Structural and free energy landscape of novel mutations in ribosomal protein S1 (rpsA) associated with pyrazinamide resistance

Abstract

Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type ( pncA WT ), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncA WT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsA CTD ) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsA CTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsA CTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

Structural insights into the mechanism of RNA recognition by the N-terminal RNA-binding domain of the SARS-CoV-2 nucleocapsid phosphoprotein

“…The binding free energy demonstrated that N-NTD exhibited a decreased affinity toward RNA in MTs T57, H59, S105A, R107A, G170, F171, and Y172. Since these methods are widely used by different studies to understand the impact of mutations [45] , [46] .…”

Dynamics Insights Into the Gain of Flexibility by Helix-12 in ESR1 as a Mechanism of Resistance to Drugs in Breast Cancer Cell Lines

“…In silico, methods to predict structural implications of mutations will be beneficial in understanding mechanisms of drug resistance for quantitative estimation of the phenotypic resistance outcomes (Khan et al, 2019). To systematically understand the effects (protein stability changes, flexibitliy drift, and protein ligand interaction) of these mutations, we performed in silico saturation mutagenesis.…”

Gibbs Free Energy Calculation of Mutation in PncA and RpsA Associated With Pyrazinamide Resistance

“…Mutants were generated at positions S324F, E325K, G341R, D342N, D343N, A344P, I351F, T370P, and W403G using PYMOL (DeLano, 2002). Free energy differences between MTs and WT RpsA from our previous papers (Khan et al, 2018c(Khan et al, , 2019bRehman et al, 2019) were re-analyzed. PZA is a prodrug, activated by MTB encoded pncA into POA, targeting RpsA.…”

“…WT exhibited a more stable state as compared to mutants. POA resistance might be due the GFE states altering the affinity of RpsA (Khan et al, 2019b).…”