A novel kinase mutation in VEGFR-1 predisposes its αC-helix/activation loop towards allosteric activation: Atomic insights from protein simulation

Abstract

Vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in diverse pathologies, including cancers. Although VEGFR-1 is considered as functionally impaired kinase, its decoy characteristics make it an important regulator of VEGFR-mediated signaling, particularly in tumor angiogenesis. VEGFR-1 conveys signaling via its tyrosine kinase (TK) domain whose activation is regulated by phosphorylation of specific tyrosine residues. Thus dysregulation of VEGFR-1 signaling, as reported in most of the cancers, might be a consequence of altered phosphorylation that could be attributed to genotypic variations in its TK domain. Considering the importance of TK domain of VEGFR-1, we carried out its mutational screening in 84 clinically validated and histopathologically confirmed colorectal cancer patients. By means of direct DNA sequencing and SNP analyses, eight novel variations, including one synonymous, two deletion, one missense and four intronic variations, were reported in the TK domain of VEGFR-1. rs730882263:C>G variation specifically reported in colon cancer, representing a single-atomic change (Sulfur to Oxygen) in the predicted (p.Cys1110Ser) protein, was observed as potentially deleterious variation as assessed by multiple single-nucleotide polymorphism prediction servers. Molecular dynamics simulations of VEGFR-1 Wt and (p.Cys1110Ser) variant models revealed major conformational changes in variant protein presumptuously generating an open conformation thereby exposing the activation domain and consequently increasing the probability of phosphorylation events: a condition frequently reported in cancers.

Figure 1

MD simulations of wild-type (black) and variant (red) structures. (a) RMSD plot depicting time points having maximal variation in structures between wild-type and variant structures captured during protein simulation at (I) 13.462, (II) 15.694, (III) 24.730 and (IV) 47.700 ns. (b) Comparative RMSF plot showing fluctuations in wild-type (black) and variant (red) structures along the protein stretch together with their predicted secondary structures. Functionally important positions are highlighted in color. A full color version of this figure is available at the European Journal of Human Genetics journal online.

Figure 2

Graph depicting relative trend in (a) surface area and (b) radius of gyration (Rg) of wild-type (black) and variant (red) during 50 ns MD simulation. A full color version of this figure is available at the European Journal of Human Genetics journal online.

Figure 3

Structure of tyrosine kinase domain of VEGFR-1 depicting important regions in its catalytic core. Prepared from PDB file 3HNGA using Accelrys Discovery Studio 4.1. A full color version of this figure is available at the European Journal of Human Genetics journal online.

Figure 4

Molecular docking simulation with ATP/ADP at binding sites in the catalytic pocket of VEGF receptors showing differential binding energy pattern in tyrosine phosphorylation events*. (*Based upon ligand docking with ATP or ADP; VEGFR-2 has been shown for comparison). A full color version of this figure is available at the European Journal of Human Genetics journal online.

Figure 5

Comparison of distance spanning through the motifs of wild-type and variant tyrosine kinase domain of VEGFR-1. (a) The distance between Glycine-rich loop (green) and catalytic loop (yellow) was shortened owing to the closure of glycine loop in the variant structure; however, (b) there was no change in distance between glycine-rich loop (green) and αC-helix (brown). (c) Widening of catalytic pocket wherein the distance between the activation segment (orange) and αC-helix (brown) is significantly increased to 27.140 and 28.096 Å from the positioned Lys 861 (αC-helix) to Tyr1048 and Tyr1053 (activation loop), respectively. A full color version of this figure is available at the European Journal of Human Genetics journal online.