MRC Laboratory of Molecular Biology, Cambridge, UK.
Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.
Nature. 2018 Jul;559(7713):274-278. doi: 10.1038/s41586-018-0281-1. Epub 2018 Jul 4.
The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome). Unattached kinetochores control MCC assembly by catalysing a change in the topology of the β-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31, an SAC antagonist, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2). Here, we present a mechanism that explains how TRIP13-p31 disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31-C-MAD2-CDC20 complex reveal that p31 recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2 within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2 to push upwards on, and simultaneously rotate, the globular domains of the p31-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 β-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.
有丝分裂过程中基因组稳定性的维持是通过纺锤体组装检查点（SAC）来协调的，其效应物是有丝分裂检查点复合物（MCC），它是后期促进复合物（APC/C，也称为周期蛋白体）的抑制剂。未附着的动粒通过催化 MAD2（MCC 亚基之一）β-片层拓扑结构的变化来控制 MCC 的组装，从而产生活性闭合 MAD2（C-MAD2）构象。游离 MCC 的解组装对于 SAC 失活和染色体分离是必需的，这是一个由 AAA+ATP 酶 TRIP13 驱动的 ATP 依赖性过程。与 SAC 拮抗剂 p31 结合，TRIP13 将 C-MAD2 重塑为无活性的开放 MAD2（O-MAD2）。在这里，我们提出了一个解释 TRIP13-p31 如何拆解 MCC 的机制。TRIP13-p31-C-MAD2-CDC20 复合物的冷冻电子显微镜结构揭示，p31 将 C-MAD2 招募到 TRIP13 六聚体环上的特定位置，将 C-MAD2 的 N 端（MAD2）定位到插入 TRIP13 的轴向孔中，并扭曲 TRIP13 环以启动重塑。分子建模表明，通过在其轴向孔中夹住 MAD2，TRIP13 将其六聚体环沿着 MAD2 的连续 ATP 驱动易位与同时向上推动和旋转 p31-C-MAD2 复合物的球状结构域相耦合。这解开了 C-MAD2 的αA 螺旋的一个区域，该区域对于稳定 C-MAD2 的β-片层是必需的，从而使 C-MAD2 不稳定，有利于 O-MAD2，并使 MAD2 与 p31 分离。我们的研究提供了有关特定底物如何通过衔接蛋白被招募到 AAA+ATP 酶的见解，并提出了一个模型，说明 AAA+ATP 酶的轴向孔中的易位如何与蛋白质重塑相耦合。
