Determining the unbinding events and conserved motions associated with the pyrazinamide release due to resistance mutations of Mycobacterium tuberculosis pyrazinamidase

Olivier Sheik Amamuddy; Thommas Mutemi Musyoka; Rita Afriyie Boateng; Sophakama Zabo; Özlem Tastan Bishop

doi:10.1016/j.csbj.2020.05.009

Determining the unbinding events and conserved motions associated with the pyrazinamide release due to resistance mutations of Mycobacterium tuberculosis pyrazinamidase

Comput Struct Biotechnol J. 2020 May 18:18:1103-1120. doi: 10.1016/j.csbj.2020.05.009. eCollection 2020.

Authors

Olivier Sheik Amamuddy¹, Thommas Mutemi Musyoka¹, Rita Afriyie Boateng¹, Sophakama Zabo¹, Özlem Tastan Bishop¹

Affiliation

¹ Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa.

Abstract

Pyrazinamide (PZA) is the only first-line antitubercular drug active against latent Mycobacterium tuberculosis (Mtb). It is activated to pyrazinoic acid by the pncA-encoded pyrazinamidase enzyme (PZase). Despite the emergence of PZA drug resistance, the underlying mechanisms of resistance remain unclear. This study investigated part of these mechanisms by modelling a PZA-bound wild type and 82 mutant PZase structures before applying molecular dynamics (MD) with an accurate Fe²⁺ cofactor coordination geometry. After observing nanosecond-scale PZA unbinding from several PZase mutants, an algorithm was developed to systematically detect ligand release via centre of mass distances (COM) and ligand average speed calculations, before applying the statistically guided network analysis (SGNA) method to investigate conserved protein motions associated with ligand unbinding. Ligand and cofactor perspectives were also investigated. A conserved pair of lid-destabilising motions was found. These consisted of (1) antiparallel lid and side flap motions; (2) the contractions of a flanking region within the same flap and residue 74 towards the core. Mutations affecting the hinge residues (H51 and H71), nearby residues or L19 were found to destabilise the lid. Additionally, other metal binding site (MBS) mutations delocalised the Fe²⁺ cofactor, also facilitating lid opening. In the early stages of unbinding, a wider variety of PZA poses were observed, suggesting multiple exit pathways. These findings provide insights into the late events preceding PZA unbinding, which we found to occur in some resistant PZase mutants. Further, the algorithm developed here to identify unbinding events coupled with SGNA can be applicable to other similar problems.

Keywords: 3D, Three-dimensional; ACPYPE, AnteChamber Python Parser interface; Amber force field parameters; CHPC, Center for High Performance Computing; COM, Center of mass; Drug resistance; Drug unbinding; FDA, Food and Drug Administration; HTMD, High throughput molecular dynamics; INH, Isoniazid; MBS, Metal binding site; MCBP, Metal Center Parameter Builder; MD, Molecular dynamics; MDR-TB, Multidrug-resistant tuberculosis; Missense mutations; Molecular dynamics simulations; PBC, Periodic boundary conditions; PDB, Protein Data bank; POA, Pyrazinoic acid; PZA, Pyrazinamide; PZase, Pyrazinamidase; QM, Quantum Mechanics; RIF, Rifampicin; SGNA, Statistically guided network analysis; Statistically guided network analysis; TB, Tuberculosis; VAPOR, Variant Analysis Portal; WHO, World Health Organization; WT, Wild type.