Ruprecht-Karls-Universität Heidelberg, Zentrum für Molekulare Biologie der Universität Heidelberg Heidelberg, Germany.
Front Mol Biosci. 2014 Jun 10;1:4. doi: 10.3389/fmolb.2014.00004. eCollection 2014.
The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90β (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre- or post-hydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.
Hsp90 家族的分子伴侣在所有真核细胞中都是必不可少的。它们协助许多不同的蛋白质（称为客户）进行晚期折叠步骤和成熟，这些蛋白质在序列或结构上没有关联。Hsp90 与其客户的相互作用似乎与一系列构象变化有关。使用氢交换质谱（HX-MS），我们研究了人 Hsp90β（hHsp90）和酵母 Hsp82（yHsp82）的结构动力学。我们发现，真核 Hsp90s 比以前研究的大肠杆菌同源物（EcHtpG）更具灵活性，并且核苷酸引起的变化更小。在存在共伴侣的情况下，yHsp82 中获得更稳定的构象。四肽重复（TPR）结构域蛋白 Cpr6 在 yHsp82 中引起的酰胺质子保护模式与以前研究的 TPR 结构域蛋白 Sti1 不同。在同时存在 Sti1 和 Cpr6 的情况下，观察到保护水平介于 Sti1 和 Cpr6 诱导的变化之间。令人惊讶的是，没有检测到同位素峰的双峰分布，这表明两种共伴侣以相似的方式影响 Hsp90 二聚体的两个亚基。共伴侣 Sba1 先前在晶体结构中与 Hsp90 的 ATP 水解活性构象结合，这使得无法区分 Sba1 通过稳定前水解或后水解步骤来抑制 Hsp90 的 ATP 酶活性的方式。我们的 HX-MS 实验现在表明，Sba1 结合导致 ATP 结合盖的保护，这表明它通过减缓产物释放来抑制 Hsp90 的 ATP 酶活性。这一假设通过单轮 ATP 酶测定得到了验证。总之，我们的数据表明，真核 Hsp90s 中构象状态之间的能量障碍要小于 EcHtpG，并且除了核苷酸之外，共伴侣对于稳定特定构象状态也是必要的。
