Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

Marina Mapelli, Fabian V. Filipp, Giulia Rancati, Lucia Massimiliano, Luigi Nezi, Gunter Stier, Robert S. Hagan, Stefano Confalonieri, Simonetta Piatti, Michael Sattler, Andrea Musacchio

Research output: Contribution to journalArticle

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-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.

Original languageEnglish
Pages (from-to)1273-1284
Number of pages12
JournalEMBO Journal
Volume25
Issue number6
DOIs
Publication statusPublished - Mar 22 2006

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Keywords

  • Cdc20
  • Centromere
  • Kinetochore
  • Mitosis
  • Mitotic arrest deficient
  • Spindle assembly checkpoint

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Mapelli, M., Filipp, F. V., Rancati, G., Massimiliano, L., Nezi, L., Stier, G., Hagan, R. S., Confalonieri, S., Piatti, S., Sattler, M., & Musacchio, A. (2006). Determinants of conformational dimerization of Mad2 and its inhibition by p31comet. EMBO Journal, 25(6), 1273-1284. https://doi.org/10.1038/sj.emboj.7601033