Kinetic analysis of Mad2-Cdc20 formation: conformational changes in Mad2 are catalyzed by a C-Mad2-ligand complex.
Structural changes in the mitotic arrest deficient protein 2 (Mad2) have been proposed to be essential for spindle checkpoint function. Current models for checkpoint activation propose that a C-Mad2-Mad1 core complex at unattached kinetochores is required for the structural activation through a process involving the interaction of two Mad2 conformers: a closed conformer bound to Mad1 or Cdc20 and an open conformer unbound to these ligands. To gain a molecular understanding of the mechanisms that accelerate the structural transition between the open and closed Mad2 conformations, we constructed a unique in vitro homogeneous Mad2 activity assay that specifically reports C-Mad2-Cdc20 formation. Using this assay we were are able to directly establish that (a) O-Mad2 transforms into a C-Mad2-Cdc20 complex >300-fold slower than unliganded C-Mad2, (b) a stable C-Mad2-Mad1 core complex catalyzes the transformation of O-Mad2 into a Cdc20-bound C-Mad2 complex, (c) a C-Mad2-Cdc20 complex can promote its own transformation of O-Mad2 into a Cdc20-bound C-Mad2 complex, and (d) the binding interaction between unliganded C-Mad2 and Cdc20 cannot be catalyzed by a C-Mad2-Mad1 core complex. Our data are consistent with the "Mad2 template" catalytic model in which a C-Mad2 template facilitates the binding of O-Mad2 to Cdc20 and supports a mechanism of C-Mad2-Cdc20 formation away from Mad1 containing kinetochores. Furthermore, our unique homogeneous Mad2 assay could be translated into a screening platform to identify small molecule drug-like compounds that directly modulate C-Mad2-Cdc20 formation.
Lad
Latesh
L
Cytokinetics Inc, South San Francisco, California 94080, USA. llad@cytokinetics.com
Lichtsteiner
Serge
S
Hartman
James J
JJ
Wood
Kenneth W
KW
Sakowicz
Roman
R
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008024:Ligands; D064246:Mad2 Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D008941:Spindle Apparatus / Q000737:chemistry* / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D008024:Ligands; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; C577488:CDC20 protein, human