Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation.

ClpB, a bacterial Hsp100, is a ring-shaped AAA+ chaperone that can reactivate aggregated proteins in cooperation with DnaK, a bacterial Hsp70, and its co-factors. ClpB subunits comprise two AAA+ modules with an interstitial rod-shaped M-domain. The M-domain regulates ClpB ATPase activity and interacts directly with the DnaK nucleotide-binding domain (NBD). Here, to clarify how these functions contribute to the disaggregation process, we constructed ClpB, DnaK, and aggregated YFP fusion proteins in various combinations. Notably, i) DnaK activates ClpB only when the DnaK substrate-binding domain (SBD) is in the closed conformation, affording high DnaK-peptide affinity; ii) although NBD alone can activate ClpB, SBD is required for disaggregation; and iii) tethering aggregated proteins to the activated ClpB obviates SBD requirements. These results indicate that DnaK activates ClpB only when the SBD tightly holds aggregated proteins adjacent to ClpB for effective disaggregation.

ClpB是一种细菌热休克蛋白100（Hsp100），是一种环形的AAA +分子伴侣，它能够与细菌热休克蛋白70（Hsp70）DnaK及其辅因子协同作用，重新激活聚集的蛋白质。ClpB亚基包含两个AAA +模块以及一个间隙的杆状M结构域。M结构域调节ClpB的ATP酶活性，并与DnaK的核苷酸结合结构域（NBD）直接相互作用。在此，为了阐明这些功能如何对解聚过程起作用，我们构建了ClpB、DnaK以及聚集的黄色荧光蛋白（YFP）融合蛋白的多种组合。值得注意的是：i）只有当DnaK的底物结合结构域（SBD）处于闭合构象，即具有高的DnaK - 肽亲和力时，DnaK才能激活ClpB；ii）尽管单独的NBD能够激活ClpB，但解聚过程需要SBD；iii）将聚集的蛋白质连接到激活的ClpB上则无需SBD。这些结果表明，只有当SBD将聚集的蛋白质紧密地保持在ClpB附近以便有效解聚时，DnaK才能激活ClpB。