Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
School of Life Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
Structure. 2021 Aug 5;29(8):899-912.e4. doi: 10.1016/j.str.2020.12.008. Epub 2021 Jan 13.
Resolution 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.
在冷冻电子显微镜（cryo-EM）技术上的进展，现在提供了可视化天然存在的蛋白质耐药突变的结构影响的可能性，也提供了理解小分子药物结合机制的可能性。分枝杆菌中的多功能血红素酶 KatG 对于异烟肼（INH）的激活是必不可少的，INH 是治疗结核病的一线前药。我们提出了一种冷冻电子显微镜方法，用于 KatG 和 INH 耐药变体的结构和功能表征。报道了在存在 INH 的情况下 161 kDa KatG 二聚体的冷冻电镜结构，分辨率达到 2.7 Å，允许观察潜在的 INH 结合位点。此外，还报道了从临床分离株中鉴定出的两种 INH 耐药变体 W107R 和 T275P 的冷冻电镜结构。结合电子吸收光谱学，我们的冷冻电镜方法揭示了这些耐药变体如何导致血红素环境的紊乱，从而阻止血红素的摄取和保留，深入了解 INH 耐药性的机制。
