Abstract
SummaryResolution advances in cryoelectron microscopy (cryo-EM) now offer the possibility to visualize structural effects of naturally occurring resistance mutations in proteins and also of understanding the binding mechanisms of small drug molecules. In Mycobacterium tuberculosis the multifunctional heme enzyme KatG is indispensable for activation of isoniazid (INH), a first-line pro-drug for treatment of tuberculosis. We present a cryo-EM methodology for structural and functional characterization of KatG and INH resistance variants. The cryo-EM structure of the 161 kDa KatG dimer in the presence of INH is reported to 2.7 Å resolution allowing the observation of potential INH binding sites. In addition, cryo-EM structures of two INH resistance variants, identified from clinical isolates, W107R and T275P, are reported. In combination with electronic absorbance spectroscopy our cryo-EM approach reveals how these resistance variants cause disorder in the heme environment preventing heme uptake and retention, providing insight into INH resistance.
Highlights
Within the past decade the cryoelectron microscopy ‘‘Resolution Revolution’’ has dramatically advanced the field of structural biology (Kuhlbrandt, 2014)
In this study we utilize the outlined advances in cryoelectron microscopy (cryo-EM) to study both resistance mutations and drug binding in the KatG protein from Mycobacterium tuberculosis, which has been implicated in drug resistance within this pathogen
We reveal that, for two isonicotinic acid hydrazide (INH) resistance variants, W107R and T275P (Gagneux et al, 2006; Heym et al, 1995), significant structural disorder relating to heme uptake and retention is the likely cause for INH resistance
Summary
Within the past decade the cryoelectron microscopy (cryo-EM) ‘‘Resolution Revolution’’ has dramatically advanced the field of structural biology (Kuhlbrandt, 2014). Cryo-EM technology is continually evolving, with improvements in microscope optics, software for data analysis, sample preparation, and electron detectors, high-resolution structures are being determined routinely. These advances are making it possible to solve the structures of large complex macromolecular assemblies, as well as proteins smaller than 100 kDa and to visualize proteins within living cells using cryoelectron tomography (Rivera-Calzada and Carroni, 2019). Cryo-EM advances make it possible to visualize a single amino acid substitution within a protein structure, which is of tremendous value when investigating point mutations implicated in drug resistance (Kim et al, 2019). It is well established that INH requires activation by KatG (Figure 1), a heme-dependent catalase-peroxidase enzyme that can utilize and degrade hydrogen peroxide (H2O2) either through functioning as a catalase (Equation 1) or as a peroxidase (Equation 2)
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