Structural and Molecular Characterization of Drug Resistance-Associated PncA and EmbB Mutations in Mycobacterium Tuberculosis

Abstract

Drug resistance in Mycobacterium tuberculosis is driven by genetic variation that can alter protein structure and function. This study characterized sequence variants in pncA and embB from clinical M. tuberculosis isolates and evaluated their predicted structural consequences using molecular modelling. Clinically suspected pulmonary tuberculosis specimens collected at the Centre for Chest and Respiratory Diseases, Erbil, from July 2024 to March 2025 were investigated by GeneXpert MTB/RIF and Line Probe Assay, followed by PCR and bidirectional Sanger sequencing of pncA and embB. Sequences were aligned with the H37Rv reference genome and variants were assessed against the WHO catalogue of resistance-associated mutations. Missense variants were modelled with Phyre2, validated using ProSA-web, evaluated for changes in stability and flexibility with DynaMut2, and examined by molecular docking using AutoDock Vina. Three-dimensional models of wild-type and mutant proteins were generated successfully, with ProSA-web Z-scores within the range of experimentally determined protein structures. Most evaluated substitutions produced negative ΔΔG values, indicating predicted destabilization of the corresponding protein structures. Structural models showed local changes in atomic interactions around selected pncA and embB substitutions. These findings indicate that sequence variation in pncA and embB can produce measurable predicted changes in protein stability and local structure, supporting the use of integrated sequence and structural analyses for investigating molecular mechanisms of drug resistance in M. tuberculosis.