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bioRxiv · 10.1101/2024.03.03.583158

Molecular mechanism of Mad2 conformational conversion promoted by the Mad2-interaction motif of Cdc20

Yu, C. W. H.·Fischer, E. S.·Greener, J. G.·Yang, J.·Zhang, Z.·Freund, S. M. V.·Barford, D.
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Abstract

During mitosis, unattached kinetochores trigger the spindle assembly checkpoint by promoting assembly of the mitotic checkpoint complex, a heterotetramer comprising Mad2, Cdc20, BubR1 and Bub3. Critical to this process is the kinetochore-mediated catalysis of an intrinsically slow conformational conversion of Mad2 from an open (O-Mad2) inactive state to a closed (C-Mad2) active state bound to Cdc20. These Mad2 conformational changes involve substantial remodelling of the N-terminal {beta}1 strand and C-terminal {beta}7/{beta}8 hairpin. In vitro, the Mad2- interaction motif (MIM) of Cdc20 (Cdc20MIM) triggers rapid conversion of O- to C-Mad2, effectively removing the kinetic barrier for MCC assembly. How Cdc20MIM directly induces Mad2 conversion remains unclear. In this study we demonstrate that the Cdc20MIM-binding site is inaccessible in O-Mad2. Time-resolved NMR and molecular dynamics simulations show how Mad2 conversion involves sequential conformational changes of flexible structural elements in O-Mad2, orchestrated by Cdc20MIM. Conversion is initiated by the {beta}7/{beta}8 hairpin of O-Mad2 transiently unfolding to expose a nascent Cdc20MIM-binding site. Engagement of Cdc20MIM to this site promotes release of the {beta}1 strand. We propose that initial conformational changes of the {beta}7/{beta}8 hairpin allows binding of Cdc20MIM to a transient intermediate state of Mad2, thereby lowering the kinetic barrier to Mad2 conversion.

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Yu, C. W. H., Fischer, E. S., Greener, J. G., Yang, J., Zhang, Z., Freund, S. M. V., Barford, D.. 2024-03-03. Molecular mechanism of Mad2 conformational conversion promoted by the Mad2-interaction motif of Cdc20. https://doi.org/10.1101/2024.03.03.583158

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