title: Insights into RpoB clinical mutants in mediating rifampicin resistance in Mycobacterium tuberculosis.
abstract: Rifampicin (RIF) an essential first-line anti-tuberculosis (TB) drug, resistance to RIF is a potential threat to TB control program and widely considered as surrogate marker for detection of multi-drug resistant-TB (MDR-TB), molecular understanding of which is the utmost need of the hour. Mutations at RIF resistance-determining region (RRDR) of 81-bp in the rpoB gene coding for β subunit or RpoB protein is the major cause of RIF resistance in Mycobacterium tuberculosis (MTB). Mutation at positions 526 and 531 are generally associated with high-level RIF resistance and at codons 516, 521 and 533 with low-level resistance. Thus, in order to understand the interactions between the clinical mutants (MTs) of RpoB and RIF which are responsible for mediating both levels of RIF resistance from MTB. In the present study, models of wild type (WT) and seven MTs (D516V, L521M, H526D, H526R, H526Y, S531L and L533P) of RpoB from MTB were generated using crystal structure of 2A68 and 4KBM as templates, for deducing 3 domains structure. Molecular docking between RpoB proteins and RIF was carried out, which showed higher values for WT compared to MTs. The high score in WT may be due to the presence of favorable interactions with RIF and MT-L521M which lacks in other MTs. Molecular dynamics (MD) simulation was performed for over 10 nanoseconds, which suggest the root mean square deviation (RMSD) was more and root mean square fluctuation (RMSF) was less in WT compared to MTs. The ligand RMSD exhibited very unique deviation with the MT-D516V compared to other MTs and WT. The RMSF for MTs such as H526R-H526D, L521M and D516V were higher for residues such as 152, 265, 352, 402, 513, 552, and 577 compared to WT. Hydrogen bond interactions at RIF binding site after MD simulations were found comparatively lower in WT than MTs. Similarly, the binding energy of WT was observed to be lesser in comparison to MTs. All MTs demonstrated certain (2Å) degree of structural deviation from the WT. Overall, these results suggest that RIF binding ability shows differences between WT and MTs, which could be because of different substitutions affecting the conformation of the MT proteins, leading to changes in binding interactions with RIF, eventually to the cause of RIF resistance.
authors.affiliation: Centre for Biomedical Informatics, National Institute for Research in Tuberculosis, India. Electronic address: nusrath.a@nirt.res.in.
authors.forename: Ameeruddin
authors.initials: A
authors.lastname: Nusrath Unissa
authors.affiliation: Centre for Biomedical Informatics, National Institute for Research in Tuberculosis, India.
authors.forename: Sameer
authors.initials: S
authors.lastname: Hassan
authors.affiliation: Centre for Biomedical Informatics, National Institute for Research in Tuberculosis, India.
authors.forename: Venkatesan
authors.initials: V
authors.lastname: Indira Kumari
authors.affiliation: Department of Integrative Biology, Vellore Institute of Technology, India.
authors.forename: Ravi
authors.initials: R
authors.lastname: Revathy
authors.affiliation: Centre for Biomedical Informatics, National Institute for Research in Tuberculosis, India.
authors.forename: Luke Elizabeth
authors.initials: LE
authors.lastname: Hanna
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D015203:Reproducibility of Results; D012293:Rifampin / Q000737:chemistry / Q000494:pharmacology*; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin
keywords: Docking; Dynamics; Molecular modeling; Mutants; Mycobacterium tuberculosis; RIF resistance; RpoB