A new Mad2-interacting domain of Cdc20 is critical for the function of Mad2-Cdc20 complex in the spindle assembly checkpoint.
Interaction between Mad2 and Cdc20 (cell division cycle 20) is a key event during spindle assembly checkpoint activation. In the past, an N-terminal peptide containing amino acid residues 111-150 of Cdc20 was shown to bind Mad2 much better than the full-length Cdc20 protein. Using co-localization, co-immunoprecipitation and peptide inhibition analysis with different deletion mutants of Cdc20, we identified another Mad2-binding domain on Cdc20 from amino acids 342-355 within the WD repeat region. An intervening region between these two domains interferes with its Mad2 binding when present individually with any of these two Mad2-binding sites. We suggest that these three domains together determine the overall strength of Mad2 binding with Cdc20. Functional analysis suggests that an optimum Mad2 binding efficiency of Cdc20 is required during checkpoint arrest and release. Further, we have identified a unique polyhistidine motif with metal binding property adjacent to this second binding domain that may be important for maintaining the overall conformation of Cdc20 for its binding to Mad2.
Mondal
Gourish
G
Human Genetics and Genomics Division, Indian Institute of Chemical Biology, Kolkata-700 032, India.
Baral
Rathindra N
RN
Roychoudhury
Susanta
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D064246:Mad2 Proteins; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D008840:Microfilament Proteins; D012097:Repressor Proteins; C118067:WDR1 protein, human; C577488:CDC20 protein, human; C033223:polyhistidine; D006639:Histidine; D009532:Nickel