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

Abstract

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.

Keywords: Cdc20; HORMA domain; Mad2; NMR; cell cycle; metamorphic proteins; molecular dynamic simulations; spindle assembly checkpoint.