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

During mitosis, unattached kinetochores trigger the spindle assembly checkpoint by promoting the 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 remodeling of the N‐terminal β1 strand and C‐terminal β7/β8 hairpin. In vitro, the Mad2‐interaction motif (MIM) of Cdc20 (Cdc20 ^MIM ) triggers the rapid conversion of O‐Mad2 to C‐Mad2, effectively removing the kinetic barrier for MCC assembly. How Cdc20 ^MIM directly induces Mad2 conversion remains unclear. In this study, we demonstrate that the Cdc20 ^MIM ‐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 Cdc20 ^MIM . Conversion is initiated by the β7/β8 hairpin of O‐Mad2 transiently unfolding to expose a nascent Cdc20 ^MIM ‐binding site. Engagement of Cdc20 ^MIM to this site promotes the release of the β1 strand. We propose that initial conformational changes of the β7/β8 hairpin allow binding of Cdc20 ^MIM to a transient intermediate state of Mad2, thereby lowering the kinetic barrier to Mad2 conversion.