From the Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453.
From the Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453
J Biol Chem. 2019 Apr 19;294(16):6387-6396. doi: 10.1074/jbc.RA118.007050. Epub 2019 Feb 20.
Hsp70 and Hsp90 chaperones are critical for protein quality control in the cytosol, whereas organelle-specific Hsp70/Hsp90 paralogs provide similar protection for mitochondria and the endoplasmic reticulum (ER). Cytosolic Hsp70/Hsp90 can operate sequentially with Hsp90 selectively associating with Hsp70 after Hsp70 is bound to a client protein. This observation has long suggested that Hsp90 could have a preference for interacting with clients at their later stages of folding. However, recent work has shown that cytosolic Hsp70/Hsp90 can directly interact even in the absence of a client, which opens up an alternative possibility that the ordered interactions of Hsp70/Hsp90 with clients could be a consequence of regulated changes in the direct interactions between Hsp70 and Hsp90. However, it is unknown how such regulation could occur mechanistically. Here, we find that the ER Hsp70/Hsp90 (BiP/Grp94) can form a direct complex in the absence of a client. Importantly, the direct interaction between BiP and Grp94 is nucleotide-specific, with BiP and Grp94 having higher affinity under ADP conditions and lower affinity under ATP conditions. We show that this nucleotide-specific association between BiP and Grp94 is largely due to the conformation of BiP. When BiP is in the ATP conformation its substrate-binding domain blocks Grp94; in contrast, Grp94 can readily associate with the ADP conformation of BiP, which represents the client-bound state of BiP. Our observations provide a mechanism for the sequential involvement of BiP and Grp94 in client folding where the conformation of BiP provides the signal for the subsequent recruitment of Grp94.
热休克蛋白 70（Hsp70）和热休克蛋白 90（Hsp90）伴侣对于细胞质中的蛋白质质量控制至关重要，而细胞器特异性的 Hsp70/Hsp90 同工蛋白则为线粒体和内质网（ER）提供类似的保护。细胞质中的 Hsp70/Hsp90 可以顺序作用，Hsp70 与客户蛋白结合后，Hsp90 选择性地与 Hsp70 结合。这一观察结果长期以来表明，Hsp90 可能更倾向于与处于折叠后期的客户蛋白相互作用。然而，最近的研究表明，即使在没有客户蛋白的情况下，细胞质中的 Hsp70/Hsp90 也可以直接相互作用，这为一种替代可能性开辟了道路，即 Hsp70/Hsp90 与客户蛋白的有序相互作用可能是 Hsp70 和 Hsp90 之间直接相互作用受调控变化的结果。然而，目前尚不清楚这种调控是如何发生的。在这里，我们发现 ER 中的 Hsp70/Hsp90（BIP/Grp94）可以在没有客户蛋白的情况下形成直接复合物。重要的是，BIP 和 Grp94 之间的直接相互作用是核苷酸特异性的，在 ADP 条件下，BIP 和 Grp94 的亲和力更高，而在 ATP 条件下，BIP 和 Grp94 的亲和力更低。我们表明，BIP 和 Grp94 之间的这种核苷酸特异性结合主要归因于 BIP 的构象。当 BIP 处于 ATP 构象时，其底物结合结构域会阻止 Grp94；相反，Grp94 可以很容易地与 BIP 的 ADP 构象结合，这代表了 BIP 与客户蛋白结合的状态。我们的观察结果为 BIP 和 Grp94 参与客户蛋白折叠的顺序参与提供了一种机制，其中 BIP 的构象为随后 Grp94 的募集提供了信号。
