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Abstract:
The spindle assembly checkpoint (SAC) monitors chromosome attachment to
spindle microtubules. SAC proteins operate at kinetochores, scaffolds
mediating chromosome-microtubule attachment. The ubiquitous SAC
constituents Mad1 and Mad2 are recruited to kinetochores in
prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate
anaphase onset. Its function is counteracted by p31(comet) (formerly
CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2
(Open) to C-Mad2 (Closed). A Mad1-boundC-Mad2 template, to which O-Mad2
binds prior to being converted into Cdc20-bound C-Mad2, assists this
process. A molecular understanding of this prion-like property of Mad2
is missing. We characterized the molecular determinants of the
O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the
binding interface in terms of symmetric and asymmetric components.
Mutation of individual interface residues abrogates the SAC in
Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that
O-Mad2 undergoes a major conformational rearrangement upon binding
C-Mad2, suggesting that dimerization facilitates the structural
conversion of O-Mad2 required to bind Cdc20. We also show that the
negative effects of p31(comet) on the SAC are based on its competition
with O-Mad2 for C-Mad2 binding.