Mechanistic insight into TRIP13-catalyzed Mad2 structural transition and spindle checkpoint silencing.

The spindle checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the checkpoint protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2-Cdc20 is incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding protein p31^comet and the ATPase TRIP13 promote MCC disassembly and checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31^comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31^comet-Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31^comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13-p31^comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region.

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