Biophysics, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Molecular Genetics of Ageing, Max Planck Institute for Biology of Ageing, Cologne, 50931, Germany.
Nat Commun. 2023 Mar 18;14(1):1529. doi: 10.1038/s41467-023-37235-z.
The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.
纺锤体组装检查点（SAC）通过产生一种称为有丝分裂检查点复合物（MCC）的效应分子来保护基因组在细胞分裂过程中的稳定性。MCC 由两个亚基组成：BUBR1:BUB3 和 CDC20:MAD2，而 CDC20:MAD2 的形成是 MCC 组装的限速步骤。最近的研究表明，CDC20 与 SAC 蛋白 MAD1 和 BUB1 的协同结合显著加速了 CDC20:MAD2 的形成。然而，这种加速的分子基础尚不完全清楚。在这里，我们证明了 MAD1 在 C 端附近保守铰链处的结构灵活性对于催化 MCC 组装至关重要。这种 MAD1 铰链使 MAD1:MAD2 复合物能够在体内形成折叠构象。重要的是，铰链的截断会降低体外 MCC 组装和体内 SAC 信号转导的速率。相反，保留铰链灵活性的突变保留了 SAC 信号转导，表明铰链的结构灵活性，而不是特定的氨基酸序列，对于 SAC 信号转导很重要。我们将这些观察结果总结为“编织模型”，解释了 MAD1:MAD2 的折叠构象如何促进 CDC20:MAD2 的组装。
