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  • The EMBO Journal (2006) 25, 1273 - 1284
  • doi:10.1038/sj.emboj.7601033

Published online: 9 March 2006

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

Marina Mapelli1,2, Fabian V Filipp3, Giulia Rancati4, Lucia Massimiliano1,2, Luigi Nezi1, Gunter Stier3, Robert S Hagan5, Stefano Confalonieri2, Simonetta Piatti4, Michael Sattler3 and Andrea Musacchio1,2

  1. Department of Experimental Oncology, European Institute of Oncology, Milan, Italy
  2. The FIRC Institute of Molecular Oncology Foundation, Milan, Italy
  3. European Molecular Biology Laboratory, Heidelberg, Germany
  4. Dipartimento di Biotecnologie e Bioscienze, Universita' di Milano-Bicocca, Milano, Italy
  5. Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA, USA

Correspondence to:

Andrea Musacchio, Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, Milan 20141, Italy. Tel.: +39 02 5748 9871; Fax: +39 02 5748 9851; E-mail: andrea.musacchio@ifom-ieo-campus.it

Received 2 December 2005; Accepted 9 February 2006

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.

  • Keywords:

    • Cdc20,
    • centromere,
    • kinetochore,
    • mitosis,
    • mitotic arrest deficient,
    • spindle assembly checkpoint