title: Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer.
abstract: In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C(Cdc20) inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2-C-Mad2 (C-C) or asymmetric O-Mad2-C-Mad2 (O-C) dimers. We also report the crystal structure of the symmetric C-C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells. Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
authors.forename: Maojun
authors.initials: M
authors.lastname: Yang
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Chyong-Jy
authors.initials: CJ
authors.lastname: Liu
authors.forename: Diana R
authors.initials: DR
authors.lastname: Tomchick
authors.forename: Mischa
authors.initials: M
authors.lastname: Machius
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
authors.forename: Hongtao
authors.initials: H
authors.lastname: Yu
authors.forename: Xuelian
authors.initials: X
authors.lastname: Luo
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D064246:Mad2 Proteins; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D050505:Mutant Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human