A molecular device for the redox quality control of GroEL/ES substrates

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal alpha-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.Hsp60伴侣蛋白（Hsp60 chaperonins）及其Hsp10辅助因子（Hsp10 cofactors）可在所有活细胞中协助蛋白质折叠，是分子伴侣（molecular chaperones）的经典范例。尽管针对其结构与作用机制已开展了大量研究，但这类伴侣蛋白如何促进蛋白质折叠的关键问题仍未得到解答。在此，我们报道细菌来源的Hsp60伴侣蛋白GroEL可与伴侣氧化还原蛋白（chaperedoxin）CnoX形成稳定且具有功能相关性的复合物——CnoX是一种兼具分子伴侣与氧化还原功能的蛋白质。GroES（即Hsp10辅助因子）与GroEL的结合会诱导CnoX的释放。冷冻电子显微镜术（cryo-electron microscopy）为GroEL-CnoX复合物提供了关键结构信息，结果显示CnoX通过一段高度保守的C端α螺旋结合于GroEL的底物结合位点外侧。此外，我们还鉴定出了结合于GroEL的CnoX与GroEL底物形成混合二硫键的复合物，这表明CnoX可能作为GroEL的氧化还原质量控制辅助模块发挥功能。与CnoX具有相似结构特征的蛋白质存在于真核生物中，这提示Hsp60分子伴侣的这类辅助模块在进化过程中具有保守性。