Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly

• Published in: Nature communications Vol. 13; no. 1; pp. 6381 - 18
• Main Authors: Fischer, Elyse S., Yu, Conny W. H., Hevler, Johannes F., McLaughlin, Stephen H., Maslen, Sarah L., Heck, Albert J. R., Freund, Stefan M. V., Barford, David
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 26.10.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 101/28; 101/6; 631/45/612/1223; 631/535/1258/1259; 631/535/878; 631/57; 82/58; 82/83; Anaphase; Anaphase-promoting complex; Assembly; BUBR1 protein; Catalysis; Cdc20 Proteins - metabolism; Cell Cycle Proteins - metabolism; Conversion; Dimerization; Humanities and Social Sciences; Kinases; Kinetochores; Kinetochores - metabolism; M Phase Cell Cycle Checkpoints; Mad2 Proteins - metabolism; Mitosis; multidisciplinary; Phosphorylation; Scaffolds; Science; Science (multidisciplinary); Spindle Apparatus - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-34058-2

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. Formation of the mitotic checkpoint complex (MCC) is catalysed by a phosphorylation-dependent scaffold. This work provides structural details of how a tripartite Mad1:Bub1:Cdc20 complex presents Cdc20 to Mad2, triggering open-to-closed conversion of Mad2 to assemble the MCC.