Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 761001, Israel.
Department of Plant Molecular Biology, Faculty of Biology and Medicine, University of Lausanne, CH-1015 Lausanne, Switzerland.
ACS Chem Biol. 2021 Apr 16;16(4):775-785. doi: 10.1021/acschembio.1c00156. Epub 2021 Mar 19.
ClpB is a tightly regulated AAA+ disaggregation machine. Each ClpB molecule is composed of a flexibly attached N-terminal domain (NTD), an essential middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains. The NTD is not well-characterized structurally and is commonly considered to serve as a dispensable substrate-binding domain. Here, we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB's NTD and reveal its unexpected autoinhibitory function. We find that the NTD fluctuates on the microsecond time scale, and these dynamics result in steric hindrance that limits the conformational space of the MD to restrict its tilting. This leads to significantly inhibited ATPase and disaggregation activities of ClpB, an effect that is alleviated upon binding of a substrate protein or the cochaperone DnaK. This entropic inhibition mechanism, which is mediated by ultrafast motions of the NTD and is not dependent on any strong interactions, might be common in related ATP-dependent proteases and other multidomain proteins to ensure their fast and reversible activation.
ClpB 是一种紧密调节的 AAA+解聚机器。每个 ClpB 分子由一个灵活连接的 N 端结构域（NTD）、一个通过倾斜激活机器的必需中间结构域（MD）和两个核苷酸结合结构域组成。NTD 在结构上没有很好的表征，通常被认为是一个可有可无的底物结合结构域。在这里，我们使用单分子 FRET 光谱学直接监测 ClpB 的 NTD 的实时动力学，并揭示其出人意料的自动抑制功能。我们发现 NTD 在微秒时间尺度上波动，这些动力学导致空间位阻，限制了 MD 的构象空间使其无法倾斜。这导致 ClpB 的 ATPase 和解聚活性受到显著抑制，而当结合底物蛋白或共伴侣 DnaK 时，这种抑制作用会得到缓解。这种由 NTD 的超快运动介导的熵抑制机制，不依赖于任何强相互作用，可能在相关的 ATP 依赖的蛋白酶和其他多结构域蛋白中很常见，以确保它们的快速和可逆激活。
