Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint

Abstract

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2–Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1–Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled‐coil to form two connected sub‐complexes with Mad2. The Mad2 C‐terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular ‘safety belts’. We show that Mad1 is a competitive inhibitor of the Mad2–Cdc20 complex, and propose that the Mad1–Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1–Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1–Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1–Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C‐terminal tail is required for ligand release from Mad2, and that the ‘safety belt’ mechanism may prolong the lifetime of Mad2–ligand complexes.

The spindle checkpoint, aneuploidy, and cancer

“…Both solution and crystal structures of Mad2 in association with this motif have been solved (Luo et al, 2002;Sironi et al, 2002). Mad1 binding to Mad2 induces the same conformational changes in Mad2 as does the Cdc20 peptide (Luo et al, 2002;Sironi et al, 2002). Thus, Cdc20 and Mad1 binding to Mad2 are mutually exclusive.…”

Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

“…It has been shown that Mad2, a protein whose interaction with the APC/C activator Cdc20 is essential for the mitotic checkpoint in vivo (10,11) and inhibits the activity of APC/C Cdc20 in vitro (12,13). Mad2 exists in two conformations, of which the closed conformer preferentially binds Cdc20 (14,15). Elegant models have been proposed according to which the activation of the checkpoint promotes binding of Cdc20 to the closed conformer of Mad2 and thus sequesters Cdc20 from APC/C (refs.…”

Modeling dual pathways for the metazoan spindle assembly checkpoint

“…Moreover, f luorescence recovery after photobleaching (FRAP) experiments have revealed that some of these proteins, including Mad2, BubR1, and Cdc20, turnover rapidly at unattached kinetochores (6,9). Furthermore, the available evidence suggests that Mad2 exists in two forms: open (O-Mad2) and closed (C-Mad2) (10,11), with the closed form adopted when bound to Cdc20. Production of C-Mad2-Cdc20 may be catalyzed by the kinetochorebound C-Mad2-Mad1 complex.…”