Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer.
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.
Yang
Maojun
M
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
Li
Bing
B
Liu
Chyong-Jy
CJ
Tomchick
Diana R
DR
Machius
Mischa
M
Rizo
Josep
J
Yu
Hongtao
H
Luo
Xuelian
X
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
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