Determinants of conformational dimerization of Mad2 and its inhibition by p31comet.
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 p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-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 p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.
Mapelli
Marina
M
Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.
Filipp
Fabian V
FV
Rancati
Giulia
G
Massimiliano
Lucia
L
Nezi
Luigi
L
Stier
Gunter
G
Hagan
Robert S
RS
Confalonieri
Stefano
S
Piatti
Simonetta
S
Sattler
Michael
M
Musacchio
Andrea
A
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D019281:Dimerization; D006801:Humans; D018386:Kinetochores; D064246:Mad2 Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins; D011487:Protein Conformation; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; C470309:MAD2L1BP protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human