Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06520, United States.
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, United States.
J Mol Biol. 2022 Sep 15;434(17):167540. doi: 10.1016/j.jmb.2022.167540. Epub 2022 Mar 23.
Understanding allostery in the Mycobacterium tuberculosis low molecular weight protein tyrosine phosphatase (MptpA) is a subject of great interest since MptpA is one of two protein tyrosine phosphatases (PTPs) from the pathogenic organism Mycobacterium tuberculosis expressed during host cell infection. Here, we combine computational modeling with solution NMR spectroscopy and we find that Q75 is an allosteric site. Removal of the polar side chain of Q75 by mutation to leucine results in a cascade of events that reposition the acid loop over the active site and relocates the catalytic aspartic acid (D126) at an optimal position for proton donation to the leaving aryl group of the substrate and for subsequent hydrolysis of the thiophosphoryl intermediate. The computational analysis is consistent with kinetic data, and NMR spectroscopy, showing that the Q75L mutant exhibits enhanced reaction kinetics with similar substrate binding affinity. We anticipate that our findings will motivate further studies on the possibility that MptpA remains passivated during the chronic state of infection and increases its activity as part of the pathogenic life cycle of M. tuberculosis possibly via allosteric means.
理解分枝杆菌低分子量蛋白酪氨酸磷酸酶（MptpA）的变构作用是一个非常有趣的课题，因为 MptpA 是在宿主细胞感染期间表达的两种来自致病生物体结核分枝杆菌的蛋白酪氨酸磷酸酶（PTP）之一。在这里，我们将计算建模与溶液 NMR 光谱学相结合，发现 Q75 是一个变构位点。通过突变将 Q75 的极性侧链去除为亮氨酸，导致一系列事件发生，使酸性环重新定位到活性位点上方，并使催化天冬氨酸（D126）重新定位到有利于质子供体到底物的离去芳基的最佳位置，以及随后的硫膦酰中间物的水解。计算分析与动力学数据和 NMR 光谱学一致，表明 Q75L 突变体表现出增强的反应动力学，具有相似的底物结合亲和力。我们预计，我们的发现将激发进一步研究 MptpA 在感染的慢性状态下是否仍然失活，并作为结核分枝杆菌致病生命周期的一部分通过变构方式增加其活性的可能性。
