The Mad2 spindle checkpoint protein has two distinct natively folded states.
The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/C(Cdc20). We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.
Luo
Xuelian
X
Department of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Tang
Zhanyun
Z
Xia
Guohong
G
Wassmann
Katja
K
Matsumoto
Tomohiro
T
Rizo
Josep
J
Yu
Hongtao
H
D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D016213:Cyclins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D064246:Mad2 Proteins; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins; D015183:Restriction Mapping; D008941:Spindle Apparatus / Q000235:genetics; D014162:Transfection
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human