Chaperonins Hsp60s are required for cellular vitality by assisting protein folding in an ATP-dependent mechanism. Although conserved, the human mitochondrial mHsp60 exhibits molecular characteristics distinct from the GroEL, with different conformational assembly and higher subunit association dynamics, suggesting a different mechanism. We previously found that the pathological mutant mHsp60 exhibits enhanced subunit association stability and ATPase activity. To provide structural explanations for the V72I mutational effects, here we determined a cryo-EM structure of mHsp60. Our structural analysis combined with molecular dynamic simulations showed mHsp60 with increased inter-subunit interface, binding free energy, and dissociation force, all contributing to its enhanced subunit association stability. The gate to the nucleotide-binding (NB) site in mHsp60 mimicked the open conformation in the nucleotide-bound state with an additional open channel leading to the NB site, both promoting the mutant’s ATPase activity. Our studies highlight the importance of mHsp60’s characteristics in its biological function.

中文翻译：

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冷冻电镜结构和分子动力学模拟解释了病理伴侣蛋白突变体稳定性和 ATP 活性的增强

伴侣蛋白 Hsp60 通过以 ATP 依赖性机制协助蛋白质折叠来维持细胞活力。尽管保守，但人类线粒体 mHsp60 表现出与 GroEL 不同的分子特征，具有不同的构象组装和更高的亚基关联动力学，表明存在不同的机制。我们之前发现病理突变体 mHsp60 表现出增强的亚基关联稳定性和 ATPase 活性。为了提供 V72I 突变效应的结构解释，我们确定了 mHsp60 的冷冻电镜结构。我们的结构分析与分子动力学模拟相结合表明 mHsp60 具有增加的亚基间界面、结合自由能和解离力，所有这些都有助于其增强的亚基缔合稳定性。 mHsp60 中核苷酸结合 (NB) 位点的门模拟了核苷酸结合状态下的开放构象，并有一个额外的通向 NB 位点的开放通道，两者都促进了突变体的 ATP 酶活性。我们的研究强调了 mHsp60 特性在其生物学功能中的重要性。