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bioRxiv · 10.1101/2022.05.16.492081

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

Fischer, E. S.·Yu, C. W.·Hevler, J. F.·McLaughlin, S. H.·Maslen, S. L.·Heck, A. J.·Freund, S. M.·Barford, D.
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Abstract

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 is a complex of Mad2, Cdc20 and BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a catalytic 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 conversion of O-Mad2 to C-Mad2 and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. In this study 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 (Mad1CTD). We additionally identified a folded state of the Mad1:Mad2 complex that suggests how the Cdc20:Mad1CTD interaction brings the Mad2- interacting motif (MIM) of Cdc20 (Cdc20MIM) into close proximity with O-Mad2. We also show that Cdc20MIM is sufficient to catalyse Mad2 conversion and its entrapment by the Mad2 safety- belt. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

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Fischer, E. S., Yu, C. W., Hevler, J. F., McLaughlin, S. H., Maslen, S. L., Heck, A. J., Freund, S. M., Barford, D.. 2022-05-16. Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly. https://doi.org/10.1101/2022.05.16.492081

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