence to cleave off the affinity

tag for crystallization purposes. A vector map of the MARK2 expression plasmid is

shown in Fig. 3.3.

Fig. 3.3: Vector map of the MARK2 expression plasmid. MARK2 fragment (N39 – K364) was cloned into the Gateway expression vector pDEST17 by using standard Gateway cloning procedures (Invitrogen). The expression clone contains an N-terminal 6 x histidine tag for the affinity purification, TEV protease recognition site to cleave off the affinity tag and ampicillin resistance gene for selection. pBR322 is the origin of replication. This map was produced by using Vector NTI software V9.0 (Invitrogen).

MARK2N39 to K364

AmpR

bla

6x HisTEV Protease recognition site

ATG

pRB

322

MARK2N39 to K364

MARK2N39 to K364

AmpR

bla

6x HisTEV Protease recognition site

ATG

pRB

322

MARK2N39 to K364

Results 45

3.4 Expression and purification

BL21 AI cells (Table 2.1) were transformed with the MARK expression plasmid by

using the chemical transformation protocol (2.2.2). Transformed cells were initially

grown at 37°C and induced either overnight at 24°C or for 5 hours at 30°C by adding

arabinose to a final concentration of 0.2% at OD600 ≈ 0.6. The cells were harvested by

centrifugation and lysed by passing two times through a French pressure cell.

Fig. 3.4: Purification of MARK2 protein. The eluted fractions of the Ni-NTA column (A), the Mono S column (B) and the gel filtration column (C) were analyzed with SDS PAGE. M, molecular weight markers. The pure peak fractions from the gel filtration column were pooled, concentrated and used for crystallization trials. The other mutants and the SeMet labelled proteins were purified in the same way as wild type protein.

M Ni-NTA column fractionsM Ni-NTA column fractions

M MonoS column fractionsM MonoS column fractions

M G200 column fractionsM G200 column fractions

Results 46

The expressed proteins were purified in four steps:

1. Ni-NTA affinity chromatography

2. TEV protease cleavage to remove the His6-tag

3. Ion exchange chromatography

4. Gel filtration chromatography

All procedures were carried out at 4ºC to minimize the protein degradation. After every

step, purity was evaluated by SDS-PAGE (Laemmli, 1970).

The MARK2 protein eluted as a monomer from the gel filtration column (Sephadex

G200, HR16/60 Amersham Pharmacia) and the pure peak fractions were pooled and

concentrated to ~20 mg/ml using a Ultrafree-30 Centricon Concentrators (Amicon).

The concentrated protein solution was then aliquoted in 0.2 ml PCR tubes and shock

frozen in liquid nitrogen. The final yield of purified protein was estimated

approximately 8 mg per liter of culture. Fig. 3.5 shows the bar diagram of the purified

and crystallized construct of MARK2 and the sites of mutations.

Fig. 3.5: Bar diagram of the crystallized construct of MARK2. Residue numbers refer to the rat MARK2 (Swiss-Prot entry O08679). The fragment used is identical in rat and human MARK2. CD, common docking domain; UBA, ubiquitin associated domain. This construct includes an N-terminal glycine (G38) left over from TEV protease cleavage of a His6 tag. Mutants: K82R - K82 is a key residue for positioning the ATP. Mutating this lysine (K) to arginine (R) inhibits the ATPase activity of the kinase; T208A/S212A - T208 phosphorylation by MARKK or LKB1 is necessary for MARK activation and either phosphorylation or mutation of S212 inhibits the MARK kinase activity. These two residues were mutated to alanine which mimics the un-phosphorylated state (Timm et al., 2003; Lizcano et al., 2004).

304

kinase domain UBA

T208

39 362

YK364G

53

K82CD linker

315

304304

kinase domain UBA

T208

39 362

YK364G

39 362362

YK364G

5353

K82CD linker

315

CD linker

315315

Results 47

3.5 Kinase activities of the purified proteins

In order to show that the purified wild type kinase is active and to elucidate the effect

of mutations, the kinase activities of the proteins were measured. For all the purified

proteins, kinase activities were assayed as described in the methods (2.3.8) by using

the TR1 peptide as a substrate. This substrate peptide was derived from the first repeat

of the KXGS motifs of human tau protein and contains the crucial phosphorylation site

S262.

The kinase assay shows (Fig. 3.6) that the wild type protein has a basal activity, even

without the phosphorylation of T208, but the other mutants T208A/S212A and K82R

show very low activity or no activity, as expected.

Fig. 3.6: Kinase activities of the MARK2 constructs. Relative activities were assayed using a substrate peptide from the first repeat of tau containing S262 in the KXGS motif (TR1 peptide NVKSKIGSTENLK); cpm- counts per minute; data show averages of 4 experiments (error bars = standard error of the mean).

Results 48

3.6 Preparation of selenomethionine labelled protein

In order to determine the phases, the selenomethionine (SeMet) labelled protein of the

MARK2 double mutant (T208A/S212A) was expressed and purified. The expression

was carried out in the methionine auxotrophic E. coli strain B834 (Table 2.1) by using

modified M9 medium supplemented with all amino acids with the exception of

methionine that was replaced by SeMet (40 mg per liter of medium; Acros Organics).

The purification procedure was essentially the same as described for the unlabelled

proteins. The incorporation of SeMet was estimated by mass spectrometry by checking

the increase of the molecular weight of the protein. The mass spectrometry results

suggested that, the labeling efficiency was ~100%, i.e. all the 10 methionine residues

per molecule were labelled with SeMet (Fig. 3.7).

Fig. 3.7: Mass spectrometry analysis of (a) wild type and (b) SeMet labelled double mutant (T208A/S212A) proteins of MARK2. The molecular weight increased in the SeMet labelled protein corresponding to the labeling efficiency (nearly 100%, i.e. 10 SeMet residues per molecule); m/z - mass by charge ratio.

3.7 Crystallization

Initial crystallization trials were performed by the sitting drop vapour diffusion

technique in CrystalQuick 96 well sitting drop plates using the commercially available

screening kits (Hampton Research, Jena Biosciences). Crystal Screen Index, Crystal

Screen I, Crystal Screen II, Crystal Screen Lite, PEG/ION Screen, Grid Screens from

Hampton Research and High throughput Crystal Screen I & II from Jena Biosciences

30000 40000 50000 60000 70000 80000

30000 40000 50000 60000 70000 80000

Chip195-G

Chip195-H

0

2.5

5 37604.6+H

75224.1+

0

2.5

5

76170.9+2H 76170.

MARK2 wild type

MARK2(SeMet)

m/z

37604.4

38085.4

a

bInte

nsity

30000 40000 50000 60000 70000 80000

30000 40000 50000 60000 70000 80000

Chip195-G

Chip195-H

0

2.5

5 37604.6+H

75224.1+

0

2.5

5

76170.9+2H 76170.

MARK2 wild type

MARK2(SeMet)

m/z

37604.4

38085.4

a

bInte

nsity

Results 49

were used. 1 µl of protein (10 mg /ml) was mixed with 1 µl of reservoir solution and

this mixture was equilibrated against 100 µl of reservoir solution. The screening

yielded small crystals in several conditions. All these conditions were further

optimized by the hanging drop vapour diffusion method using 24 well VDX Plates.

Protein concentration, precipitant concentration, pH, salt concentration, temperature

and detergents to avoid nonspecific nucleation were taken into account for

optimization.

After optimization, suitable crystals could be grown by mixing 2 µl of protein

(~20 mg/ml) with 2 µl of a reservoir solution containing 7-10 % PEG 3350, 0.1 M Bis-

Tris pH 6.5, 0.2 M ammonium sulfate or 7-10 % PEGMME 5000, 0.1 M Bis-Tris pH

6.5, 7.5 % tacsimate at room temperature.

Fig. 3.8: Images of crystals of MARK2 wild type and mutant proteins. (a, b – wild type protein, c – K82R mutant, d – T208A/S212A mutant). Crystallization conditions for a: 7-10 % PEGMME 5000, 0.1 M Bis-Tris pH 6.5, 7.5 % tacsimate; Crystallization conditions for b, c, d: 7-10 % PEG 3350, 0.1 M Bis-Tris pH 6.5, 0.2 M ammonium sulfate.

a b

c d

a b

c d

Results 50

Pictures of Hexagonal, rod shaped MARK2 crystals are shown in Fig. 3.8. Crystals for

the mutants were also obtained by using the wild type crystallization conditions.

Normally the wild type and K82R mutant protein formed crystals in two to three weeks

in the presence of DTT and removal of DTT from these setups reduced the time needed

for the crystal growth. The double mutant (T208A/S212A) protein formed crystals in

two to six days in the absence of DTT. SeMet labelled protein crystals were grown

under similar conditions.

3.8 Data collection and phasing

X-ray diffraction data were collected with synchrotron radiation at the beamline of the

X13 Consortium for Protein Crystallography at HASYLAB (DESY, Hamburg). Before

data collection, crystals were soaked in cryoprotectant solution (15 % PEG 3350, 0.1

M Bis-Tris, 0.2 M ammonium sulfate and 15% glycerol or 10 % PEGMME 5000, 0.1

M Bis-Tris, 7.5 % tacsimate and 15% glycerol) for a few minutes and rapidly cooled to

100 K in a stream of cold nitrogen (Oxford Cryosystems).

Fig. 3.9: X-ray diffraction pattern from a wild type MARK2 crystal. X-ray crystal diffraction measured at the beamline of the X13 consortium for protein crystallography (HASYLAB, DESY, Hamburg). The oscillation range was 1°, wavelength 0.802 Å and diffraction images were recorded with a MAR-CCD detector with a distance of 250 mm between the crystal and detector. The edge of the image corresponds to ~2.5 Å.

Results 51

Diffraction images were recorded with a MAR CCD detector and the distance between

the detector and crystal was 250mm. Datasets were measured from several crystals of

the wild type construct and the double mutant and from one crystal of the K82R

mutant.

Data reduction and statistical analysis were performed with programs DENZO,

XDISPLAYF, and SCALEPACK of the HKL data processing system V1.97.2

(Otwinowski and Minor, 1997). Programs of the CCP4 package (Collaborative

Computer Project Number 4, 1994) and its graphical user interface CCP4i (Potterton et

al., 2003) were used for further analysis, phasing, and model refinement. The best

dataset for each crystal type was used for structure determination and refinement

(Table 3.1).

All crystals had a similar shape (hexagonal rods) and belonged to space group P61.

However, with regard to the cell dimensions, the crystals fall into two distinct classes

differing by the length of the c-axis (hexagonal axis). The double mutant consistently

crystallized in the form with the long c-axis (c = 106.0 Å, sd = 0.37 Å, based on 15

crystals including crystals grown from SeMet protein and crystals soaked with heavy

atom salts), while the wild type crystallized in both forms, with a preference for the

short c-axis (c = 99.7 Å, sd = 0.23 Å, 8 out of 10 crystals). The crystal of the K82R

mutant had also a short c-axis (c = 99.66 Å).

Results 52

Data collection and refinement statistics Construct wild type K82R mutant T208A, S212A T208A, S212A double mutant double mutant Se-methionine Space group P61 P61 P61 P61 Cell constants [Å]: a = b 120.3 121.2 118.8 119.3

c 99.5 99.7 105.4 105.7 Resolution range [Å] 46.14 - 2.90 51.78 - 3.10 46.30 - 2.50 73.88 - 2.80 Data collection High resolution shell [Å] 2.95 - 2.90 3.15 - 3.10 2.54 - 2.50 2.85 - 2.80 No of observations 122003 88139 214839 132608 No of unique reflections 18222 14814 29259 21073 Completeness [%] 99.9 (100) 98.2 (99.3) 99.9 (100) 99.9 (100) Redundancy 6.6 (6.6) 5.7 (4.0) 7.1 (7.1) 6.2 (4.8) Rsym 0.067 (0.626) 0.109 (0.639) 0.099 (1.066) 0.102 (0.576) <I>/<sigI> 27.8 ( 3.3) 14.0 ( 2.0) 17.9 ( 2.0) 17.1 ( 2.8) Refinement High resolution shell [Å] 2.976 - 2.90 3.185 - 3.10 2.57 - 2.50 2.875 - 2.80 No of reflections working set 17281 14072 27745 19981 test set 942 740 1514 1092 R 0.195 (0.335) 0.200 (0.287) 0.198 (0.295) 0.203 (0.313) Rfree 0.270 (0.429) 0.251 (0.385) 0.268 (0.359) 0.262 (0.353) No of residues (total 654) 589 589 604 604 No of atoms (total) 4664 4667 5071 4885 No of water molecules - - 158 - B-factors individ., isotrop. overall, TLS individ., isotrop. individ., isotrop. NCS restraints 3 groups 3 groups - 3 groups MC tight, SC loose MC, SC tight MC tight, SC loose

Table 3.1: Summary of data collection and structure refinement

TLS, TLS refinement assuming 6 rigid groups (minor and larger lobes, and UBA domains for two molecules); MC, main chain atoms; SC, side chain atoms. Values in parentheses are for the high resolution shells.

Results 53

3.8.1 Molecular replacement

The phases of the structure factors of a protein crystal can be determined by the

method of molecular replacement (MR) if the structure of a homologous protein is

available, which can be used as a search model. In this method, the homologous probe

structure is fitted into the unit cell of the unknown structure and the phases resulting

from this are used as an initial guess for the phases of the unknown structure. Here, the

crystal structure of other protein kinases like PKA, PKB, GSK3β, PHK and CHK1

were used as initial models, because of their sequence similarity within the kinase

domain.

As initial attempts to determine the phases by molecular replacement failed, heavy

atom derivatives were searched for in order to solve the phase problem by MIR or

MAD. Several datasets of crystals soaked with heavy atom salts were collected and

analyzed by inspection of difference Patterson maps, but without clear results. In the

same vein, a selenomethionine derivative of the double mutant was prepared and

eventually crystallized. In the meantime, however, a potential MR solution was found

with the program PHASER Version 1.2 (Storoni et al., 2004) finding two molecules in

the asymmetric unit (corresponding to a Matthews parameter of 2.9 Å3/Da) with the

structure of the kinase domain of human checkpoint kinase Chk1 (PDB-ID: 1IA8;

(Chen et al., 2000)) as search model (sequence identity 36%). Both of the

enantiomorphic space groups P61 and P65 were allowed for MR solutions. The best

solution was found in space group P61 with a log-likelihood gain of 10 for the first

molecule and 33 for the second (Z-score = 12). Confidence in the correctness of this

solution was strengthened by the fact that calculations repeated with datasets from wild

type and mutant crystals and with modified search structures led to equivalent

solutions.

3.8.2 Heavy atom derivatives of MARK2 crystals

Two of the crystals of the double mutant that had been soaked for several hours in cryo

buffers supplemented with 1mM of potassium tetra-chloro-aurate and ytterbium

nitrate, respectively, were found to have heavy atoms incorporated, each at two sites

(one site per molecule), the sites of the gold atoms being distinct from the ytterbium

Results 54

sites. Using only the positions of the heavy atom sites as determined with the help of

MR phases, heavy atom parameters were refined and experimental phases were

calculated with MLPHARE (Otwinowski, 1991), including the anomalous signals of

both derivatives. Occupancies of the heavy atoms were found to range between 0.27

and 0.40. The overall figure of merit of the phases calculated with MLPHARE was

0.26 (using reflections up to 3 Å resolution). Experimental phases from MLPHARE

were improved with DM (Cowtan, 1994) by solvent flattening, histogram mapping,

and NCS (non-crystallographic symmetry) averaging using automatically generated

NCS masks and NCS operator refining, starting with the NCS operator derived from

the MR solution. The overall mean figure of merit after phase extension to 2.7 Å with

DM was 0.79.

3.8.3 Model building and refinement

Manual model building was performed with the graphics software TURBO-FRODO

(Roussel and Cambillau, 1989) and O (Jones et al., 1991). The electron density map

calculated with phases obtained with DM allowed to recognize a couple of secondary

structure elements, mostly helices. Some of them could easily be identified by

comparison with the Chk1 catalytic domain as they were located at the positions to be

expected according to the MR solution found by PHASER, others were displaced from

the MR solution by 1-2 Å. In addition to that, a small globular domain consisting of

three short helices was visible, that turned out to represent the UBA domain. A model

was built and completed to about 80 % by several rounds of manual model building,

local real space refinement with RSRef2000 (Korostelev et al., 2002), and phase

combination of the partial model with experimental phases by using SIGMAA (Read,

1986), followed by density modification with DM. The two molecules were treated

independently. When the completeness of the model was about 80%, cycles of

automatic refinement and manual rebuilding and completion were started, first using

CNS 1.1 (Brunger et al., 1998) for simulated annealing, switching to REFMAC5

(Murshudov et al., 1997) at later stages of refinement. Cross validation was enabled

throughout model building and refinement by separating 5 percent of the reflections for

calculation of free R-factors.

Results 55

3.8.3 Crystals of selenomethionine labelled MARK2

Before completion of the refinement, crystals of the selenomethionine derivative of the

double mutant became available. Though not required for phasing any more at this

time, X-ray diffraction data were collected for these crystals to check the model by

determining the positions of the selenium atoms. The dataset proved to be of higher

quality than any of the datasets obtained so far, extending to a resolution of 2.5 Å

(mean I/σI > 2). Thus, the structure of the selenomethionine derivative was the first of

four MARK2 structures that was refined to the end, using the partially refined model of

the double mutant as start model. The final model contains 604 of 654 residues in two

independent molecules, 20 Se atoms in SeMet residues, and 158 water molecules.

Eight residues have been modeled with dual conformation, amongst them four of the

selenomethionine side chains (MSE 104A/B and MSE 187A/B). In each of the

molecules, ten or eleven residues at the N-termini and 13 residues close to the catalytic

cleft have not been modeled due to weak or un-interpretable electron density, three

residues have missing side chains (Glu260A, Arg264A, Arg268A).

The final R-factor was 0.198 (Rfree = 0.268) using all reflections up to 2.5 Å.

Structures of the double mutant with native methionine, as well as those of the wild

type and the K82R mutant were modeled after the SeMet derivative. To reduce model

bias, "composite omit maps" were calculated with CNS prior to refinement. In all

cases, the same subset of reflections was used for cross validation. To avoid over

fitting, NCS restraints were applied for all structures except the SeMet derivative. All

structures were refined with restrained individual isotropic B-factors, except for the

K82R mutant which has been refined with overall B-factor and TLS parameters for six

groups (N-terminal and C-terminal lobes of the catalytic domain and UBA domains,

three groups per molecule). The refinement statistics and further details are

summarized in Table 3.1. The quality of the models was checked with the programs

PROCHECK (Laskowski et al., 1993) and WHAT_CHECK (Hooft et al., 1996) using

DSSP secondary structure assignments (Kabsch and Sander, 1983). Inter atomic

contacts and solvent accessible surface areas were calculated with the software

modules CONTACT and AREAIMOL of the CCP4 package.

Results 56

3.9 Overall structure of the MARK2 catalytic and UBA domains

The four crystal structures of the MARK2 constructs are very similar to each other

with respect to both the crystal packing and the folding of the individual molecules.

The general structure of the kinase and UBA domain will be described using the SeMet

double mutant as the reference structure, if not stated otherwise, since this is the

structure of highest resolution (2.5 Å) among the four that have been determined so far.

Significant differences between the structures will be mentioned when necessary.

3.10 Structure of the catalytic domain

The crystal structures contain two molecules per asymmetric unit. In the case of the

SeMet double mutant they have been refined independently. Fig. 3.10 shows Cα-traces

of both molecules in superposition.

Fig. 3.10: Folding of the catalytic and UBA domain of MARK2. Stereo view of an overlay of Cα-traces of molecules A and B in the asymmetric unit, based on the SeMet double mutant T280A/S212A (A blue, B with different colors depending on the distance to A, distance increasing from blue to red). The superposition was calculated using residues 135-309 (C-lobe). Not shown are residues 38-47, 193-205 (activation loop, indicated by dashed line), and 363-364. Besides the initial residues (48-51), the largest shift in Cα positions occurs in the UBA domain (maximum 1.87 Å at residue M335). This Figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

50 50

60 6080 80

70 70

90 90

180 180

100 100110 110

120 120

140 140

130 130

150 150160 160

170 170

220 220

190 190

210 210

230 230

320 320

240 240250 250

260 260

270 270

280 280290 290300 300

310 310

330 330340 340

350 350

360 360

50 50

60 6080 80

70 70

90 90

180 180

100 100110 110

120 120

140 140

130 130

150 150160 160

170 170

220 220

190 190

210 210

230 230

320 320

240 240250 250

260 260

270 270

280 280290 290300 300

310 310

330 330340 340

350 350

360 360

Results 57

The MARK2 kinase domain possesses a bi-lobe structure like all other protein kinases.

The cleft between the two lobes is the presumed site of nucleotide binding and

catalytic activity. The smaller, N-terminal lobe (residues ~ 53 to 130) consists of five

β-strands and a long α-helix (helix C in the nomenclature of PKA (Knighton et al.,

1991a)), whereas the large, C-terminal lobe (residues ~135 to 304) is composed mainly

of helices. A structural sequence alignment with other kinases is presented in Fig. 3.11,

including the notation for the structural elements used in this thesis.

P-loop hinge40 50 60 64 70 80 82 90 100 110 116 120 130 135

RnMARK2 [ 1Y8G ] NS A T SADEQPH I GNYR L L K T I GKGNFAK V K L ARH I L TGK E V AVK I I DK TQL N - - - - - - - S S S LQK L F REVR I MK V L N - HPN I V K L FE V I E T - - - - - - E K T L Y L VME Y A SGGE122 130 140 144 150 160 162 170 181 191 197 201 211 216

HsAurora-A [ 1OL5 ] E SK KRQWA L ED F E I GRP L GKGKFGNV Y L ARE KQS K F I L A LKV L F K AQL E K AG - - - - - - V EHQL RREV E I QSH L R - HPN I L R LYGY F HD - - - - - - A T RV Y L I L E Y A P LGT107 110 120 130 140 144 150 160 162 170 181 191 197 201 211 216

HsAurora-A [ 1MUO ] ENNP E E E L AS KQKNE E SK KRQWA L ED F E I GRP L GKGKFGNV Y L ARE KQS K F I L A LKV L F K AQL E K AG - - - - - - V EHQL RREV E I QSH L R - HPN I L R LYGY F HD - - - - - - A T RV Y L I L E Y A P LGT1 6 16 20 26 36 38 46 55 65 71 75 85 90

HsCHK1 [ 1IA8 ] MA V P F VEDWD L VQT L GEGAYGE VQL A VNRV T E E A V AVK I VDMKRA VD - - - - - - - - CP EN I K KE I C I NKML N - HENV V K FYGHRRE - - - - - - GN I QY L F L E YCSGGE1 10 16 26 30 36 46 48 56 73 84 90 94 104 109

OcPHKg [ 1QL6 ] T RDA A L PGSHS T HGF YENY E P K E I L GRGV S SV VRRC I HKP T CK E Y AVK I I DV TGGGS F S A E EVQE L RE A T L KEVD I L RK V SGHPN I I QL KD T Y E T - - - - - - N T F F F L V F D LMK KGE1 10 20 30 34 40 50 52 59 69 79 85 89 100 104 108

RnERK2 [ 2ERK ] MA A A AA AGP EMVRGQV F DVGPRY T N L S Y I GEGAYGMVCS A YDN L NK VRV A I KK I S - P F EH - - - - - - - QT YCQR T L RE I K I L L R F R - HEN I I G I ND I I R - A P T I EQMKDV Y I VQD LM - E T D1 11 21 31 35 41 51 53 61 71 81 87 91 103 107 111

MmMAPKp38 [ 1LEW ] MSQERP T F YRQE L NK T I WE V PERYQN L S PVGSGAYGS VCA A F D T K TGHRV AVKK L SRP FQS - - - - - - - I I HA KR T YRE L R L L KHMK - HENV I GL L DV F T P ARS L E E F NDV Y L V T H LM - GAD1 10 20 30 40 50 54 60 70 72 80 91 101 107 111 117 121 126

MmPKA [ 1ATP ] GNA A A A K KGSEQE S V K E F L A K A K ED F L K KWE T PSQNT AQL DQF DR I K T L GTGSFGRVML V KHKE SGNHY AMK I L DKQKV V K L K - - - - - - Q I EH T L NEKR I L QA VN - F P F L V K L E F S F KD - - - - - - NSN L YMVME Y V AGGE

helix A beta1 beta2 beta3 helix B helix C beta4 beta5

activation segmentcatalytic loop N-anchor activation loop C-anchor

Mg-bind loop P+1 loop140 150 160 170 174 180 190 193 200 208 210 217 220 230 240 250 260

RnMARK2 [ 1Y8G ] V F DY L V AHGRMK E K E ARA K F RQ I V SA VQYCHQK F I VHRDL K A ENL L L DADMN I K I ADFGF SNE F T FGN - - - K L DT - F CGS P P Y AAPE L FQGK K - - - - - YDGP E VDVWS L GV I L Y T L V SGS L P F DG - QN L K E L RERV L RGK221 231 241 251 255 261 271 274 281 288 290 297 300 309 319 329 339

HsAurora-A [ 1OL5 ] V YRE L QK L S K F DEQR T A T Y I T E L ANA L S YCHS KRV I HRD I K P ENL L LGS AGE L K I ADFGWS VHA P S S - - - - RR TT - L CGT L DY LPPEM I EGRM - - - - - - HDE K VD L WS L GV L CY E F L VGK P P F E A - NT YQE T Y KR I SRVE221 231 241 251 255 261 271 274 281 288 290 297 300 309 319 329 339

HsAurora-A [ 1MUO ] V YRE L QK L S K F DEQR T A T Y I T E L ANA L S YCHS KRV I HRD I K P ENL L LGS AGE L K I ADFGWS VHA P S S - - - - RR TT - L CGT L DY LPPEM I EGRM - - - - - - HDE K VD L WS L GV L CY E F L VGK P P F E A - NT YQE T Y KR I SRVE95 105 115 125 129 135 145 148 155 166 168 175 178 188 198 208 219

HsCHK1 [ 1IA8 ] L F DR I E PD I GMP E PDAQR F F HQLMAGV V Y L HG I G I T HRD I K P ENL L L DERDN L K I SDFGL A T V F RYNNRER L L NK - MCGT L P Y VAPE L L KRRE - - - - - F HAE P VDVWSCG I V L T AML AGE L PWDQP SDSCQE Y SDWK E KK114 124 134 144 148 154 164 167 174 182 184 191 194 209 219 229 239

OcPHKg [ 1QL6 ] L F DY L T E K V T L S E K E T RK I MRA L L EV I CA L HK L N I VHRDL K P EN I L L DDDMN I K L TDFGF SCQL DPGE - - - K L RE - VCGT P S Y LAPE I I ECSMNDNHPGYGK E VDMWS TGV I MY T L L AGS P P FWH - RKQMLML RM I MSGN113 122 132 142 146 152 162 165 172 183 186 193 196 206 216 226 236

RnERK2 [ 2ERK ] L YK L L K TQ - H L SNDH I CY F L YQ I L RG L K Y I HS ANV L HRDL K P SNL L L N T T CD L K I CDFGL ARVADPDHDH TGF L TE Y VA T RWYRAPE I ML NS K - - - - - GY T K S I D I WSVGC I L A EML SNRP I F PG - KHY L DQL NH I LG I L116 125 135 145 149 155 161 165 168 175 180 183 190 193 203 213 223 233

MmMAPKp38 [ 1LEW ] L NN I V KCQ - K L T DDHVQF L I YQ I L RG L K Y I HS AD I I HRDL K P SNL A VNEDCE L K I LDFGL ARH T DDE - - - - - - MTGY VA T RWYRAPE I ML NWM - - - - - HYNQT VD I WSVGC I MA E L L TGRT L F PG - T DH I DQL K L I L R L V131 141 151 161 165 171 181 184 191 197 199 206 209 218 228 238 248

MmPKA [ 1ATP ] MF SH L RR I GR F S E PHAR F Y A AQ I V L T F E Y L HS L D L I YRDL K P ENL L I DQQGY I QV TDFGF A KRV KG - - - - - R TWT - L CGT P E Y LAPE I I L S KG - - - - - - YNK A VDWWA L GV L I Y EMA AGY P P F F A - DQP I Q I Y E K I V SGK

helix D helix E beta6 beta7 beta8 beta9 helix EF helix F helix G

CD domain UBA domain270 280 290 300 310 320 330 340 350 360 364

RnMARK2 [ 1Y8G ] YR I P F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - YMS T DCEN L L K K F L I L NP S KRG - - - - - T L EQ I MKDRWMNVGHEDDE L K P Y V E P L PDY KDPRR T E LMV SMGY T RE E I QDS L VGQRYNE VMA T Y L L L GY K349 359 369 379 389 399 403

HsAurora-A [ 1OL5 ] F T F PD - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F V T EGARD L I SR L L KHNP SQRP - - - - - ML REV L EHPW I T ANS S K P SNCQNK E S AS KQS349 359 369 379 389 399 403

HsAurora-A [ 1MUO ] F T F PD - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F V T EGARD L I SR L L KHNP SQRP - - - - - ML REV L EHPW I T ANS S K P SNCQNK E S AS KQS229 241 251 261 271 281 289

HsCHK1 [ 1IA8 ] T Y L NP - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - WK K I DS A P L A L L HK I L V ENP S AR I - - - - - T I PD I K KDRWYNK P L K KGA KRPRV T SGGV S E S P SG249 263 273 283 293 298

OcPHKg [ 1QL6 ] YQFGS P E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - WDDY SDT V KD L V SRF L V VQPQKRY - - - - - T A E EA L AHP F FQQY V V E E VRH F243 253 263 273 283 287 297 307 317 327 337 347 358

RnERK2 [ 2ERK ] - - - GS P SQED L NC I I N L K ARNY L L S L PHKNK V PWNR L F PNADS K A L D L L DKML T F NPHKR I - - - - - E V EQA L AHP Y L EQY YDP SDEP I A E A P F K F DME L DD L P K E K L KE L I F E E T AR FQPGYRS240 250 260 270 280 284 294 304 314 323 333 343 353 360

MmMAPKp38 [ 1LEW ] - - - GT PGA E L L K K I S S ES ARNY I QS L AQMP KMN F ANV F I GANP L A VD L L E KML V L DSDKR I - - - - - T A AQA L AHA Y F AQYHDPDDEP V AD - P YDQS F E SRD L L I DEWKS L T YDE V I S F V P P P L DQE EME S258 268 278 288 293 303 313 323 333 343 350

MmPKA [ 1ATP ] VR F P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - H F S SD L KD L L RN L LQVD L T KRFGN L KNGVND I KNHKWF A T T DW I A I YQRK V E A P F I P K F KGPGD T SN F DDY E E E E I RV S I NE KCGK E F T E F

helix H helix I helix J Fig. 3.11: Structural sequence comparison of MARK2 and related kinases. Secondary structure is color coded (orange = α-helices, pink = 310 helices, yellow = β strands, numbered in the usual kinase convention. Special elements are boxed (P-loop in active site, hinge between lobes, catalytic loop containing RD motif, activation segment with N-anchor, P+1 loop, and C-anchor, CD domain, UBA domain). Blue residues are not visible due to disorder.

The two lobes are linked by a flexible stretch of amino acids around glycines G134 and

G135. Furthermore, there are hydrogen bonds between the two lobes, connecting

residues in the loop between helix C and strand β4 (small lobe) with residues in helix E

and the loop between β7 and β8 (large lobe). Both, the covalent link and the H-bond

interactions are restricted to a narrow region at the back of the catalytic cleft and could

serve as a hinge that allows the small lobe to librate, thereby opening and closing the

catalytic cleft. Fig. 3.12 shows the electron density map around the catalytic cleft of the

SeMet double mutant, superimposed with the final model.

Results 58

Fig. 3.12: Electron density map around the catalytic cleft of the SeMet double mutant (stereo view). Wire-frame representation of weighted 2Fo-Fc maps calculated with REFMAC5 (Murshudov et al., 1997). Electron density contour levels are 1 σ (cyan) and 4 σ (green). Superimposed to the electron density is the final model. View into the catalytic cleft of molecule A centered on A192 which precedes the DFG motif of the activation segment. The DFG motif and the activation segment up to K205 are not part of the model. Electron density near A192 is not accounted for in the final model because this region could not be built unambiguously. The electron density for the Se atom of M104 is delocalized with peaks of ~3 σ and ~5 σ at two different sites. The Se atom of M129 is well localized with a peak electron density of about 10σ. The figure was prepared with graphics software O (Jones et al., 1991).

3.10.1 Conformation of the activation loop

While most of the activation loop is disordered in the wild type and the K82R

structures (D193-C210), the T208A/S212A mutant reveals five more residues at the

end of the activation loop (L206-C210). This includes the P+1 loop (F209-A217)

which is thought to recognize the substrate by specific interaction with the residue

following the phosphorylation site. The end of the activation segment (P213-E219

including the APE motif) is well defined in all MARK2 structures. The conformation

of the structured parts of the activation segment indicates that it folds away from the C

helix, in the opposite direction to most active kinases (Nolen et al., 2004), and occupies

the area below the P-loop (G60-A65), similar to Aurora-A kinase in its inactive state

(Fig. 4.1, and see Discussion).

M129 M129

A192

M104 M104

A192

H173 H173N180 N180

M129 M129

A192

M104 M104

A192

H173 H173N180 N180

Results 59

3.10.2 Intermolecular disulfide bridge

In the double mutant structures, the C-terminal anchor of the activation loop is

stabilized by an intermolecular disulfide bridge between cysteines C210 of two

adjacent molecules (Fig. 3.13). Cysteine C210 is located in the midst of T208A and

S212A, the sites that distinguish the double mutant from the wild type. Obviously, the

disulfide bridge stabilizes of the C-terminal portion of the activation loop in the double

mutant. Formation of the disulfide bridge is probably a crystallisation artifact.

Fig. 3.13: Electron density map around the disulfide bridge of the SeMet double mutant (stereo view). Wire-frame representation of weighted 2Fo-Fc maps calculated with REFMAC5 (Murshudov et al., 1997). Electron density contour level is 1σ. View on the C-terminal end of the activation segment. Residues L206-S212A were omitted before calculation of the map. The view is perpendicular to the two-fold NCS axis relating molecules A and B. Close to the center are cysteines C210 of both molecules, which form a disulfide bridge in the structures of the MARK2 double mutant. The figure was prepared with graphics software O (Jones et al., 1991). The two molecules covalently linked by a disulfide bridge are related by non-

crystallographic symmetry (NCS) with the center of the S-S bond located at a proper,

two-fold rotation axis. It appears that the S-S bridge is essential for crystallization, as

crystals do not form in the presence of DTT. This holds for all variants of the protein

although the disulfide bridge is visible only in the T208A/S212A structure.

L206 L206

F138(B)

C210 C210

P213 P213

F138(B)

A208 A208

L206 L206

F138(B)

C210 C210

P213 P213

F138(B)

A208 A208

Results 60

3.10.3 Dimerization

The MARK2 crystals contain two molecules per asymmetric unit (A and B) which

interact via multiple contacts and form a dimer with a proper two-fold non-

crystallographic symmetry (Fig. 3.14). The catalytic domains in a dimer face each

other with their active sites.

Fig. 3.14: Intermolecular contacts in MARK2 dimers (stereo view). Ribbon diagrams of the wild type dimer (a, total view) and the double mutant dimer (b, close-up of the central part) viewed along the non-crystallographic two-fold symmetry axis (molecule A blue, B purple). Residues involved in intermolecular contacts (at least one atom closer than 4 Å to an atom of the other molecule) are in stick model representation. In panel a, contact residues are color-coded after their distance from cyan (4 Å) to red (~2 Å). The shortest contact (2.07 Å distance) is between cysteines C210 in the double mutant (panel b) which form an interchain disulfide bridge. No disulfide bridge is observed in the wild type structure due to disorder. Most of the

a

b

A A

B B

helix G helix G

P-loop P-loop

helix C helix C

UBAdomain

UBAdomain

R261 R261

S92 S92

K96 K96

D175 D175Y226 Y226E219 E219

D207 D207

P214 P214

C210 C210

a

b

A A

B B

helix G helix G

P-loop P-loop

helix C helix C

UBAdomain

UBAdomain

R261 R261

S92 S92

K96 K96

D175 D175Y226 Y226E219 E219

D207 D207

P214 P214

C210 C210

Results 61

contact residues are in two zones: C-terminal anchor of the activation loop and the following loop preceding helix F (zone 1, residues 206-227) and helix G and part of its N-terminal loop (zone 2, residues 251-261). In the double mutant, zone 1 comprises 15 contact residues in the range from D207 to D227, zone 2 all but one residue in the range D251-R261. For the wild type the corresponding numbers are lower (9 res. in zone 1, 5 res. in zone 2), due to disordered residues L206 to C210. Residues S92, S93, and K96 at the N-terminus of helix C form another cluster of contact residues (zone 3). Helix G and the preceding loop (zone 2) in one molecule inserts into the space between zones 1 and 3 of the other molecule, making extensive contacts with both of these zones and approaching the RD motif (R174, D175) in the catalytic loop. This Figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

The most variable and disordered portion of the activation segment is close to center of

the dimer, encircled by the four lobes of the catalytic domains. Interactions between

the monomers are concentrated in three zones (Fig. 3.14). Zone 1 in the C-lobe and

zone 3 in the N-lobe of one molecule form a wide-open entrance to the catalytic cleft.

Helix G of the other molecule (zone 2) inserts into this space, making contacts to both

rims. In the T208A/S212A mutant, 30 residues of each molecule are involved in intra-

dimer contacts, while the wildtype and K82R mutant show only 18 contact residues,

and the S-S bond at C210 is not visible. Thus, the dimer-forming interaction in these

constructs appear to be weakened compared to the double mutant.

3.11 Structure of the UBA domain

The UBA domain (Y323-K362) is a small, globular domain that consists of three short

helices (α1-α3). Helices α1 and α3 are roughly antiparallel (folding reminiscent of a

"U", Fig. 3.16 & 3.17). This conformation is unexpected since the helices of other

UBA domains solved so far (Fig. 3.15, structural sequence alignment) alternate so that

α1 and α3 are almost parallel to each other (as in an "N"). Fig. 3.17 compares stereo

views of the MARK2 UBA domain (yellow) with the UBA domain of HHR23A

(green, PDB-ID: 1IFY; (Mueller and Feigon, 2002)) which is representative for the

other UBA domains listed in Fig. 3.15.

In MARK2, the UBA domain binds to the N-lobe of the catalytic domain close to the

hinge, opposite to the cleft (i.e. at the "back side"). The interaction is predominantly

hydrophobic and mainly due to helix α3. It involves residues Y351, M335, A356,

L359, L360, and L361 of the UBA domain and residues L115, F116 at the beginning

Results 62

of β4 in the catalytic domain (Fig. 3.18). In addition, Y351 in the UBA domain is

hydrogen bonded to the side chain amino group of K114 and to the carbonyl oxygen of

K105 at the end of helix C. Other hydrophobic interactions involve residues at the N-

terminus of the N-lobe (Y53) and at the β2-β3 turn, e.g. L74 interacts with M335 and

Y337 between helices α1 and α2 of the UBA domain (Fig. 3.18) which belong to the

MGF/Y motif characteristic for UBA domains. The solvent accessible surface area of

the isolated UBA domain (residues 325 to 363 only) is 2890 Å2; a fraction of 23%

(660 Å2) of the surface area can be attributed to hydrophobic residues (Val, Ile, Leu,

Met, Phe, Trp, and Cys, if present) (Fig. 3.16). About one third of the hydrophobic

surface (230 Å2) is buried by binding to the catalytic domain. 194 206 230 243

Hs TDRD3 UBA 14,79 [ 1WJI ] G V Y R E L V D E K A L K H I T E M - G F - - S K E A S R Q A L M D N G N N - L E A A L N V L L T S N K Q K P V M G P P156 174 198 204

Hs HHR23A UBA1 14,72 + [ 1IFY ] T L V T G S E Y E T M L T E I M S M - GY - - E R E R V V A A L R A S Y N N - P H R A V E Y L L T G I P G651 683 707 710

At UBP14 UBA2 14,23 [ 1WIV ] L L S H M D D P D I D A P I S H Q T S D I D Q S S V D T L L S F - G F - - A E D V A R K A L K A S G G D - I E K A T DWV F N N P N342 374 398 401

At RSGI RUH-014 UBA 13,75 [ 1VG5 ] S R Q A P I A N A A V L P Q S Q G R V A A S E E Q I Q K L V A M - G F - - D R T Q V E V A L A A A D D D - L T V A V E I L M S Q Q A2 14 39 52

Mm U33K UBA 13,20 [ 1WHC ] M A E L T A L E S L I E M - G F - - P R G R A E K A L A L T G N Q G I E A A M DW L M E H E D D P D V D E P L319 331 355 363

Hs HHR23A UBA2 12,88 + [ 1DVO ] Y I Q V T P Q E K E A I E R L K A L - G F - - P E S L V I Q A Y F A C E K N - E N L A A N F L L S Q N F D D E318 331 355 363

Hs HHR23A (P333E) UBA2 12,88 + [ 1F4I ] Y I Q V T P Q E K E A I E R L K A L - G F - - E E S L V I Q A Y F A C E K N - E N L A A N F L L S Q N F D D E381 403 427 430

Hs UBAP1 UBA1 12,19 [ 1WGN ] A Y S E L QM L S P S E R Q C V E T V V NMG - Y - - S Y E C V L R A M K K K G E N - I E Q I L D Y L F A H G Q328 343 368 373

Sc Dsk2p UBA 12,14 + [ 1WR1 ] T R P P E E R Y E H Q L R Q L N DM - G F - F D F D R N V A A L R R S G G - S V Q G A L D S L L N G D V

469 501 525 538

Mm NUB1 UBA3 12,09 [ 1VEG ] N P H MWW L Q D A D P E N N S R Q A S P S Q E S I N Q L V YM - G F - - D T V V A E A A L R V F G G N - V Q L A A Q T L A H H G G S L P P D L Q F 594 626 651 664

At UBP14 UBA1 11,41 [ 1VEK ] G E E L L P D G V P E E V M E S A Q P V A N E E I V A Q L V S M - G F - - S Q L H C Q K A A I N T S N A G V E E A M NW L L S H M D D P D I D A P I323 336 350 359 364

Rn MARK2 UBA 9,94 [ 1Y8G ] . . . P Y V E P L P D Y K D P R R T E L M V S M - GY - - T R E E I Q D S L V G Q R Y N - - E VMA T Y L L L G Y K387 405 431 436

Hs p62 UBA 9,61 + [ 1Q02 ] P P E A D P R L I E S L S Q M L S M - G F S D E G GW L T R L L Q T K N Y - D I G A A L D T I Q Y S K H

1 29 54

Mm RSGI RUH-013 UBA 7,58 [ 1VDL ] M T V E Q N V L Q Q S A A Q K H Q Q T F L N Q L R E I T G I N - D A Q I L Q Q A L K D S N G N - L E L A V A F L T A K N A K T P P Q E E T

77 92 116

Archaea AENAC * * [ 1TR8 ] M E I P E D D I E L V M N Q T G A - - S R E D A T R A L Q E T G G D - L A E A I M R L S137 154 177

Sc SWA2P * * + [ 1PGY ] A L V D E V K D M E I A R L M S L - G L - - S I E E A T E F Y E N D V T - - Y E R Y L E I L K S K Q K E1 17 42

Rn p47 * * + [ 1V92 ] M A E E R Q D A L R E F V A V T G A - - E E D R A R F F L E S A GWD L Q I A L A S F Y E D G G 561 579 603

Hs TAP * * [ 1OAI ] P T L S P E Q Q E M L Q A F S T Q S GM N - - L EW S Q K C L Q D N NWD - Y T R S A Q A F T H L K A K G E I P E V A F M K

alpha 1 alpha 2 alpha3

Fig. 3.15: Structural sequence alignment of UBA domains. Parts of the sequences determined by NMR or X-ray analysis are highlighted by grey and coloured background (orange for helices α1, α2, and α3, pink for other helices; boundaries determined with Promotif (Hutchinson and Thornton, 1996)). Leading or trailing residues on white background were not part of the constructs. The parts enclosed by the black line correspond to the UBA domain identified by Prosite scans (Release 19.2 (Bucher and Bairoch, 1994)). Residues at the C-terminus of MARK2 are printed in purple to indicate that the structural alignment breaks down for this part of the sequence: starting with R350, the polypeptide chain diverges from the common trace of the other UBA structures resulting in an orientation of the final helix that is reversed compared to normal UBA domains. Residues that have been shown to interact with ubiquitin are marked by blue underlines (for HHR23A, PDB-ID 1DVO, only residues of the primary interaction site are highlighted). In the case of MARK2, residues in contact with the N-lobe of the kinase domain are marked. Columns: source, protein name, domain, Prosite score, PDB-ID. A plus sign following the Prosite score indicates that binding to ubiquitin has been reported. The list is sorted according to the Prosite

Results 63

score, sequences at the end of the list are not recognized as UBA domains (no score), although they are structurally similar.

Fig. 3.16: A potential protein–protein binding interface of UBA domains is built from hydrophobic residues on the surface. (a) Ribbon representation of UBA(1) of the HHR23A (green; PDB-ID: 1IFY; (Mueller and Feigon, 2002)). (b) Surface representation of UBA(1) using the following color coding: red, acidic amino acid residues Glu and Asp; blue, basic amino acid residues Arg and Lys, orange, polar amino acid residues Asn, Gln, His, Ser and Thr; white, hydrophobic residues Ala, Gly, Phe, Ile, Pro, Met, Leu, Tyr and Val. The major accessible residues on the hydrophobic surface, Met173, Gly174, Tyr175, Leu199 and Ile202 are marked. The surface area of the hydrophobic surface patch is about 470 Å2, which corresponds to ~ 17% of the total surface area of about 2830 Å2. (c) Ribbon representation of UBA domain of MARK2 (orange), it is shown in same orientation as in (a). (d) For comparison, the surface of UBA domain of MARK2 is shown in the same orientation as in (b), same color coding. The key residues on the hydrophobic surface, Met335, Gly336, Tyr337, Leu360 and Leu361 are marked. The surface area of the hydrophobic surface is about 660 Å2, which corresponds to ~23% of the total surface area of about 2890 Å2. This Figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

N

C

Y175 G174

M173

L199

I202

a

b

C

N

c

M335

G336Y337

L361L360

d

α1

α2

α3α1

α2

α3

UBAMARK2UBAHHR23A

N

C

Y175 G174

M173

L199

I202

a

b

C

N

c

M335

G336Y337

L361L360M335

G336Y337

L361L360

d

α1

α2

α3α1

α2

α3

UBAMARK2UBAHHR23A

Results 64

Fig. 3.17: (stereo view) Overlay of the MARK2 UBA domain with UBA of HHR23A. The MARK2 UBA domain (yellow) is overlaid with UBA of HHR23A (green; PDB-ID: 1IFY; (Mueller and Feigon, 2002)) after least-squares superposition of 9 residues in helix α1. Residues M335 and Y337 of the MGY motif are shown in stick model representation. Helix α2 of MARK2 is tilted outwards by about 20° compared to HHR23A. This is accompanied by a change in the main chain conformation of the MGY loop that translates into a ~30° inward rotation of the aromatic ring of tyrosine Y337. At the end of helix α2, the peptide chains bend in different directions, in such a way that helix α3 ends up at almost the same position but with reversed orientation. This figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

Most UBA domains contain one or two leucines near the end of α3 (three in the case

of MARK2, L359-L361). The first one (corresponding to L359) is highly conserved. It

is important for the internal cohesion of the UBA domain by fitting into a hydrophobic

pocket formed by residues of α1-α2 and the MGF/Y motif. In MARK2, the conserved

L359 lies on the outside of the reversed helix α3 and makes hydrophobic contacts with

the N-lobe of the catalytic domain. Instead of L359, L361 forms hydrophobic intra-

UBA interactions with the side chains of helix α1 (R328, L331, M332, and M335 of

N

C

Cα1

N

C

Cα1

N

C

C

α1

α2

α3

N

C

C

α1

α2

α3UBAMARK2 UBAHHR23A

N

C

Cα1

N

C

Cα1

N

C

C

α1

α2

α3

N

C

C

α1

α2

α3

N

C

Cα1

N

C

Cα1

N

C

Cα1

N

C

Cα1

N

C

C

α1

α2

α3

N

C

C

α1

α2

α3

N

C

C

α1

α2

α3

N

C

C

α1

α2

α3UBAMARK2 UBAHHR23AUBAMARK2 UBAHHR23A

Results 65

the MGF/Y motif). The pocket normally occupied by the conserved leucine is

narrowed by a ~30° inward tilt of Y337. The remaining space is filled with the side

chain of V354 at the start of α3 which – because of the inversion of the helix – ends up

roughly at the same place as the conserved leucine in normal UBA structures.

Fig. 3.18: (stereo view) Details of the binding interactions between the UBA domain and the N-lobe of the catalytic domain of MARK2. All three leucines at the end of helix α3 are involved in hydrophobic interactions with the N-lobe. The final leucine (L361) is also engaged in hydrophobic interactions with helix α1, and plays an important role for the cohesion of the UBA domain. In normal UBA structures it is the almost invariant leucine (L359 in MARK) that is most important for the internal interactions. This Figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

M335

L359

Y337

F116

L361

L360

α3

β2-β3 turn

M335

L359

Y337

F116

L361

L360

α3

β2-β3 turn

UBA UBA

N-lobe N-lobe

helix Chelix Cβ1 β1

α1 α1

α2 α2

α3 α3

α1 α1

M335

L359

Y337

F116

L361

L360

α3

β2-β3 turn

M335

L359

Y337

F116

L361

L360

α3

β2-β3 turn

UBA UBA

N-lobe N-lobe

helix Chelix Cβ1 β1

α1 α1

α2 α2

α3 α3

α1 α1

Results 66

3.12 UBA linker and common docking domain for kinase activators

The UBA domain is linked to the catalytic domain by ~20 residues (~305 to 322, Fig.

3.10, 3.19). The first half contains a motif similar to the "common docking" motif (CD)

of MAP kinases, characterized by a cluster of negative surface charges (DxxD/E,

(Tanoue et al., 2000). In MARK2 the motif E309DDE312 and surrounding residues folds

into a loop similar to the CD domain of MAP kinases (Fig. 3.19).

Fig. 3.19: Common docking domain and ED site of MAP kinases compared to MARK2. The structures of (a) MARK2 and (b) ERK2 (PDB-ID: 2ERK, (Canagarajah et al., 1997)) are shown in the same orientations after least-squares superposition of 35 residues from helix E to the catalytic loop. The common docking domain (CD, in red) according to Tanoue and Nishida (Tanoue and Nishida, 2003) is C-terminal to the kinase domain and corresponds in MARK to the first half of the tether connecting the kinase domain to the UBA domain (residues ~305-315). The C-terminal extensions following the CD domain (linker and UBA domain in MARK2) are shown in purple. Characteristic for the CD domain is a cluster of negatively charged residues exposed to the surface, located in a bulge at the end of the catalytic domain (stick model representation). This Figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

MARK2

ERK2

CD

ED

CDED

UBA

helix E

helix E

helix Ehelix E

a

b

MARK2

ERK2

CD

ED

CDED

UBA

helix E

helix E

helix Ehelix E

MARK2

ERK2

CD

ED

CDED

UBA

helix E

helix E

helix Ehelix E

a

b

Results 67

Together with the "ED site" (corresponding to residues A185-D186 in MARK2) at the

tip of the β7-β8 turn, the CD domain seems to form a docking groove for upstream and

downstream signalling molecules on the back surface of the catalytic domain opposite

to the active site (Tanoue and Nishida, 2003), although the exact location of the

docking groove is still a matter of debate (Chang et al., 2002). The presence of these

features in MARK2 suggests a similar function, but the putative docking partners are

not known so far.

Fig. 3.20: Electron density map around the linker region of the SeMet double mutant (stereo view). View on the C-terminal part of the peptide stretch that links the UBA domain to the catalytic domain. Residues Y316 to K324 were omitted before calculation of the 2Fo-Fc map. Although there is virtually no contact to the catalytic domain or to another molecule, the electron density is well defined. The figure was prepared with graphics software O (Jones et al., 1991).

The second half of the stretch tethering the UBA and catalytic domain ("linker", ~315-

322) assumes an extended conformation. Remarkably, the linker has little contact to

the lobes of the catalytic domain, in fact L320 and adjacent residues are surrounded by

interstitial water. Accordingly, the B-factors are high in this region (main chain B-

factor about 60), but the electron density was sufficiently well defined (Fig. 3.20) to

trace the linker unambiguously all the way from the end of the catalytic domain up to

the UBA domain. However, the loose attachment suggests the possibility that the

linker and UBA domain could swing away from the catalytic domain and thus alter the

regulatory state of the domains.

N108

Y323 Y323

V317

D322 D322

L320 L320

V317

N108N108

Y323 Y323

V317

D322 D322

L320 L320

V317

N108

Discussion 68

4 DISCUSSION

MARK kinases constitute a subfamily of the AMPK/Snf1 family of kinases within the

CAMK group of Ser/Thr kinases (Manning et al., 2002). Apart from a highly

conserved catalytic domain these kinases are unique in that they contain a UBA

domain adjacent to the catalytic domain and a KA1 domain at the C-terminus (Fig.

1.3). MARK occurs in 4 isoforms and several splicing variants. Homologues of MARK

include the kinase PAR-1 in C. elegans and D. melanogaster and KIN-1 in S.

cerevisiae which are involved in the generation of embryonic polarity and cell

morphology.

Solving the MARK2 structure by molecular replacement (MR) proved difficult and did

not result in a definite solution using structures of kinases like PKA, PKB, GSK3β, and

PHK in various forms as search models. A solution was found with the apo structure of

the kinase domain of the checkpoint kinase CHK1. Like MARK, CHK1 is a member

of the CAMK group of protein kinases (Manning et al., 2002) and it has a similar

substrate specificity: both kinases phosphorylate Cdc25C at S216 and prefer a basic

residue at the P-3 position (Muller et al., 2001; Peng et al., 1998). Although the search

model derived from CHK1 accounts only for part of the MARK2 structure, and a

substantial fraction of the large lobe (~30-60 residues) folds differently in both

structures, CHK1 was superior to other model structures as it has a wide-open cleft,

similar to MARK2. Besides the structural differences between the search model and

the target, another reason for the difficulties with MR may be due to a special feature

of the crystal packing. The two molecules of a dimer are related by proper two-fold

NCS, and a rotation of the dyad axis by ~7° would transform the space group from P61

to P6122.

By structural comparison of MARK2 with other kinases using the program CE

(Shindyalov and Bourne, 1998)), Aurora-A and Aurora-B were consistently found at or

close to the top of the ranking, even above CHK1, which is more closely related to

MARK by sequence and which has been used for molecular replacement. The kinase

domain of Aurora-A has been solved in an inactive form (in complex with adenosine;

PDB-ID: 1MUO; (Cheetham et al., 2002)), as well as in active and "half-activated"

Discussion 69

Fig. 4.1: Comparison of MARK2 and Aurora-A kinase domains. a, d: MARK2 double mutant (yellow), b, e: inactive Aurora-A (blue, PDB-ID: 1MUO), c, f: Aurora-A in the fully activated state (green, PDB-ID: 1OL5) with a framgent of the activating protein TPX2 (gray). In all panels, the catalytic loop is red and the activation segment is purple. Catalytically important residues are shown in stick model representation. All three structures are in the same orientation by least-squares superposition of their catalytic loops (residues 171-183 in MARK2, 252-264 in Aurora-A). a-c: Front view, showing the open (a, b; inactive state) or closed (c, active state) cleft between the lobes. In the active state (c) the activation loop is ordered and points to the right side, in the inactive state, it is disordered and presumably leans to the left side. In a and b, plausible conformations of the invisible part of the activation loop are indicated by dotted lines. d-f: Side view, showing a close-up of the nucleotide binding site with catalytic loop and helix C; the P-loop has been omitted to allow an unobstructed view on the invariant ion pair (K82 and E100 in MARK2, K162 and E181 in Aurora-A) that coordinates α- and β-phosphates of the bound nucleotide in the active state (f). In the inactive structures (d, e) the ion pair interaction is disrupted. Part of the catalytic loop around the active aspartate (D175 in MARK2, D256 in Aurora-A) and the preceding arginine (R174 in MARK2, R255 in Aurora-A) is represented by its Cα-trace to show the subtle differences in the conformations of MARK2 and Aurora-A. In the active state (c, f) the arginine of the conserved RD motif is hydrogen bonded to phosphothreonine pT288. This figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

K82

E100

S210

A208

R174D175

helix C

helix E

K82

E100

S210

A208

R174D175

helix C

helix E

K82

E100

R174

D175

helix C

helix F

K82

E100

R174

D175

helix C

helix F

D256

K162

E181

R255

D256

K162

E181

R255

K162 E181

pT288

K162 E181

pT288

a

d e f

D256

K162

E181

R255

b

D256

K162

E181

R255

b

K162

E181

pT288

cTPX2

K162

E181

pT288

cTPX2

Discussion 70

forms (ATPγS complexes with/without a fragment of the activating protein TPX2;

PDB-ID: 1OL5 and 1OL7, resp. (Bayliss et al., 2003)) and, thus, lends itself as a

paradigm for the discussion of the MARK2 structure.

4.1 Activation segment

The inactive and the fully activated form of Aurora-A mainly differ by the

conformation of the activation segment and by a 6.7° tilt of the minor lobe (Fig. 4.1b,

c). In the active form, the activation loop passes underneath the helix C, similar to

other active kinase structures, the tip pointing to the right side in Fig. 4.1, top row. In

the inactive form, it is partially disordered; the N- and C-termini of the activation

segment indicate that the activation loop points in the opposite direction, passing close

below the P-loop (named for its binding to the phosphate moiety of the nucleotide, Fig.

4.1b). In the MARK2 structures, only the C-terminal residues of the activation segment

are visible. They adopt a conformation similar to the inactive form of Aurora-A,

suggesting that the activation loop of inactive MARK2 also folds to the left side (Fig.

4.1a). By folding in this way, cysteine residues C210 of the two monomers can meet

and form a disulfide bridge.

4.2 Activation loop of MARK2 interferes with substrate binding

The structure of phosphorylated c-AMP dependent protein kinase (PKA) has been

determined in complexes with a peptide substrate or with a peptide inhibitor, both

derived from protein kinase inhibitor PKI (Knighton et al., 1991b; Madhusudan et al.,

2002; Fig. 4.2 a). There are only minor differences in the overall fold of the kinase

domain and the mode of peptide binding. The activation loop, being phosphoralyted at

threonine T197 (T208 in MARK), is well defined and folds into a hairpin-like

conformation underneath the helix C, as it is typical for active kinase. The ternary

structure with the peptide substrate and with ADP and aluminium fluoride (AlF3)

shows that the substrate binds in a groove on the surface of the large lobe

(Madhusudan et al., 2002). The C-terminal part of the peptide substrate, comprising the

last seven or eight residues including the phosphorylation site, adopts an extended

conformation. The peptide interacts mainly with residues of the large lobe. The P-site

Discussion 71

Fig. 4.2: Active site of the cAMP dependent protein kinase A compared with MARK2. (a) transition state mimick of PKA (PDB-ID: 1L3R; (Madhusudan et al., 2002) in blue with substrate peptide derived from PKI in green and ADP.AlF3 (catalytic loop red, activation segment purple). (b) MARK2 double mutant (yellow, except for the catalytic loop and the activation segment) with the peptide substrate and ADP.AlF3 complex of PKA overlaid in faint colors (superposition by least-squares fit of the catalytic loops, res. 171-183 in MARK2, res. 162-174 in PKA). PKA is in a closed, active conformation; the P-loop is "down" and hydrogen bonded to the β-phosphate. The activation loop folds back and forth underneath the C-helix. The phosphorylatable serine S21 of the substrate is located in line with the β-phosphate and the aluminum fluoride, properly positioned for phosphotransfer. In MARK2, the catalytic cleft is open: the P-loop is in "up" position, more than 8 Å above that of the PKA structure (measured at the tip). The C-terminus of the activation segment occupies the same area as the peptide substrate in PKA as shown by the position of the phosphorylatable T208 (mutated to Ala), which is close to S21 of the substrate peptide (Cα distance ~3.5 Å). This figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

ADP

AlF3

PKI

P-loophelix C

activation segment

S21

a

P-loophelix C

activation segment

catalytic loop

T208A

b

ADP

AlF3

PKI

P-loophelix C

activation segment

S21

a

P-loophelix C

activation segment

catalytic loop

T208A

b

Discussion 72

serine is positioned between the tip of the Gly-rich loop and the C-terminal end of the

activation loop. In the ternary complex with ADP and AlF3, mimicking the transition

state, aluminium fluoride is located between the β-phosphate and the hydroxyl group

of the P-site serine. Thus, the P-site serine is properly positioned for phospho-transfer.

Superposition of the PKA-substrate complex with the structure of the MARK2 double

mutant reveals that the activation loop in the "inactive" conformation of MARK2

occludes the space required for substrate binding (Fig. 4.2b). Thus, in the inactive state

observed in the MARK2 structure the substrate cannot bind in a productive way

because of steric interference with the activation loop. It may also be notable that

threonine T208, the primary phosphorylation site of the activation loop, is close to the

position of the P-site serine in the PKA-substrate complex (CA distance 3.5 Å).

Phosphorylase kinase (PHK) provides another example of a kinase domain whose

structure has been solved in a ternary complex with ATP analogue and substrate

peptide (Lowe et al., 1997). The substrate analogue, an artificial heptapeptide related to

the natural substrate and to the so-called optimal peptide substrate (Songyang et al.,

1996), binds to the kinase domain in a similar conformation as PKI to PKA. PHK is

constitutively active; and accordingly PHK has a glutamate (E182) in the activation

loop at the site corresponding to the primary phosphorylation site, T208 in MARK2.

Thus, it is not surprising that the activation loop folds into a conformation

characteristic for the active state. In a ternary complex with AMPPNP and the peptide

substrate (PDB-ID: 2PHK; (Lowe et al., 1997)), E128 forms a salt bridge with arginine

R148 (R174 in MARK2) which helps to lock the catalytic loop in a enzymatically

competent shape. Remarkably, in a binary complex of the inactive mutant E182S with

AMPPNP, the activation loop and, in fact, the complete kinase domain adopt a

conformation very similar to the constitutively active complex with bound substrate

(PDB-ID: 1QL6; (Lowe et al., 1997)). This is explained by a sulfate ion at a position

close to the carboxylate group of E182 in the wild type stabilizing the active

conformation by ion pair interactions with arginine R148 and lysine K72 in helix C

(R99 in MARK2).

Discussion 73

The substrate peptide binds to the PHK catalytic site in extended conformation,

analogous to the PKA-substrate complex. However, there are significant differences in

the P+2 and P+3 positions, which are probably important for substrate specificity.

These residues form a short, antiparallel beta-sheet with the C-terminal portion of the

activation segment. As in the case of the cAMP dependent protein kinase PKA,

binding of the substrate to MARK2 in the same position as in PHK would result in a

steric clash with residues 208 to 210 of the MARK2 – at least according to the double

mutant, where this segment of the activation loop is stabilized by an intradimer

disulfide bridge between cysteines C210. While residues 208 to 210 are disordered and

not well defined in the structure of MARK2 wild type, they are compelled by the

connectivity of the peptide chain to occupy the same area. In any case, it can be

concluded that threonine T208, the primary phosphorylation site of MARK2, is close

to the catalytic centre, approaching the place that seems to be reserved for the P-site

serine or threonine of the substrate.

4.3 Catalytic cleft and nucleotide binding site

The catalytic cleft in the MARK2 structure is extremely wide open, compared to other

active or inactive kinases. It is 1-2 Å wider than that of Aurora-A (inactive form),

judged by the distance between β1 (N-lobe) and β6 (C-lobe). In the activated form of

Aurora-A (as in other active kinases), helix C contributes to nucleotide binding by the

conserved glutamic acid E181 (E100 in MARK2) that forms a salt bridge with K162

(K82 in MARK2), a strictly conserved lysine in strand β3 (Fig. 4.1f). E181 positions

this lysine for proper coordination of the nucleotide's α- and β-phosphates. In the

inactive form of Aurora-A (Fig. 4.1e), the salt bridge is interrupted by the N-terminal

anchor of the activation loop (DFG motif), especially by phenylalanine F275.

In MARK2, the side chains of K82 and E100 are not aligned correctly for interaction

with the nucleotide (Fig. 4.1d). In the double mutant, these residues adopt a

conformation similar to that observed for the inactive Aurora-A kinase domain (Fig.

4.1e), in the wild type structure and especially in that of the K82R mutant, the side

chains of K82 and E100 are less well ordered (by B-factor) or even invisible. It is not

surprising that long side chains facing the activation loop are affected by disorder. This

Discussion 74

applies also to methionine M104 which reveals a double conformation that could be

identified due to the high electron density of the selenium atom in the SeMet structure.

There is no hint at a specific interaction between K82 and E100 in any of the four

MARK2 structures. Thus, it is not surprising that the single site mutation K82R has no

effect on the overall structure.

4.4 Conformation of the catalytic loop

Activation of Aurora-A involves phosphorylation of threonines T287 and T288 in the

activation loop. In the fully activated state, the phospho group of pT288 (primary

phosphorylation site, corresponding to T208 in MARK2) is engaged in ion pair

interactions with R255 in the catalytic loop (R174 in MARK2), adjacent to the

catalytically active aspartate D256 (D175 in MARK2, Fig. 4.1f). This interaction

stabilizes the catalytic loop and positions the aspartate towards the attacking OH-group

of the substrate. Remarkably, the conformation of the catalytic loop in the inactive

form of Aurora-A is very similar to that of the fully activated form, although there is

no phosphothreonine to interact with R255 (Fig. 4.1e). In MARK2, the overall fold and

conformation of the catalytic loop is the same; there are, however, significant

differences that culminate at the RD motif (R174, D175; Fig. 4.1d-f). In contrast to

Aurora-A, the side chain of D175 is too far from N180 (N261 in Aurora-A) further

down the catalytic loop to form a hydrogen bond which is important for coordination

of a divalent cation. The absence of the hydrogen bond between D175 and N180 is

probably a consequence of the unusual main chain conformation of the RD motif.

Possibly the catalytic loop adopts several, slightly different conformations which

cannot be described adequately by a single conformation. This would agree with the

assumption that the catalytic loop needs stabilization by interaction with the primary

phospho-site. The unusual conformation of the RD motif in MARK2 could also be

induced by interaction of the two monomers of the dimer: helix G of one molecule

protrudes toward the catalytic loop of the other molecule, with the side chain of N254

at the tip of helix G approaching the catalytic aspartate (minimum distance 3.3 Å; Fig.

3.13).

Discussion 75

4.5 Dimerization

Similar to MARK2, the catalytic core of PHK forms dimers in crystals of the ternary

complex with AMPPNP and peptide substrate (PDB-ID: 2PHK; (Lowe et al., 1997)).

The PHK dimer consists of two molecules that are related by two-fold crystal

symmetry. The molecules associate head to tail, burying the active sites and the

substrate molecules in the centre of the dimer (Fig. 4.3). The heptapeptide substrates

are in close proximity to each other, reminiscent of the segments of the activation loop

that are visible and cross-linked in the structure of the MARK2 double mutant. Thus,

PHK dimers bear a striking resemblance to MARK2 dimers, although the orientation

of the molecules in the dimers is not exactly the same: the conformation of the

MARK2 dimer is less compact, and the symmetry is not perfect (non-crystallographic

symmetry). As in the case of MARK2, dimerization of the PHK ternary complex was

unexpected, since dimers of the complex could not be identified in solution.

Nevertheless, possible functions of dimerization were proposed, including the

possibility of a cooperative effect on substrate binding (Lowe et al., 1997).

Dimerization of kinase domains in crystal structures has also been observed for other

kinases (e.g. ERK2, PAK1). However, these dimers are of a different nature, involving

contacts with domains or segments outside the catalytic core, and therefore they are not

comparable with the case of MARK2. In crystals of the doubly phosphorylated kinase

domain of the MAP kinase ERK2, dimerization of symmetry related molecules by

interactions of the C-terminal extensions has been observed (PDB-ID: 2ERK;

(Canagarajah et al., 1997)). The C-terminal extension of MAP kinases spans both

domains of the kinases core and terminates in a helix that interacts with helix C in the

form of an antiparallel coiled coil. Dimerization occurs via hydrophobic contacts at the

start of the terminal helix, in the vicinity of the catalytic cleft. However, the catalytic

clefts of the dimer remain exposed to the surface and are separated by a distance of

about

Discussion 76

Fig. 4.3: Dimerization of phosphorylase kinase (PHK) and MARK2. View perpendicular and parallel to the symmetry axis of PHK dimers (top) and dimers of MARK2 double mutant (bottom). In the case of PHK (PDB-ID: 2PHK; complex with AMPPNP and substrate peptide; (Lowe et al., 1997)) the monomers are related by two-fold crystal symmetry, whereas the MARK2 dimer is not exactly symmetric. The two molecules of a dimer are yellow and green, peptide substrate in the PHK and activation segment in MARK2 (both close to the centre of symmetry) are red. This figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

50 Å. In the autoinhibited structure of PAK1 (PDB-ID: 1F3M; (Lei et al., 2000))

dimerization occurs via interactions of the autoregulatory fragment (80 residues further

N-terminal of the catalytic domain) cocrystallized with the catalytic domain. Eight

residues at the N-termini of two autoregulatory fragments form an antiparallel β-

ribbon, while the C-terminal residues of each fragment insert into the catalytic site of

two kinase domain molecules, preventing substrate binding and proper folding of the

activation loop.

PHKPHK

MARK2MARK2

Discussion 77

4.6 C-terminal extension of the kinase core

Many kinases comprise C-terminal extensions of the catalytic core that wrap around

the core domain and terminate in a subdomain that binds to the N-lobe. They are

probably involved in regulation of the kinase activity, comparable to regulatory

proteins of other kinases, like cyclins for CDKs or TPX2 and INCENP in the case of

Aurora-A and B, respectively. In PKA, for instance, the C-terminal extension spirals

up in a right-handed rotation, in the MAP kinases ERK2 and p38 it winds in the

opposite direction around the core domain; in either case, the terminal subdomain ends

up at a similar location, close to helix C of the N-lobe.

In MARK2, a corresponding extension consists of the UBA domain which is linked to

the catalytic domain by a stretch of about 20 amino acids, comprising a bulge with a

cluster of negatively charged residues (~N305-P315) and a straight section heading for

the UBA domain (linker, ~P315-D322). This is roughly similar to ERK2 except that

the UBA domain binds at some distance to the C helix (Fig. 3.18). In MAP kinases, the

bulge residues (~L311-P321 in ERK2 and ~F308-P318 in p38) have been proposed as

a common docking domain ("CD domain") for many upstream and downstream

interaction partners (Tanoue et al., 2000; Tanoue and Nishida, 2003). The similarity in

position, conformation and amino acid composition suggests that the bulge in the

structure of MARK2 may also play a role in protein-protein recognition.

The UBA domain is linked to the potential CD domain by a stretch of about seven

residues in extended conformation. As the UBA domain binds opposite to the catalytic

cleft, at the back of the hinge region, closure of the catalytic cleft by rotation of the N-

lobe (with the UBA domain attached on it) around the hinge would require further

elongation of the linker (e.g. by unfolding of the CD domain) or detachment of the

UBA domain from the N-lobe. Thus it seems, that the crystal structure of MARK2

represents a state with the kinase domain locked in an open (inactive) conformation. A

similar mechanism for regulation of the kinase activity has been proposed for Aurora-

B (Sessa et al., 2005). In the crystal structure of Aurora-B with part of the activating

protein INCENP, the C-terminal tail of the kinase domain assumes an extended

conformation and connects back to the N-lobe, similar to MARK2. In the case of

Aurora-B, the contact to the N-lobe is mediated by INCENP, in the case of MARK2 by

Discussion 78

the UBA domain. The INCENP peptide consists of three helices that wind around the

N-lobe. Interestingly, the second helix (B) binds to the same groove at the surface of

the N-lobe as the UBA domain (Fig. 4.5). Thus, in a superposition of the structures,

helix B of INCENP and helix α3 of the UBA domain would overlap to a large extent.

4.7 UBA domain and regulation of MARK2

Fig. 4.4: UBA domain and regulation of MARK2 (stereo view) UBA domain of MARK2 overlaid with that of Dsk2p in complex with ubiquitin. The UBA domains are green (Dsk2p, PDB-ID: 1WR1; (Ohno et al., 2005)) and purple (MARK2), ubiquitin red, and MARK2 kinase domain yellow. In the overlay by least-squares fit of 10 Cα atoms of helices α1, the ubiquitin locates above the MARK2 kinase domain, with a small overlap in the β2-β3 region of the N-lobe. This figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

ubiquitin

helix C

β1

β4

UBAMARK2UBADsk2p

ubiquitin

helix C

β1

β4

UBAMARK2UBADsk2p

ubiquitin

helix C

β1

β4

UBADsk2p

UBAMARK2

ubiquitin

helix C

β1

β4

UBADsk2p

UBAMARK2

Discussion 79

MARK kinases are unique in that they contain a UBA domain adjacent to their

catalytic domain. The role of this domain is unknown, but suggestive of a ubiquitin-

related function, such as protein degradation or others (Buchberger, 2002). Other

possibilities include an autoregulatory role, reminiscent of Ca/calmodulin regulated

kinases whose C-terminal tail binds into the catalytic cleft, members of the PAK or

MAP kinase familes which have extra helices that bind to the N-lobe. In MARK2 the

UBA domain binds opposite to the catalytic cleft, at the back of the hinge region. It is

tethered to the C-lobe by an extended linker and binds to the N-lobe by hydrophobic

interactions. It is thus conceivable that this type of binding locks the catalytic domain

in an open, inactive conformation. In this picture, binding of the UBA domain to

ubiquitin (or another interacting protein) could induce dissociation from the N-lobe and

relieve the strain on the catalytic domain which would then be free to switch to the

active, closed conformation.

Superposition of the MARK2-UBA structure with published UBA-ubiquitin

complexes (Fig. 4.4, (Ohno et al., 2005)) shows an overlap between the catalytic

domain and ubiquitin, indicating that binding of the two would be mutually exclusive.

On the other hand superposition of the MARK2-UBA structure with the model of UBA

- K48 linked di-ubiquitin complex (Fig. 4.5, (Varadan et al., 2005)) suggests that

proximal ubiquitin which has more contacts with α2 of UBA might have steric clashes

with helix C of MARK catalytic domain. This implies a possible role of the UBA

domain in regulation of kinase activity. Another possibility for regulation of MARK2

activity via the UBA domain could make use of the tight connection with the common

docking domain: binding of an activating protein to this domain could induce a

conformational change, pulling the UBA domain away from the N-lobe.

Discussion 80

Fig. 4.5: Comparison of MARK2 and Aurora-B kinase domains and possible interaction of MARK2 UBA with K48 linked di-ubiquitin. a: UBA domain of MARK2 overlaid with that of HHR23A in complex with K48 linked di-ubiquitin chain (PDB-ID: 1ZO6; (Varadan et al., 2005)). The UBA domain of MARK2 is coloured orange and the kinase domain is coloured blue. The distal ubiquitin is coloured green and the proximal ubiquitin is coloured red. b: crystal structure of Aurora-B kinase domain complexed with INCENP (PDB-ID: 2BFX; (Sessa et al., 2005)). The kinase domain is coloured blue and the activator INCENP coloured red. This figure was prepared with Deep View Swiss-PDB Viewer (Guex and Peitsch, 1997) and POVray for Windows (Persistence of Vision Pty. Ltd. (2004), Persistence of Vision Raytracer Version 3.5, retrieved from http://www.povray.org/).

While the MARK sequence complies with the Prosite profile of the UBA domain (Fig.

3.14), (Hofmann and Bucher, 1996)), the crystal structure reveals an unexpected

conformation as the third helix is inverted compared to the known structures. It is

conceivable that the inversion of helix α3 is evoked by interaction with the N-lobe,

while the free UBA domain (after detachment from the catalytic domain) could adopt

the normal conformation. Alternatively, the unusual conformation of the UBA domain

could be a specific feature of MARK2 and related kinases. The structures of "normal"

UBA domains vary considerably, complementary to the variable interactions with

mono- or polyubiquitin, and the different linkage modes of polyubiquitin (Chim et al.,

2004). This opens a range of potential regulatory interactions which awaits further

analysis.

a bUBA

proximalubiquitin

distalubiquitin

INCENP

helix Chelix C

a ba bUBA

proximalubiquitinproximalubiquitin

distalubiquitin

distalubiquitin

INCENP

helix Chelix C

Summary 81

5 Summary

MAP/microtubule affinity regulating kinases (MARKs) are a family of protein

serine/threonine kinases which have been identified by their ability to phosphorylate

the microtubule-associated proteins tau, MAP2 and MAP4. Phosphorylation of the

neuronal MAP tau on S262 dramatically reduces its microtubule binding capacity and

leads to the formation of neurofibrillary tangles, which is a hallmark of Alzheimer’s

disease.

Homologues of MARK include the kinase PAR-1 in C. elegans and D. melanogaster

and KIN-1 in S. cerevisiae which are involved in the generation of embryonic polarity

and cell morphology respectively. Compared to other kinases, MARK is a relatively

large protein (~720 amino acids) which contains an N-terminal leader sequence, a

typical kinase catalytic domain, an ubiquitin associated domain (UBA), a spacer and a

tail domain containing the KA1 (kinase associated) motif characteristic for this family

of kinases ending with the ELKL motif.

A stable fragment of MARK2 was identified by limited proteolysis, which contains the

catalytic and ubiquitin associated domains. This fragment was crystallized in several

variants. Crystals of the wild type construct and of two inactive mutants K82R and

T208A/S212A were analyzed. K82 is essential for catalysis, T208 is the primary

phosphorylation site in the activation loop which controls access of the substrate and

S212 was also found to be phosphorylated in MARK2 from brain. The three variants

and the selenomethionine labelled protein were crystallized in the hexagonal space

group P61. Two distinct crystal forms were observed which differ by the length of the

c-axis: 106.0 ± 0.37 Å (sd, n = 15) for the double mutant, 99.7 ± 0.23 Å (sd, n = 8) for

the wild type and the K82R mutant; the wild type was found in both forms. All crystal

structures are similar in crystal packing and folding of the molecules.

The structure of the catalytic domain shows the small and large lobes typical of

kinases. The substrate cleft between the lobes is wide open both in the inactivated and

the wild type structures. In the crystal, two kinase moieties form a dimer, facing each

other with the catalytic cleft such that helix G of one molecule inserts into the cleft of

Summary 82

the other, similar to the dimers of phosphorylase kinase. This prevents cleft closure and

a conformation reminiscent of the active state.

The UBA domain is attached via a taut linker to the large lobe of the kinase domain

and leans against a hydrophobic patch on the back of the small lobe. The UBA

structure is unusual in that the orientation of its third helix is inverted, relative to

previous structures. The ubiquitin-binding interface is partially masked by the

interaction with the kinase domain, implying that detachment and/or conformational

changes are necessary for activation of ubiquitin-dependent signaling. The linker

sequence also contains a peptide motif similar to the "common docking domain" of the

MAP kinase family, which could serve as an anchor for partners in the signaling

cascade of MARK/PAR-1.

References

83

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7 Appendix

7.1 Abbreviations

(v/v) Volume per volume

(w/v) Weight per volume

Å Angstrom (0.1 nm)

ADP Adenosine- 5’- diphosphate

AI Arabinose inducible

AIEX Anion exchange chromatography

Amp Ampicillin

APP Amyloid precursor protein

ATP Adenosine- 5’- triphosphate

Ax UV Absorbance at x nm

CaMKII Calmodulin dependent protein kinases II

cAMP 3’5’ cyclic Adenosine monophosphate

Cdc25C Cell division cycle 25C

CHAPS 3-(3-Cholamidopropyl) dimethylamino-1-propanesulphonate

CHK1 Check point kinase 1

Chl Chloramphenicol

CIEX Cation exchange chromatography

C-TAK1 Cdc25C-associated kinase 1

Dcx Doublecortin

DESY Deutsches Elektronen Synchrotron

DNTP Deoxynucleotide triphosphate

DORIS Double Storage Ring (Doppelring-Speicher)

DTT Dithiothreitol

EDTA Ethylendiaminetetraacetate

EGTA Ethylenglycol-bis-(2-aminoethylether)-N, N, N’, N’-tetra acetic acid

EMK ELKL motif kinase

Exu Exuperantia

GST Glutathione-S-transferase

HEPES N-2-Hydroxyethyl-piperazine-N-2-ethanesulfonic acid

His6-tag 6x histidine tag

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IEX Ion exchange chromatography

IMAC Immobilized metal chelating chromatography

IPTG Isopropyl-D-β-galactopyranoside

KA1 Kinase associated domain 1

Kan Kanamycin

kDa Kilodalton(s)

krpm 1000 revolutions per minute

KSR1 Kinase suppressor of Ras 1

LB Luria-Bertani

MAD Multi wavelength anomalous dispersion

MAP Microtubule-associated protein

MARK MAP/microtubule affinity regulating kinase

MARKK MAP/microtubule affinity regulating kinase kinase

MIR Multiple isomorphous replacement

MME Monomethylether

MR Molecular replacement

HHR23A Human homologue of RAD23 A

MT Microtubules

NCS Noncrystallography symmetry

Ni-NTA Nickel-nitrilotriacetic acid

NMR Nuclear magnetic resonance spectroscopy

ODx Optical density at x nm

PAGE Polyacrylamide gel electrophoresis

PAK5 p21 activated kinase 5

PAR Partitioning defective

PDB Protein Data Bank

PEG Polyethylenglycol

PHK Phosphorylase Kinase

pI Isoelectric point

PIPES Piperazine-N,N’- bis- (2-ethanesulfonic) acid

PKA Protein kinase A

PKB Protein kinase B

PMSF Phenylmethylsulfonylfluoride

PTPH1 Protein tyrosine phosphatase H1

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SeMet Selenomethionine

TAO-1 Thousand and one aminoacids protein kinase 1

TE Tris-EDTA

TEV Tobacco etch virus

Tm Melting temperature

Tris Tris- (Hydroxylmethyl)-aminomethane

Triton X-100 Polyoxyethylen-(9-10)-p-t-octylphenol

Tween 20 Polyoxyethylen-sorbitanmonolaurate

UBA Ubiquitin-associated domain

7.2 List of figures Figure 1.1: Phylogenetic tree of the complete superfamily of human protein kinases Figure 1.2: Bar diagram of human tau and its phosphorylation sites for different

protein kinases Figure 1.3: Domain organization of MARK2 Figure 1.4: Multiple sequence alignment of MARK/PAR-1 kinase family

members Figure 1.5: Structure of UBA(2) domain from HHR23A Figure 2.1: Overview of the Gateway cloning technology Figure 2.2: Set up of crystallization trials using: A) the sitting or B) the hanging

drop vapour diffusion technique Figure 3.1: Summary of different constructs of MARK2 and their solubility upon

prokaryotic expression Figure 3.2: Limited proteolysis of MARK2 Figure 3.3: Vector map for the MARK2 expression plasmid Figure 3.4: Purification of MARK2 protein Figure 3.5: Bar diagram for the crystallized construct of MARK2 Figure 3.6: Kinase activities of the MARK2 constructs

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Figure 3.7: Mass spectrometry analysis of wild type and SeMet labeled double mutant (T208A/S212A) proteins of MARK2

Figure 3.8: Images of crystals of MARK2 wild type and mutants Figure 3.9: Diffraction pattern of a wild type MARK2 crystal Figure 3.10: Folding of the catalytic and UBA domain of MARK2 Figure 3.11: Structural sequence comparison of MARK2 and related kinases Figure 3.12: Electron density map around the catalytic cleft of the SeMet double

mutant Figure 3.13: Electron density map around the disulfide bridge of the SeMet double

mutant Figure 3.14: Intermolecular contacts in MARK2 dimers Figure 3.15: Structural sequence alignment of UBA domains Figure 3.16: A potential protein–protein binding interface of UBA domains is

built from hydrophobic residues on the surface Figure 3.17: Overlay of the MARK2 UBA domain (yellow) with UBA of

HHR23A Figure 3.18: Details of the binding interactions between the UBA domain and the

N-lobe of the catalytic domain of MARK2 Figure 3.19: Common docking domain and ED site of MAP kinases compared to

MARK2 Figure 3.20: Electron density map around the linker region of the SeMet double

mutant Figure 4.1: Comparison of MARK2 and Aurora-A kinase domains Figure 4.2: Active site of the cAMP dependent protein kinase A compared with

MARK2 Figure 4.3: Dimerization of phosphorylase kinase (PHK) and MARK2 Figure 4.4: UBA domain and regulation of MARK2 Figure 4.5: Comparison of MARK2 and Aurora-B kinase domains and possible

interaction of MARK2 UBA with K48 linked di-ubiquitin

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7.3 List of tables Table 1.1: Genes encoding MARK/PAR-1/KIN1 kinases in yeast and animal

species Table 1.2: Isoforms, localization and substrates of mammalian MARK kinases Table 2.1: Cell strains and feature list Table 2.2: Summary of the vectors used in this study Table 2.3: Summary of reactions and nomenclature of the Gateway cloning

technology Table 2.4: Solutions for preparing SDS-PAGE gels Table 3.1: Summary of data collection and structure refinement 7.4 Oligonucleotides Oligonucleotides for Gateway cloning Adaptor Primers: AttB1-f 5’-GGGG ACA AGT TTG TAC AAA AAA GCA GGC T-3’ AttB2-r 5’-GGGG AC CAC TTT GTA CAA GAA AGC TGG GT-3’ MARK2 Gene specific primers: SP-6 5’-AA AAA GCA GGC TTC GAA AAC CTG TAT TTT CAG GGC ATG TCC AGC GCT CGG ACC C -3’ SP-8 5’-AA AAA GCA GGC TTC GAA AAC CTG TAT TTT CAG GGC ATG TAC AGA CTC CTT AAG ACC ATT GGC AAG G-3’ SP-10 5’-AGA AAG CTG GGT CTT A CTT GTA GCC AAG GAG CAG ATA GGT AGC -3’

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SP-12 5’-AGA AAG CTG GGT CTT A ATT CAT CCA CCG ATC TTT CAT AAT TTG C-3’ SP-13 5’-AA AAA GCA GGC TTC GAA AAC CTG TAT TTT CAG GGC AAC TCA GCC ACC TCT GCT GAC G-3’ SP-14 5’-AGA AAG CTG GGT CTT ACT TTA GCT CGT CAT CCT CAT GGC C-3’ SP-15 5’-AGA AAG CTG GGT CTT ACT TGT AGT CAG GGA GTG GCT CCA C-3’ Oligonucleotides for sequencing For Gateway entry clones: attL1f 5’-TCGCGTTAACGCTAGCATGGATCTC-3’ attL2r 5’-GTAACATCAGAGATTTTGAGACAG-3’ For Gateway Expression Clones: T7pp 5’- TAATACGACTCACTATAGGG-3’ T7tp 5’- GCTAGTTATTGCTCAGCGG-3’ GSTsq 5’- CCATCCTGACTTCATGTTGTATG-3’ For MARK2 Sequences: K15 5’- CAACTCAGCCACCTCTGC -3’ K11 5’- ACTGAGAAGACGCTCTAC -3’ K18 5’- GATGCTGATATGAACATC-3’

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K25 5’- GAACCTCAAGGAGCTAC-3’ K27 5’- GAAGAGATCCAGGACTC-3’ K31 5’- ACCTCTGGCCTACCAG-3’ K35 5’- ACAACGCAGAAAATAAG-3’ K36 5’- CTTCTGTTTGTGCT-3’ K45 5’- CCAGACCGAACTAATTT-3’ K28 5’- AAGATGCTGCCAGAGGCCCC-3’ K22 5’- CAGTGGGAGATGGAGGT-3’ 7.5 Purification buffers Cell lysis Buffer: 50 mM Hepes- NaOH pH 7.2 at RT, 300 mM NaCl, 5% Glycerol Ni-NTA Buffer A: 50 mM Hepes- NaOH pH 7.2 at RT, 300 mM NaCl, 5% Glycerol Ni-NTA buffer B: 50 mM Hepes- NaOH pH 7.2 at RT, 300 mM NaCl, 1000 mM Imidazole, 5% Glycerol Dialysis Buffer for TEVprotease cleavage: 50 mM Hepes- NaOH pH 7.2 at RT, 250 mM NaCl,5% Glycerol, 1 mM EGTA, 1 mM DTT Dilution Buffer: 50 mM Hepes- NaOH pH 7.2 at RT, 5% Glycerol MonoS buffer A: 50 mM Hepes- NaOH pH 7.2 at RT, 100 mM NaCl, 5% Glycerol MonoS buffer B: 50 mM Hepes- NaOH pH 7.2 at RT, 1000 mM NaCl, 5% Glycerol

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Gel filtration buffer: 50 mM Bis-TRIS SO4 pH 6.5 at RT, 250 mM NaCl, 5% Glycerol Protease Reaction buffer: 50 mM Tris-Cl pH8.0 at RT, 200 mM NaCl, 5 mM CaCl2 7.6 List of Coordinates The atomic coordinates for the MARK2 structures have been deposited in the Protein Data Bank (PDB) with the following accession codes. 1Y8G - Crystal structure of the kinase MARK2/PAR-1: T208A/S212A inactive double mutant (Selenomethionine derivative) 1ZMU - Crystal structure of the kinase MARK2/PAR-1: wild type 1ZMV - Crystal structure of the kinase MARK2/PAR-1: K82R mutant 1ZMW - Crystal structure of the kinase MARK2/PAR-1: T208A/S212A

inactive double mutant

Acknowledgements

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8. Acknowledgements

First of all, I would like to express my sincere gratitude to Prof. Eckhard Mandelkow

for giving me the opportunity to carry out this interesting project and providing all the

facilities promptly to my work. His stimulating and lively discussions have always

been very helpful. Also, special thanks to Dr. Eva Maria Mandelkow for her constant

encouragement and guidance throughout this work.

I pay my sincere thanks to Dr. Alexander Marx, who helped me in crystallographic

data analysis. Without him this work would not have been possible.

I also thank Dr. Jens Müller, Dr. Jacek Biernat, Dr. Michael Brüggert and Dr. Martin

von Bergen for their generous support, guidance and help during this work and

Dr. Thomas Timm for critical reading of my thesis.

I would like to thank Prof. C. Betzel, Dr. M. Perbandt and Dr. W. Rypniewski for

beam time allocation and helpful discussions throughout this work. I also thank Dr.

Young-Hwa Song, EMBL for her help during this work.

I would like to thank Narayana Kalkura, Olivia, Sadasivam and Vijayalakshmi for their

constant friendship and understanding during these years. I would like to thank all the

lab members, who provided a pleasant and humorous atmosphere in the lab and whose

friendship is invaluable.

Finally, I would like to thank my parents and family members for their constant

support, encouragement and understanding during these years.

Saravanan Panneerselvam

Curriculum Vitae

105

9. Curriculum Vitae Name : Saravanan Panneerselvam Home address : 7, Ayya Swamy Street,

Kamatchi Amman pet, Gudiyatham, India, 632 602.

Date of Birth : 07.05.1978 Nationality : Indian University studies 1995-1998 : Bachelor of Science (Biochemistry),

Islamiah Arts and Science College Vaniyambadi, India.

1998-2000 : Master of Science (Biochemistry)

Department of Biochemistry and Molecular Biology, University of Madras, Chennai, India.

Doctoral studies 04.2002-12.2005 : Cytoskeleton Research Unit MPASMB, c/o DESY, Hamburg, Germany Supervisor : Prof. Eckhard Mandelkow Thesis title : Crystal structure of the catalytic and ubiquitin-

associated domains of the protein kinase MARK2 / PAR-1 from Rattus norvegicus

Curriculum Vitae

106

Publications: Saravanan Panneerselvam, Alexander Marx, Eva-Maria Mandelkow, Eckhard Mandelkow (2006) Structure of the catalytic and ubiquitin-associated domains of the protein kinase MARK / Par-1. Structure, 14(2), 173-183.

Declaration

107

10. Declaration (Erklärung)

I declare that I have carried out this thesis by myself and have not used external help

except where explicitly indicated.

This thesis was not submitted to any other university.

I did not make any earlier attempt to submit this work as a doctoral thesis.

Saravanan Panneerselvam Hamburg, 02nd December, 2005

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig und ohne fremde Hilfe

verfasst, andere als die angegebenen Quellen und Hilfsmittel nicht benutzt und die den

verwendeten Werken wörtlich oder inhaltlich entnommenen Stellen als solche

kenntlich gemacht habe.

Ferner versichere ich, dass ich diese Dissertation noch an keiner anderen Universität

eingereicht habe, um ein Promotionsverfahren eröffnen zu lassen.

Hiermit erkläre ich auch, dass ich keine anderen früheren Versuche gemacht habe, die

Arbeit zur Promotion einzureichen.

Saravanan Panneerselvam Hamburg, 02. December 2005

Table of Contents i

1 Introduction 1 1.1 The protein kinase superfamily 1 1.2 Classification of the protein kinase superfamily 1

1.3 Identification of MAP/microtubule affinity regulating kinase (MARK) 3

1.3.1 Homologues of MARK 6

1.3.2 Activation of MARK 9

1.3.3 Inhibition of MARK 9

1.3.4 Proteins interacting with MARK 10

1.4 Ubiquitin binding domains 11

1.4.1 The ubiquitin-associated domain (UBA) 11

1.4.2 Structure of the UBA domain 12

1.4.3 The UBA domain of the MARK 13

1.5 Aim of the work 14

2 Materials and methods 15

2.1 Materials 2.1.1 Chemicals 15

2.1.2 Enzymes 15

2.1.3 Bacterial strains 16

2.1.4 Cloning vectors 17

2.1.5 Expression vectors 17

2.1.6 Media 17


2.1.7.1 Crystallization supplies and tools 18

2.1.7.2 Crystallization solutions 19

2.1.8 Equipment and accessories 19


2.2.1 Culture and storage of E. coli strains 20

2.2.2 Transformation of E. coli strains 20

2.2.3 Isolation of plasmid DNA 21

2.2.4 Determination of DNA concentration and purity 21

Table of Contents ii

2.2.5 Ligation reaction 21

2.2.6 Restriction analysis of DNA 22

2.2.7 DNA sequencing 22

2.2.8 Mutagenesis of DNA 23

2.2.9 Gene cloning using the Invitrogen Gateway technology 24

2.3 Protein methods

2.3.1 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) 29

2.3.2 Protein expression and purification 30

2.3.3 Cell lysis and solubility test 30

2.3.4 Chromatography 31

2.3.4.1 Ni-NTA affinity chromatography 31

2.3.4.2 GST affinity chromatography 31

2.3.4.3 Anion and cation exchange chromatography 32

2.3.4.4 Gel filtration chromatography 32

2.3.5 Determination of concentration of proteins 33

2.3.6 Concentrating the protein solution 33

2.3.7 Protein kinase assay 33


2.3.9 N-terminal sequencing 34

2.3.10 Mass spectrometry 34

2.3.11 Detailed protocols on purification of MARK2 wild type and mutant proteins 35

2.3.11.1 Expression 35

2.3.11.2 Purification 35

2.3.11.3 Selenomethionine labelling 36


2.4.1 Crystallization techniques 36

2.4.2 Cryoprotection of crystals 38

2.4.3 Data collection 38

2.4.4 Methods for phase determination 39

Table of Contents iii

3 Results 41 3.1 Cloning of different constructs of MARK2 41

3.2 Identification of structurally folded part of MARK2 42


3.2.2 N-terminal sequencing and mass spectrometry analysis 43

3.3 Cloning of the stable fragment of MARK2 44

3.4 Expression and purification 45

3.5 Kinase activities of the purified protein 47

3.6 Preparation of selenomethionine labelled protein 48

3.7 Crystallization 48

3.8 Data collection and structure determination 50

3.8.1 Molecular replacement 53

3.8.2 Heavy atom derivatives of MARK2 crystals 53

3.8.3 Model building and refinement 54

3.8.4 Crystals of selenomethionine labelled MARK2 55

3.9 Overall structure of the MARK2 catalytic and UBA domains 56

3.10 Structure of the catalytic domain 56

3.10.1 Conformation of the activation loop 58

3.10.2 Intermolecular disulfide bridge 59

3.10.3 Dimerization 60

3.11 Structure of the UBA domain 61

3.12 UBA linker and common docking domain for kinase activators 66

4 Discussion 68

4.1 Activation segment 70

4.2 Activation loop of MARK2 interferes with substrate binding 70

4.3 Catalytic cleft and nucleotide binding site 73

4.4 Conformation of the catalytic loop 74

4.5 Dimerization 75

4.6 C-terminal extension of the kinase core 77

4.7 UBA domain and regulation of MARK2 78

Table of Contents iv

5 Summary 81

6 References 83

7 Appendix 96 7.1 Abbreviations 96

7.2 List of figures 98

7.3 List of tables 100

7.4 Oligonucleotides 100

7.5 Purification buffers 102

7.6 List of Coordinates 103

8 Acknowledgements 104

9 Curriculum vitae 105

10 Declaration 107

crystal structure of the catalytic and ubiquitin

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