In silico analysis predicting effects of deleterious SNPs of human RASSF5 gene on its structure and functions

Abstract

Ras association domain-containing protein 5 (RASSF5), one of the prospective biomarkers for tumors, generally plays a crucial role as a tumor suppressor. As deleterious effects can result from functional differences through SNPs, we sought to analyze the most deleterious SNPs of RASSF5 as well as predict the structural changes associated with the mutants that hamper the normal protein-protein interactions. We adopted both sequence and structure based approaches to analyze the SNPs of RASSF5 protein. We also analyzed the putative post translational modification sites as well as the altered protein-protein interactions that encompass various cascades of signals. Out of all the SNPs obtained from the NCBI database, only 25 were considered as highly deleterious by six in silico SNP prediction tools. Among them, upon analyzing the effect of these nsSNPs on the stability of the protein, we found 17 SNPs that decrease the stability. Significant deviation in the energy minimization score was observed in P350R, F321L, and R277W. Besides this, docking analysis confirmed that P350R, A319V, F321L, and R277W reduce the binding affinity of the protein with H-Ras, where P350R shows the most remarkable deviation. Protein-protein interaction analysis revealed that RASSF5 acts as a hub connecting two clusters consisting of 18 proteins and alteration in the RASSF5 may lead to disassociation of several signal cascades. Thus, based on these analyses, our study suggests that the reported functional SNPs may serve as potential targets for different proteomic studies, diagnosis and therapeutic interventions.

Figure 1

Protein structure of RASSF5 (adapted and modified from Shuofeng et al. 2018).

Figure 2

Schematic diagram summarizing the study.

Figure 3

Evolutionary conservancy of RASSF5 produced by Consurf.

Figure 4

Structural alteration of the wild type residue P350 by the mutant R350 illustrated by Project Hope. The wild type residue is presented as green and the mutant residue is shown in red.

Figure 5

Structural Effect of P350R on the native structure of RASSF5 presented by Swiss PDB Viewer. (a) P350 forms three H-bonds with T347 (3.21 Å), E353 (3.26 Å), and V354 (2.92 Å), which are indicated by green discontinuous lines. (b) R350 forms a clash with V354 (1.53 Å) presented by pink discontinuous line along with the three H-bonds.

Figure 6

Molecular docking analysis of wild type and mutant peptide of P350R with H-Ras protein. (a) Native peptide sequence (P350). (b) Superimposed image of native peptide sequence and H-Ras protein. E353 forms two H-bonds with Gly60, one H-bond with G13 and another with G15. L347 forms one H-bond with E31. (c) Mutant peptide sequence (R350). (d) Superimposed image of mutant peptide sequence with H-Ras protein. Three new H-bonds are formed with the H-Ras protein where G353 forms two H-bonds with T35, and D33. Moreover, D351 forms single H-bond with K147.

Figure 7

Post translational modification (methylation and phosphorylation) sites predicted by GPS-MSP 1.0, NetPhos 3.1 and GPS 3.0 (using IBS software).

Figure 8

Protein interaction network of RASSF5 protein.