Bachochin Max J, McGuire Kelly L, Cook Brian D, Ye Qiaozhen, Silletti Steve, Corbett Kevin D, Komives Elizabeth A, Herzik Mark A
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, 92093 USA.
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, 92093 USA.
bioRxiv. 2025 Jul 23:2025.07.19.665690. doi: 10.1101/2025.07.19.665690.
Tom70 mediates mitochondrial protein import by coordinating the transfer of cytosolic preproteins from Hsp70/Hsp90 to the translocase of the outer membrane (TOM) complex. In humans, the cytosolic domain of Tom70 (Tom70c) is entirely -helical and comprises modular TPR motifs divided into an N-terminal chaperone-binding domain (NTD) and a C-terminal preprotein-binding domain (CTD). However, the mechanisms linking these functional regions remain poorly understood. Here, we present the 2.04 Å crystal structure of unliganded Tom70c, revealing two distinct conformations - open and closed - within the asymmetric unit. These states are stabilized in part by interdomain crystal contacts and are supported in solution by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Principal component and dynamical network analyses reveal a continuum of motion linking the NTD and CTD via key structural elements, notably residues in helices 7, 8, and 25. Engagement of the CTD by the viral protein Orf9b interrupts this network, stabilizing a partially-closed intermediate conformation and dampening dynamics at distal NTD sites. Collectively, our findings lay the groundwork for understanding Tom70 allostery and provide a framework for dissecting its mechanistic roles in chaperone engagement, mitochondrial import, and viral subversion.
Tom70通过协调胞质前体蛋白从Hsp70/Hsp90到外膜转位酶（TOM）复合物的转移来介导线粒体蛋白导入。在人类中，Tom70的胞质结构域（Tom70c）完全由α螺旋组成，包含模块化的TPR基序，分为N端伴侣结合结构域（NTD）和C端前体蛋白结合结构域（CTD）。然而，连接这些功能区域的机制仍知之甚少。在这里，我们展示了未结合配体的Tom70c的2.04 Å晶体结构，揭示了不对称单元内的两种不同构象——开放和封闭。这些状态部分通过结构域间的晶体接触得以稳定，并在溶液中得到氢-氘交换质谱（HDX-MS）和分子动力学（MD）模拟的支持。主成分分析和动力学网络分析揭示了通过关键结构元件（特别是螺旋7、8和25中的残基）连接NTD和CTD的连续运动。病毒蛋白Orf9b与CTD的结合中断了这个网络，稳定了部分封闭的中间构象，并抑制了远端NTD位点的动力学。总的来说，我们的研究结果为理解Tom70的变构作用奠定了基础，并为剖析其在伴侣结合、线粒体导入和病毒颠覆中的机制作用提供了框架。
