Exploring the Structural and Functional Consequences of Deleterious Missense Nonsynonymous SNPs in the EPOR Gene: A Computational Approach

Abstract

Background: Mutations in the EPOR gene can disrupt its normal signaling pathways, leading to hematological disorders such as polycythemia vera and other myeloproliferative diseases.

Methodology: In this study, a range of bioinformatics tools, including SIFT, PolyPhen-2, SNAP2, SNPs & Go, PhD-SNP, I-Mutant2.0, MuPro, MutPred, ConSurf, HOPE, and Interpro were used to assess the deleterious effects of missense nonsynonymous single nucleotide polymorphisms (nsSNPs) on protein structure and function. Furthermore, molecular dynamics simulations (MDS) were conducted to assess the structural deviations of the identified mutant variants in comparison to the wild type.

Results: The results identified two nsSNPs, R223P and G302S, as deleterious, significantly affecting protein structure and function. Both substitutions occur in functionally conserved regions and are predicted to be pathogenic, associated with altered molecular mechanisms. The MDSs indicated that while the wild-type EPOR maintained optimal stability, the G302S and R223P variants exhibited substantial deviations, adversely affecting overall protein stability and compactness.

Conclusions: The computational analysis of missense nsSNPs in the EPOR gene identified two missense SNPs, R223P and G302S, as deleterious, occurring at highly conserved regions, and having substantial effects on erythropoietin receptor (EPO-R) protein structure and function, suggesting their potential pathogenic consequences.

Keywords: EPOR gene; erythropoietin receptor; nonsynonymous single nucleotide polymorphisms.

Figure 1

Flowchart outlining the identification and categorization of nonsynonymous single nucleotide polymorphisms (nsSNPs) in the EPOR gene, with each step indicating the tools used. If an nsSNP is classified as deleterious by a particular tool at any step, it progresses to the next tool or step for further analysis. In the last two steps, tools were utilized to examine and visualize structural alterations.

Figure 2

Outcomes of the conservation analysis using the ConSurf tool display sequence conservation using a color gradient. In this scheme, sky blue represents variable residues, while dark purple indicates highly conserved residues. Functional residues are marked with “f”, while structural residues are marked with “s”. Buried (b) and exposed (e) residues are also distinguished, showing their potential interactions within the protein or with external molecules. The two mutant variants (in the boxes) are situated in exposed-functionally conserved positions.

Figure 3

The Root-Mean-Square Deviation (RMSD) plot compares the structural stability of the wild-type protein with two mutant variants, G302S and R223P. The y-axis represents the RMSD in nanometers (nm), while the x-axis shows time in picoseconds (ps). The wild type is represented in black, with the G302S and R223P mutants represented in red and blue, respectively.

Figure 4

The Root-Mean-Square Fluctuation (RMSF) plot for three different protein variants, R223S (black), G302S (red), and wild type (blue). The y-axis represents the fluctuation in nanometers (nm), while the x-axis represents the atomic positions in the protein chain.

Figure 5

The Radius of Gyration (Rg) plot demonstrates the compactness of the wild-type protein compared to the G302S and R223P mutant variants. The Rg values, measured in nanometers (nm), are plotted on the y-axis, while the x-axis represents time in picoseconds (ps). The wild type is depicted in blue, with the G302S and R223P mutants shown in red and green, respectively.

Figure 6

The number of hydrogen bonds over time for wild-type (black), G302S (red), and R223S (blue) protein variants. The y-axis represents the number of hydrogen bonds, while the x-axis represents the simulation time in picoseconds (ps).

Figure 7

The solvent-accessible surface (SAS) of the protein over time for the wild type and two mutants (G302S and R223S). The SAS was calculated in nm² over a simulation period of 1000 ps.

Figure 8

Three-dimensional structure of the EPO-R protein highlighting the two mutant residues, GLY-302 and ARG-223. The protein is color coded to represent different structural regions, with GLY-302 (magenta) and ARG-223 (orange) shown in ball-and-stick representations to indicate their positions within the protein structure.