title: Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif.
abstract: CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4). The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein. We now show that the N-terminal coiled coil (residues 45-160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells. The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells. A protease-resistant domain (residues 27-168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This region is predicted to contain two smaller coiled-coil regions. The CtIP-(45-160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil. The two smaller domains, CtIP-(45-92) and CtIP-(93-160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide. The hydrodynamic radius of CtIP-(45-160) is the same as those of CtIP-(45-92) and CtIP-(93-160), implying that CtIP-(45-160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure. These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia.
authors.forename: Manu J
authors.initials: MJ
authors.lastname: Dubin
authors.forename: Philippa H
authors.initials: PH
authors.lastname: Stokes
authors.forename: Eleanor Y M
authors.initials: EY
authors.lastname: Sum
authors.forename: R Scott
authors.initials: RS
authors.lastname: Williams
authors.forename: Valentina A
authors.initials: VA
authors.lastname: Valova
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Lindeman
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
authors.forename: Jane E
authors.initials: JE
authors.lastname: Visvader
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Matthews
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000429:Alcohol Oxidoreductases; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D060588:LIM Domain Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; C114905:LMO4 protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D004706:Endodeoxyribonucleases; C410741:RBBP8 protein, human; D002918:Chymotrypsin; D014357:Trypsin