Oxometalates as artificial proteases: sequence versus region selectivity

Karen Stroobants

2

Oxometalates as artificial proteases: sequence versus region selectivity

Inorganic chemistry

Protein chemistry

« Why is protein hydrolysis important? »

« What is wrong with trypsin? »

- limited solvent compatibility- unreactive toward IDPs- small fragments

3

Often proposed solution:

The use of artificial metallopeptidases

metal ions as hydrolytic active centers

Current problems:

Low reactivity at physiological pH and temperature

Non specific interaction / Non selective cleavage

“The development of alternative cleavage strategies would greatly facilitate the study of protein structure and function.”

Grant 2006

Need for complementary and versatile cleavage agents

4

Hetero Polyoxometalates“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”

“A multitechnique study of europium decatungstate and human serum albumin molecular interaction”

(Nadjo et al.)

Conceptually new artificial cleavage agents

Iso Oxometalates

“Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate”

“Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.)

5

Hetero Polyoxometalates“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”

“A multitechnique study of europium decatungstate and human serum albumin molecular interaction”

(Nadjo et al.)

Conceptually new artificial cleavage agents

Iso Oxometalates

“Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate”

“Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.)

6

Molybdate(IV) hydrolyzes HEWL

SDS-PAGE &Edman degradation:

Asp18-Asn19Asp48-Gly49Asp52-Trp53Asp101-Gly102

pH 5.0 / 60 °C

+Ho et al. (2011) Inorg. Chem.Stroobants et al. (2013) submitted to J. Inorg. Biochem.

7

Sequence specific hydrolysis at Asp-X bonds

Interaction occurs at many positions and thus is not selective

Reaction only occurs when an Asp-X bond is accesible and thus is sequence selective

8

Side chain assistence mechanism

Polarization of the carbonyl due to [MoO4]2- binding & Internal nucleophilic attack of the Asp side chain

9

Side chain assistence mechanism

Polarization of the carbonyl due to [MoO4]2- binding & Internal nucleophilic attack of the Asp side chain

Appearance of Gly

Disappearance of Asp-Gly

ppm

10

Hetero Polyoxometalates“Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”

“A multitechnique study of europium decatungstate and human serum albumin molecular interaction”

(Nadjo et al.)

Conceptually new artificial cleavage agents

Iso Oxometalates

“Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate”

“Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.)

11

Polyoxotungstate ligand

SPECIFIC INTERACTION

Polyoxotungstates as more tunable alternative

Lewis active metal

HYDROLYTIC ACTIVITY

- Higher stability (vs. Oxomolybdate)- Known region specific interaction with protein surfaces- Not reactive as such!!!

RC

NR

O

H

OH

- Oxophilicity- Coordination number- Ligand exchange kinetics Ce(IV) ion

12

Zr(IV)-substituted POMs hydrolyze HSA

…KQNCys392//Glu393LFE…

…VESLys313//Asp314VCK…

…DDRAla257//Asp258LAK…

…NLPArg114//Leu115VRP…

Stroobants et al. (2013) Chem. Eur. J.

13

lacunary Wells Dawson

Zr(IV) substitutedWells Dawson

1:1 interaction / 1:3 interactionKa ~ 108 M-1/ Ka ~ 105 M-1

1:1 interactionKa ~ 106 M-1

Zr(IV) and Wells Dawson POM driven interactions

14

* Arg114-Leu115

known POM binding positive regionNadjo et al.

* Ala257-Asp258* Lys313-Asp314* Cys392-Glu393

regions of mixed charge, hydrolysis downstream from acidic residue

Zr(IV)-Wells Dawson exhibits region selective interaction

lacunary Wells Dawson

Zr(IV) substitutedWells Dawson

1:1 interactionKa ~ 106 M-1 1:1 interaction / 1:3 interaction

Ka ~ 108 M-1/ Ka ~ 105 M-1

15

Additional fragments upon prolonged reaction time

…VESLys313//Asp314VCK…

…DDRAla257//Asp258LAK…

Edman degradation

16

Same selectivity for secondary fragmentation

17

Same selectivity for secondary fragmentation

18

Reaction occurs- at pH 7.4- at Asp and Gluresidues

* Ala257-Asp258* Lys313-Asp314* Cys392-Glu393

regions of mixed charge, hydrolysis downstream from acidic residue

Is X-Asp/Glu hydrolysis mechanistically related to Asp-X cleavage?

Proposed assistence mechanism- does only dominate < pH 6- does only occur for Asp(not for Glu)

19

Different mechanisms for oxometalate versus metal-substituted POM reactivity

Non-specific binding

Sequence selective hydrolysis,via side chain assistence mechanism

Region selective interaction

Hydrolysis without side chain assistance, presumably via external water nucleophile delivery

20

(Poly)oxoanions hold promise as artificial proteases

Evidence for * Sequence selective Asp-X protein hydrolysis in the presence of oxomolybdate & * Region selective protein cleavage in the presence of metal-substituted polyoxotungstates

Future perspective

« Hydrolysis of membrane proteins and IDPs possible? »

Many thanks to…

My promotor – Prof. Parac-Vogt Tatjana

POM post-doc – Dr. Absillis Gregory

Edman degradation – Prof. Proost Paul & Dr. Moelants Eva

ITC – Dr. Bruylants Gilles

NMR technician – Karel Deurinckx

Master students – Laure Baeyens, Dounia Saadallah, Anja Bourgois, Vincent Goovaerts & Jeroen Lannoeye

All the colleagues of lab LBC / LIC / ULB

FWO for financial support

& you for your attention!