Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, 23298, USA.
Department of Chemistry, University of Virginia, Charlottesville, VA, 22908, USA.
Nat Commun. 2017 Oct 31;8(1):1201. doi: 10.1038/s41467-017-01310-z.
Cellular protein homeostasis depends on heat shock proteins 70 kDa (Hsp70s), a class of ubiquitous and highly conserved molecular chaperone. Key to the chaperone activity is an ATP-induced allosteric regulation of polypeptide substrate binding and release. To illuminate the molecular mechanism of this allosteric coupling, here we present a novel crystal structure of an intact human BiP, an essential Hsp70 in ER, in an ATP-bound state. Strikingly, the polypeptide-binding pocket is completely closed, seemingly excluding any substrate binding. Our FRET, biochemical and EPR analysis suggests that this fully closed conformation is the major conformation for the ATP-bound state in solution, providing evidence for an active release of bound polypeptide substrates following ATP binding. The Hsp40 co-chaperone converts this fully closed conformation to an open conformation to initiate productive substrate binding. Taken together, this study provided a mechanistic understanding of the dynamic nature of the polypeptide-binding pocket in the Hsp70 chaperone cycle.
细胞蛋白质稳态依赖于热休克蛋白 70kDa（Hsp70s），这是一类普遍存在且高度保守的分子伴侣。伴侣活性的关键是多肽底物结合和释放的 ATP 诱导变构调节。为了阐明这种变构偶联的分子机制，我们在此呈现了一个完整的人 BiP（内质网中必需的 Hsp70）在 ATP 结合状态下的新型晶体结构。引人注目的是，多肽结合口袋完全关闭，似乎排除了任何底物结合。我们的 FRET、生化和 EPR 分析表明，这种完全封闭的构象是溶液中 ATP 结合状态的主要构象，为 ATP 结合后结合的多肽底物的主动释放提供了证据。Hsp40 共伴侣将这种完全封闭的构象转化为开放构象，从而启动有效的底物结合。总之，这项研究提供了对 Hsp70 伴侣循环中多肽结合口袋的动态性质的机制理解。
