In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1^CTD). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1^CTD interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

为了响应有丝分裂中不正确的动粒-微管附着，纺锤体组装检查点（SAC）组装有丝分裂检查点复合物（MCC）以抑制后期促进复合物/环体，从而延迟进入后期。 MCC 包括 Mad2:Cdc20:BubR1:Bub3。它的组装是由 Mad1:Mad2 平台上的独立着丝粒催化的。 Mad1 结合的封闭型 Mad2 (C-Mad2) 通过自二聚化招募开放型 Mad2 (O-Mad2)。这种相互作用与 Mps1 激酶介导的 Bub1 和 Mad1 磷酸化相结合，加速了 MCC 组装，该过程需要 O-Mad2 向 C-Mad2 转化并同时结合 Cdc20。 Mad1 磷酸化如何催化 MCC 组装尚不清楚。在这里，我们表征了 Mad1 的 Mps1 磷酸化，并获得了磷酸化特异性 Mad1:Cdc20 相互作用的结构见解。这种相互作用与 Bub1 和 Mad1 的 Mps1 磷酸化依赖性关联一起，在 Mad1 的 C 末端结构域上生成 Bub1 和 Cdc20 的三联组装 (Mad1 ^CTD )。我们还确定了 Mad1:Mad2 的灵活性，这表明 Cdc20:Mad1 ^CTD相互作用如何使 Cdc20 的 Mad2 相互作用基序 (MIM) 接近 O-Mad2。因此，MCC 组装支架的 Mps1 依赖性形成可将 Cdc20 MIM 定位和定向到 O-Mad2 附近，从而催化 C-Mad2:Cdc20 的形成。