A comprehensive in silico investigation into the nsSNPs of Drd2 gene predicts significant functional consequences in dopamine signaling and pharmacotherapy

Abstract

DRD2 is a neuronal cell surface protein involved in brain development and function. Variations in the Drd2 gene have clinical significance since DRD2 is a pharmacotherapeutic target for treating psychiatric disorders like ADHD and schizophrenia. Despite numerous studies on the disease association of single nucleotide polymorphisms (SNPs) in the intronic regions, investigation into the coding regions is surprisingly limited. In this study, we aimed at identifying potential functionally and pharmaco-therapeutically deleterious non-synonymous SNPs of Drd2. A wide array of bioinformatics tools was used to evaluate the impact of nsSNPs on protein structure and functionality. Out of 260 nsSNPs retrieved from the dbSNP database, initially 9 were predicted as deleterious by 15 tools. Upon further assessment of their domain association, conservation profile, homology models and inter-atomic interaction, the mutant F389V was considered as the most impactful. In-depth analysis of F389V through Molecular Docking and Dynamics Simulation revealed a decline in affinity for its native agonist dopamine and an increase in affinity for the antipsychotic drug risperidone. Remarkable alterations in binding interactions and stability of the protein-ligand complex in simulated physiological conditions were also noted. These findings will improve our understanding of the consequence of nsSNPs in disease-susceptibility and therapeutic efficacy.

Figure 1

Schematic representation of the workflow of the study. The overall process can be summarized as a series of progressive filtration steps pursued for the identification of the most damaging nsSNPs of Drd2 and subsequent in-depth analysis of one particular nsSNP.

Figure 2

Graphical representation of evolutionary relationships between the human Drd2 and its closest counterparts by Iroki server. The human DRD2 appears to be most closely related to its homologs in Pan troglodytes and Pan paniscus.

Figure 3

Evolutionary conservation profile of amino acid residues of DRD2 as predicted by ConSurf. Almost all the nsSNPs primarily evaluated as deleterious belonged to the highly conserved regions.

Figure 4

Secondary structure analysis of native DRD2 and the mutants revealed by PDBsum. It shows the modifications in terms of alpha helices, beta strands as well as various motifs that had occurred as a result of nsSNPs.

Figure 5

Dynamut prediction of inter-atomic interactions of the native DRD2 vs the mutants. Native and mutant residues are colored in light-green and showed as sticks along with the surrounding residues involved in interaction. Dot points with several colors represent the interactions such as hydrogen bonds, ionic interactions etc.

Figure 6

Binding site prediction and visualization of the molecular docking output. (a) FTSite predicted three ligand binding sites in DRD2. (b) Ligand non-bond binding interactions between the protein residues and the ligands visualized by Discovery Studio. Dopamine docked against (I) native DRD2 (II) F389V mutant; and risperidone docked against (III) native DRD2 and (IV) F389V mutant are presented. The receptor surface is colored according to solvent accessibility surface (SAS) in an ascending degree from blue to green.

Figure 7

Results of 200 ns Molecular Dynamics Simulation by GROMACS software. Of the wild-type and F389V complexes with dopamine and risperidone: (a) RMSD values, (b) RMSF values, (c) SASA calculations and (d) Radius of gyration.

Figure 8

Binding free energy of the docked complexes during the last 20 ns of Molecular Dynamics Simulation by GROMACS software. (a) Complex of dopamine with native DRD2 (blue) and F389 V mutant (orange), (b) Complex of risperidone with native DRD2 (blue) and F389 V mutant (orange).