Insight into rifampicin resistance behind discordant mutations in RpoB of Mycobacterium tuberculosis: A molecular dynamics simulations study

The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing may lead to misdiagnosis and mistreatment. In this study, we aimed to find the effects of discordant mutations on RNA polymerase (RNAP) β-subunit (RpoB) thermodynamic properties leading RIF resistance. We selected one wild type (WT) and twelve discordant RpoB mutants (MTs), including seven singles (L430P, L430R, D435G, T444A, H445N, H445L, and H445C), four double MTs (H445W/L452P, L430P/C701W, S428R/H445Y, and S428T/D435G) and one triple MT (H445N/S491A/F424V). The WT and MTs were subjected to 500 ns all-atom molecular dynamic (MD) simulations. The results revealed considerable variations in discordant MTs and WT dynamic, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), dynamic cross-correlation matrix (DCCM), principal component analysis (PCA), and free energy using the molecular mechanics Poisson–Boltzmann surface area continuum solvation (MMPBSA) approach. The MMPBSA binding free energies revealed that discordant mutations generally decreased the binding affinity with RIF. Based on DCCM and PCA, highest negatively-correlated motion was found for L430 > P/C701 > W followed by S428T/D435G, H445W/L452, and S428/H445Y. The role of the loop 431–447 of RpoB flexibility was found to be correlated to RIF binding. MTs retain interactions with key residues (R154, R181A, V153A, P256A, G257A, E258A, R255A, D344A, R440A, I343A, L436A) seen in the WT, indicating these are crucial for structural stability. Each mutant exhibited some unique interactions that were not present in the WT, potentially altering the protein's structure and function. In the presence of RIF, the WT exhibits 42 DNA-residue interactions, one weak DNA-residue interaction, a total binding surface area of 1658 Å 2 , 27 hydrogen bonds, and 140 van der Waals interactions, with a hydrophobicity score of −1.348 which was significantly lower than the MTs. The study confirmed that the structural and dynamic changes induced by discordant mutations may significantly impact RIF binding and resistance.