O'Connor Michael S, Konovalov Kirill A, Duvall Josephine L, Jang Jinoh, Lao Yichong, Cavagnero Silvia, Huang Xuhui
Biophysics Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin.
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin.
Biophys J. 2025 Aug 26. doi: 10.1016/j.bpj.2025.08.022.
Heat shock protein 70 (Hsp70) is a molecular chaperone that plays a key role in cellular processes by assisting protein folding and preventing aggregation. During client-protein folding, Hsp70 undergoes an ATP-dependent chaperone cycle involving the opening and closing of a flexible lid. Although the open-lid and closed-lid states of Hsp70 have been studied extensively, the specific role of the lid upon its interaction with client proteins remains unclear. In this study, we generated a Markov state model from coarse-grained molecular dynamics (MD) simulations of Hsp70 spanning from open-lid to closed-lid states and sampling a flexible lid-domain conformational ensemble. Starting from metastable Hsp70 conformations with varying degrees of lid opening, we performed nuclear magnetic resonance distance restraint-assisted all-atom MD simulations in explicit solvent to investigate the folding of the SH3 client protein bound to nucleotide-free Hsp70. All-atom MD simulations were performed with SH3 bound to and released from Hsp70, with nuclear magnetic resonance restraints applied to guide SH3 folding. Our results show that SH3 folds more effectively after having sampled conformational space within the closed-lid state of Hsp70. Further analysis reveals that the closed-lid state of Hsp70 interacts with SH3 via specific and highly conserved nonpolar residues, preventing the nonnative hydrophobic collapse of the SH3 client upon release from the chaperone. This study provides insights into specific atomic-level interactions that can be targeted by future experiments to better understand the molecular mechanism of Hsp70-assisted protein folding.
热休克蛋白70（Hsp70）是一种分子伴侣，通过协助蛋白质折叠和防止聚集在细胞过程中发挥关键作用。在底物蛋白折叠过程中，Hsp70经历一个依赖ATP的伴侣循环，涉及一个柔性盖子的打开和关闭。尽管对Hsp70的开放盖子和关闭盖子状态进行了广泛研究，但盖子与底物蛋白相互作用的具体作用仍不清楚。在本研究中，我们从Hsp70从开放盖子状态到关闭盖子状态的粗粒度分子动力学（MD）模拟中生成了一个马尔可夫状态模型，并对一个柔性盖子结构域构象集合进行了采样。从具有不同程度盖子打开的亚稳态Hsp70构象开始，我们在明确溶剂中进行了核磁共振距离约束辅助的全原子MD模拟，以研究与无核苷酸Hsp70结合的SH3底物蛋白的折叠。对SH3与Hsp70结合和释放的情况进行了全原子MD模拟，并应用核磁共振约束来指导SH3折叠。我们的结果表明，SH3在对Hsp70关闭盖子状态内的构象空间进行采样后折叠得更有效。进一步分析表明，Hsp70的关闭盖子状态通过特定且高度保守的非极性残基与SH3相互作用，防止SH3底物从伴侣蛋白释放后发生非天然疏水塌缩。本研究为未来实验可针对的特定原子水平相互作用提供了见解，以更好地理解Hsp70辅助蛋白质折叠的分子机制。
