Computational Saturation Mutagenesis to predict structural consequences of systematic mutations on protein stability and rifampin interactions in the β subunit of RNA polymerase inMycobacterium leprae

In contrast to the situation with tuberculosis, rifampin resistance in leprosy may remain undetected due to the lack of rapid and effective diagnostic methods. A quick and reliable method is essential to determine the impacts of emerging detrimental mutations. The functional consequences of missense mutations within the β-subunit of RNA polymerase inMycobacterium leprae(M. leprae) contribute to phenotypic rifampin resistance outcomes in leprosy. Here we reportin-silicosaturation mutagenesis of all residues in the β-subunit of RNA polymerase to all other 19 amino acid types and predict their impacts on overall thermodynamic stability, on interactions at subunit interfaces, and on β-subunit-RNA and rifampin affinities using state-of-the-art structure, sequence and normal mode analysis-based methods. A total of 21,394 mutations were analysed, and it was noted that mutations in the conserved residues that line the active-site cleft show largely destabilizing effects, resulting in increased relative solvent accessibility and concomitant decrease in depth of the mutant residues. The mutations at residues S437, G459, H451, P489, K884 and H1035 are identified as extremely detrimental as they induce highly destabilizing effects on the overall stability, nucleic acid and rifampin affinities. Destabilizing effects were predicted for all the experimentally identified rifampin-resistant mutations inM. lepraeindicating that this model can be used as a surveillance tool to monitor emerging detrimental mutations conferring rifampin resistance in leprosy.AUTHOR SUMMARYEmergence of primary and secondary drug resistance to rifampin in leprosy is a growing concern and poses threat to the leprosy control and elimination measures globally. In the absence of an effectivein-vitrosystem to detect and monitor phenotypic rifampin resistance in leprosy, most of the diagnosis relies on detecting mutations in the drug resistance determining regions of therpoBgene that encodes the β subunit of RNA polymerase inM. leprae. Few labs in the world perform mouse food pad propagation ofM. lepraein the presence of drugs (rifampin) to determine growth patterns and confirm resistance, however the duration of these methods lasts from 8 to 12 months making them impractical for diagnosis. Understanding molecular mechanisms of drug resistance is vital to associating mutations to clinical resistance outcomes in leprosy. Here we propose anin-silicosaturation mutagenesis approach to comprehensively elucidate the structural implications of any mutations that exist or can arise in the β subunit of RNA polymerase inM. leprae. Most of the predicted mutations may not occur inM. lepraedue to fitness costs but the information thus generated by this approach help decipher the impacts of mutations across the structure and conversely enable identification of stable regions in the protein that are least impacted by mutations (mutation coolspots) which can be a choice for small molecule binding and structure guided drug discovery.