Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques
Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques
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DOI：
10.1073/pnas.1916030116
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发表时间：
2020-01-07
影响因子：
11.1
通讯作者：
Goldfarb, Daniella
中科院分区：
文献类型：
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作者：
Giannoulis, Angeliki;Feintuch, Akiva;Goldfarb, Daniella
Hsp90在细胞内稳态中起着重要作用，它通过协助多种细胞的折叠和成熟。它是一种同型二聚体，通过atp水解偶联构象变化起作用。Hsp90在没有伴侣的情况下的构象周期目前被假设为载脂蛋白Hsp90，是一个“开放”/“封闭”构象的集合。在ATP结合后，Hsp90采用一种活性的ATP结合封闭构象，其中包含ATP结合位点的n端结构域紧密接触。然而，对于adp结合的Hsp90的构象尚未达成共识，这被认为对患者的释放很重要。在这项工作中，我们利用电子顺磁共振（EPR）技术，特别是双电子-电子共振（DEER）距离测量，跟踪了酵母Hsp90在冷冻溶液中ATP水解不同阶段的构象状态。使用刚性Gd（III）自旋标记，我们发现C结构域在所有水解状态下都具有相同的距离分布。然后，我们用顺磁性的Mn（II）取代atp酶Mg（II）辅因子，利用超精细光谱跟踪水解状态，并通过Mn(II)-Mn(II) DEER测量n域间距离分布。Mn（II）自旋标记的点特征允许我们解析2种不同的封闭状态：atp结合（预水解）的距离分布最大为4.3 nm，其变宽和缩短，将adp结合状态（后水解）的平均值移至3.8 nm。这为Hsp90在溶液中的第二封闭构象状态提供了实验证据，称为“致密”。最后，通过添加钒酸盐捕获的所谓高能态，在结构上与水解后态相似。
Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact" Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.