365 C

Human Vitamin K Epoxide Reductase (hVKORC1): Search for the Functional Conformational States

Nolan CHATRON1,2, Bernard CHALMOND1, Alain TROUVE1, Etienne BENOIT2, Virginie LATTARD2, Luba TCHERTANOV1 1

Centre de Mathématiques et de Leurs Applications, Ecole Normale Supérieure de Cachan CMLA - UMR 8536 CNRS, Université Paris-Saclay, 94235 Cachan, France

2

INRA – VetAgro Sup, USC 1233, Ecole Nationale Vétérinaire de Lyon 69280 Marcy l’Etoile, France

Introduction VKDPs

Human Vitamin K Epoxide Reductase (hVKORC1): - a reticulum endoplasmic membrane protein, activating coagulation process through vitamin K recycling; - a leading target for anti-coagulation therapies, especially based on Vitamin K Agonists (VKAs) oral administration.

COAGULATION

quinone

epoxide

R

R

VKORC1

Problems: - hVKORC1 mutations  catalytic disorders or VKAs resistance (left panel); - no crystallographic data available for hVKORC1; - contradictory experimental/theoretical data  distinct membrane topology models: 3 TM-helices, 4 TM-helices (right panel), and 5 TM-helices models.

Mutations

Goals: - design a relevant hVKORC1 structural model; - describe hVKORC1 conformational space; - unravel hVKORC1 catalytic mechanism.

R=

Homology modeling

MD simulations & analysis 60,000 atoms 5 x 100 ns 2 x 500 ns  1 µs

hVKORC1 bacterial homolog (left), used as a template to build a hVKORC1 3D model (right).

Active site: C132xxC135 C43 C51

C132

C135

C51 C135

C132

PCA - First eigenvector

DSSP

VKAs

Biological data:  either a direct activation of C132 and C135;  or an electron transfer from C43 and C51 to C132 and C135.

Residue

PCA - Second eigenvector 90°

Cysteine residues in our model are protonated. Proton donor is considered as an external source.

ConfiScan

I

C132-C135

II

C43-C51 C132-C135

III

C51-C132

IV

V

C51-C132

C51-C132

I III II

IV V

Conclusions - Perspectives hVKORC1 3D model: - 4 TM helices - protonated cysteine residues  conformational space exploration MD trajectories: - 5 x 100 ns; 2 x 500 ns  1 µs - secondary structures preservation - highly flexible luminal loop (carrying C43 and C51)

ConfiScan: - similar conformations identification - free energy estimation - minimum energy conformations highlighting  identification of biologically relevant conformations concept of hVKORC1 catalytic mechanism, consistent with experimental data (scheme), suggesting an electron transfer from C43 and C51 to C132 and C135.

Ongoing and future work: MD simulations extending (until 1 µs), hVKORC1-vitamin K complexes MD simulations, the binding free energy calculations

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