Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia, USA.
Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia, USA; Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China.
J Biol Chem. 2021 Sep;297(3):101082. doi: 10.1016/j.jbc.2021.101082. Epub 2021 Aug 14.
Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the "holdase" activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain-domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDβ interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDβ contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the "holdase" activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.
热休克蛋白 110kDa（Hsp110s）是一类独特的分子伴侣，对于维持蛋白质的内稳态至关重要。Hsp110s 具有很强的分子伴侣活性，可防止蛋白质聚集（“持家酶”活性），并且还作为 Hsp70 伴侣的主要核苷酸交换因子（NEF）发挥作用。Hsp110s 包含两个功能域：核苷酸结合域（NBD）和底物结合域（SBD）。ATP 结合对于 Hsp110 的功能至关重要，并且导致 NBD 和 SBD 之间的紧密接触。然而，这种 ATP 诱导的变构偶联的分子机制仍未得到很好的定义。在这项研究中，我们对白色念珠菌中唯一的 Hsp110 Msi3 进行了生化分析，通过影响域-域界面的三个突变来剖析 Hsp110s 的独特变构偶联。所有突变均消除了 Msi3 的体内和体外功能。虽然所有突变体的 ATP 结合状态均受到破坏，但只有 NBD-SBDβ 界面的突变显示出显著的 ATP 酶活性，这表明全长 Hsp110s 具有主要被 NBD-SBDβ 相互作用抑制的 ATP 酶。此外，高亲和力的 ATP 结合出乎意料地似乎需要这些 NBD-SBD 接触。值得注意的是，所有测试的突变体的“持家酶”活性基本完整，而 NEF 活性大多受损，尽管这两种活性均严格依赖于 ATP 结合状态，表明这两种活性有不同的要求。稳定的肽底物与 Msi3 的结合导致 NBD-SBD 接触的解离，并损害了与 Hsp70 的相互作用。总而言之，我们的数据表明，Hsp110s 中异常强的 NBD-SBD 接触决定了其独特的变构偶联和生化活性。
