Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly
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
(2022) Nature Communications, volume 13, issue 1
(Article)
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 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)
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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.
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Keywords: M Phase Cell Cycle Checkpoints, Cell Cycle Proteins/metabolism, Kinetochores/metabolism, Mitosis, Catalysis, Mad2 Proteins/metabolism, Spindle Apparatus/metabolism, Cdc20 Proteins/metabolism
ISSN: 2041-1723
Publisher: Nature Publishing Group
Note: Funding Information: E.S.F. was funded by a Gates Cambridge Scholarship. This work was funded by an MRC grant (MC_UP_1201/6) and a CRUK grant (C576/A14109) to D.B. The XL-MS experiments were supported by the Horizon 2020 INFRAIA project Epic-XS (Project 823839 to A.J.R.H.). We thank K. Yan and the MRC-LMB EM facility for helping with EM data collection. J. Grimmett and T. Darling for computing and J. Shi for help with insect cell expression. We acknowledge S. Tamara (University of Utrecht) for additional cross-linking mass spectrometry experiments and T. Dendooven for advice in SEC-SAXS analyses. We thank Ajit Jogelkar and Andrea Musacchio for discussing unpublished work and for useful discussions regarding Mad1 fold-over and Mad2 conversion, and Andrea Musacchio for helpful comments on this manuscript. Some of the NMR studies were supported by the Francis Crick Institute through access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). The authors also thank the B21 SAXS beamline at the Diamond Light Source, Oxfordshire, for the measurements of mail-in SAXS samples. Funding Information: E.S.F. was funded by a Gates Cambridge Scholarship. This work was funded by an MRC grant (MC_UP_1201/6) and a CRUK grant (C576/A14109) to D.B. The XL-MS experiments were supported by the Horizon 2020 INFRAIA project Epic-XS (Project 823839 to A.J.R.H.). We thank K. Yan and the MRC-LMB EM facility for helping with EM data collection. J. Grimmett and T. Darling for computing and J. Shi for help with insect cell expression. We acknowledge S. Tamara (University of Utrecht) for additional cross-linking mass spectrometry experiments and T. Dendooven for advice in SEC-SAXS analyses. We thank Ajit Jogelkar and Andrea Musacchio for discussing unpublished work and for useful discussions regarding Mad1 fold-over and Mad2 conversion, and Andrea Musacchio for helpful comments on this manuscript. Some of the NMR studies were supported by the Francis Crick Institute through access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). The authors also thank the B21 SAXS beamline at the Diamond Light Source, Oxfordshire, for the measurements of mail-in SAXS samples. Publisher Copyright: © 2022, The Author(s).
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