Department of Biotechnology, BMS Block-1, South Campus, Panjab University, Chandigarh, India.
Department of Zoology, North Campus, University of Delhi, New Delhi, India.
Curr Comput Aided Drug Des. 2021;17(1):95-106. doi: 10.2174/1573409916666200115120051.
Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis.
In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay.
This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (-6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type.
Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings.
耐多药结核病是人类健康的主要威胁。对几种异烟肼（INH）耐药结核分枝杆菌菌株的全基因组测序揭示了几个基因的突变。Rv1592c 被证明为脂酶，其表达在异烟肼（INH）治疗期间上调。Rv1592c 位置 430 位的缬氨酸在结核分枝杆菌 INH 耐药株中经常突变为丙氨酸。
在本报告中，使用一系列计算方法来了解 Rv1592c 中 Val430-Ala 突变在 INH 耐药性中的作用。使用分子动力学方法证明突变对结构稳定性和 INH 修饰程度的影响。通过 PCR 引物行走方法在 Rv1592c 基因的 V430 位置创建突变。突变和野生型基因被克隆到大肠杆菌-分枝杆菌穿梭载体（pVV-16）中，并在分枝杆菌系统中表达。通过琼脂平板培养点和 CFUs 计数测定进行异烟肼药敏试验。
本研究表明，Rv1592c 中的 Val430 构成覆盖底物结合腔的瓣的一部分。Rv1592c 中的 Val430-Ala 突变导致瓣区位移，导致腔暴露，从而使底物进入活性位点裂谷。Rv1592c 中的 Val430-Ala 突变使其结构能量更稳定。突变体的 RMSD、RMSF 和 Rg 模拟在整个模拟期间保持整体稳定性，而天然蛋白显示出相对更多的波动。此外，对接研究表明，INH 以相当大的结合能（-6.3 kcal/mol）结合到突变体的活性口袋中。为了观察 INH 的恒定结合，复合物模拟了 50 ns。观察到模拟后，INH 仍留在口袋中，与相邻氨基酸残基的分子键网络增加。体外研究清楚地表明，与野生型相比，表达突变体的耻垢分枝杆菌在异烟肼治疗中有更好的存活率。
总的来说，这项研究首先表明，在耐药株中观察到的突变为 Rv1592c 蛋白提供了稳定性，并由于瓣位移而增加了对 INH 的亲和力，从而使其有可能被修饰。体外结果支持我们的计算机发现。
