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.

热休克蛋白60（Hsp60）伴侣蛋白及其Hsp10辅因子在所有活细胞中辅助蛋白质折叠，构成了分子伴侣的典范实例。尽管对其结构和机制进行了广泛的研究，但关于这些伴侣蛋白如何促进折叠的关键问题仍未得到解决。在本研究中，我们报告了细菌Hsp60伴侣蛋白GroEL与伴侣氧化还原酶CnoX形成一个稳定且功能相关的复合物，CnoX是一种兼具伴侣蛋白和氧化还原功能的蛋白质。GroES（Hsp10辅因子）与GroEL的结合导致CnoX的释放。冷冻电子显微镜技术为GroEL-CnoX复合物提供了关键的结构信息，显示CnoX通过一个高度保守的C端α螺旋在底物结合位点外与GroEL结合。此外，我们还鉴定了CnoX与GroEL结合形成的混合二硫键复合物，这表明CnoX可能作为GroEL的氧化还原质量控制插件发挥作用。存在于真核生物中与CnoX具有相似结构特征的蛋白质，表明Hsp60分子插件在进化过程中得到了保守。