Effect of T68A/N126Y mutations on the conformational and ligand binding landscape of Coxsackievirus B3 3C protease

Abstract

3C protease of Coxsackievirus B3 (CVB3) plays an essential role in the viral replication cycle, and therefore, emerged as an attractive therapeutic target for the treatment of human diseases caused by CVB3 infection. In this study, we report the first account of the molecular impact of the T68A/N126Y double mutant (Mutant_Bound) using an integrated computational approach. Molecular dynamics simulation and post-dynamics binding free energy, principal component analysis (PCA), hydrogen bond occupancy, SASA, R_g and RMSF confirm that T68A/N126Y instigated an increased conformational flexibility due to the loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces, which led to a decreased protease grip on the ligand (3CPI). The double mutations triggered a distortion orientation of 3CPI in the active site and decreases the binding energy, ΔG_bind (∼3 kcal mol⁻¹), compared to the wild type (Wild_Bound). The van der Waals and electrostatic energy contributions coming from residues 68 and 126 are lower for Mutant_Bound when compared with Wild_Bound. In addition, variation in the overall enzyme motion as evident from the PCA, distorted hydrogen bonding network and loss of protein–ligand interactions resulted in a loss of inhibitor efficiency. The comprehensive molecular insight gained from this study should be of great importance in understanding the drug resistance against CVB3 3C protease; also, it will assist in the designing of novel Coxsackievirus B3 inhibitors with high ligand efficacy on resistant strains.