Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation
Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation
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DOI：
10.1002/pro.4770
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发表时间：
2023-10-01
期刊：
影响因子：
8
通讯作者：
Saxena，Sunil
中科院分区：
文献类型：
--
作者：
Bogetti，Xiaowei;Bogetti，Anthony;Saxena，Sunil
人谷胱甘肽S-转移酶A1 - 1（hGSTA 1 - 1）是一种同源二聚体解毒酶，其催化活性取决于每个单体中关键C-末端螺旋α9的构象动力学。然而，两个单体在结合底物时如何相互作用的结构细节尚未得到很好的理解，hGSTA 1 - 1同二聚体的无配体状态的结构尚未得到解决。在这里，我们使用电子顺磁共振（EPR）距离测量和加权系综（WE）模拟的组合来表征原子水平上无配体状态的构象系综。EPR测量揭示了在无配体状态下的一对Cu（II）标记之间的宽距离分布，其随着配体浓度的增加而逐渐移动和变窄。这些位移表明配体结合后两个α9螺旋的相对位置发生了变化。WE模拟为酶的交替状态之间的秒-时间尺度转变生成了无偏路径，导致生成无配体状态的原子详细结构。值得注意的是，模拟提供了hGSTA 1 - 1单体之间的负协同性的直接观察，这涉及每个单体中α9的互斥对接作为活性位点上的盖子。我们确定了导致这种负协同性的残基之间的关键相互作用。负协同性可能是hGSTA 1 - 1与多种毒性底物相互作用及其随后中和所必需的。更广泛地说，这项工作证明了将EPR距离与WE罕见事件采样策略相结合的能力，以获得原子水平上蛋白质功能的机制信息。
The catalytic activity of human glutathione S‐transferase A1‐1 (hGSTA1‐1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C‐terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand‐free state of the hGSTA1‐1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand‐free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand‐free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds‐timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand‐free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1‐1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1‐1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare‐events sampling strategy to gain mechanistic information on protein function at the atomic level.