Investigating the Structural and Functional Consequences of Pathogenic SNPs on Human VEGFA Dimer: Insights from Molecular Dynamics Study

Abstract

Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis. It forms a homodimer and binds to VEGF receptors to activate signaling pathways important for blood vessel growth and maintenance. In this study, we examined the structural and functional effects of two deleterious mutants, R262Q and C266Y, using integrative computational methods. Molecular dynamics simulations and principal component analysis showed that both mutations disrupted crucial interface interactions, including H-bonds, salt bridges, and disulfide linkages. As a result, the dimer conformation became less stable, which was also supported by binding free energy analysis. In addition, structural superimposition analysis revealed that both mutations changed the receptor-binding shape. This may block normal signaling and affect biological functions. Overall, our findings provide structural insights into how these mutations affect VEGFA dimer and may help guide the development of new therapeutics.

1

Conformational stability of native, R262Q, and C266Y VEGFA dimers over the course of 1 μs MD simulations. (a) Cα RMSD profiles, (b) Rg profiles, and (c) SASA profiles.

2

a, b) Residue-wise flexibility analysis of the VEGFA dimer for native, R262Q, and C266Y systems. Secondary structure elements (α-helices and β-strands) are highlighted to indicate fluctuation regions. Zoomed-in plots of selected regions showing higher deviation, including α2−β2, β3−β4, and β4−β5 loops, where mutants exhibit increased flexibility compared to the native structure. c) Cartoon representation of the VEGFA dimer structure, with chain A (green) and chain B (magenta) showing secondary structure elements.

3

Comparative analysis of dimer interface interactions in native and mutant VEGFA dimers. Interface residues and interaction distances (in Å) are highlighted, with dashed green lines indicating hydrogen bonds and red lines denoting salt bridges (bottom). (a) Native VEGFA dimer showing key hydrogen bonds and one salt bridge (E236–R229) stabilizing the A- and B-chain interface. (b) R262Q mutant dimer exhibits increased hydrogen bonding and formation of multiple salt bridges, altering the interface geometry and potentially overcompensating for local destabilization. (c) C266Y mutant lacks the native disulfide linkage and salt bridge, forming fewer hydrogen bonds and relying on alternative polar contacts, indicating a weakened and structurally altered interface.

4

Scores plot presented five data clusters in different colors, where each dot represented one time point. Left panel; the clustering is attributable as Native (cyan), R262Q (light yellow), C266Y (red). Right panel; Loadings plot from Principal Components Analysis of the energy and structural data.

5

MM-PBSA binding energy analysis calculated from the trajectory of last 100 ns molecular dynamics simulation.

6

a, b) Structural superimposition of native VEGFA and its R262Q and C266Y mutants, each in complex with the VEGFR-1 extracellular domain. c) Close view of the superimposed VEGF-A structure with the R262Q and C266Y variants reveals transient positional shifts ranging from 2 to 10 Å. d–f) Mutation-induced conformational shift in chain positions compared to the native structure. In the native VEGFR-1-VEGFA complex, the distance between the A chain of VEGF-A and domain D2 of VEGFR-1 is ∼ 8 Å, which is moderately reduced to ∼ 6 Å in R262Q and slightly increased to ∼ 9 Å in C266Y. For chain B, the distance is ∼ 29.3 Å in the native, ∼ 28.6 Å in R262Q, and ∼ 19.2 Å in C266Y, indicating altered binding geometry.