PB1109 - In silico modeling of factor Xa – prothrombin complexes using AlphaFold-Multimer and Molecular Dynamics simulations for functional prediction

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

DV

Dejvid Veizaj

PhD Candidate
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, the Netherlands
Leiden, Zuid-Holland, Netherlands

Co-Author(s)

RV

Rianne Van Diest

Researcher
VarmX B.V., Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
DP

David A. Poole III

PostDoc
AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
KC

Ka Lei Cheung

Technician
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, the Netherlands
Leiden, Zuid-Holland, Netherlands
PR

Pieter Reitsma

Chief Scientific Officer
Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
Daniël Verhoef

Research Director
Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
DG

Daan P. Geerke

Associate Professor
AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, Amsterdam, The Netherlands;
Amsterdam, Noord-Holland, Netherlands
Mettine H.A Bos, PhD

PhD
Dept. Internal Medicine, Div. Thrombosis and Hemostasis, Leiden University Medical Center
Leiden, Zuid-Holland, Netherlands

Background: Factor (F)Xa is a plasma vitamin K-dependent serine protease that converts prothrombin to thrombin via limited proteolysis at Arg271 and Arg320. While physiologically relevant prothrombin conversion requires prothrombinase complex assembly, FXa by itself is also capable of activating prothrombin. Despite attempts to determine the structure of FXa–prothrombin complexes, the detailed information required for FXa active-site engagement correlating with function is lacking.

Aims: Develop an in silico approach to achieve 3D-models of FXa and variants thereof complexed with prothrombin cleavage sequences using AlphaFold-Multimer, and assess their interactions using Molecular Dynamics (MD) simulations.

Methods: AlphaFold-Multimer was employed to generate 3D-models of the FXa serine protease domain in complex with prothrombin Arg271 or Arg320 sequences (2–18 residues). MD (10-replicate100 ns simulations per variant) were performed using Amber23; binding free energies were estimated with the MM/PBSA approach.

Results: Using AlphaFold-Multimer, 80 3D-models were generated for each cleavage sequence, of which 74% (Arg271 sequences) or 29% (Arg320 sequences) were advantageously positioned for catalysis. Per-residue interaction analysis of MD trajectories revealed Arg271 sequence 266-RAIEGRTATSEY-277 (Figure 1) and Arg320 sequence 310-RELLESYIDGRIVE-323 (Figure 2) to drive FXa binding. Overall binding free energy computations of FXa-prothrombin sequence complexes predicted reduced binding for FXa-F174Ala/Ser variants relative to wild-type FXa, corroborating experimental findings. Evaluation of prothrombin sequence-interactive residues in FXa revealed that FXa active site residues Arg326, Gly402, and Arg405 contribute substantially to substrate binding in addition to widely-reported specificity pocket residues Asp373 and Gln376. Their relevance is further underscored since missense mutations at these positions are associated with FX deficiency. Assessment of missense variant FXa-Gln376Leu predicted reduced binding to prothrombin, reinforcing this computational model.

Conclusion(s): We developed an in silico model to predict prothrombin binding to the FXa active site, validated by experimental findings. The key residues identified in FXa for prothrombin binding will allow us to better understand FXa function.