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

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.