Nicotinamide adenine dinucleotide-induced multimerization of the co-repressor CtBP1 relies on a switching tryptophan.
The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD(+) binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp(318)) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD(+) binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp(318) switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.
From the Division of Endocrinology and.
Dana L
DL
Madison
Jacqueline A
JA
Wirz
Don
D
Siess
James R
JR
Lundblad
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D020816:Amino Acid Motifs; D001665:Binding Sites; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016296:Mutagenesis; D009243:NAD / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry*; D020798:Two-Hybrid System Techniques
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009711:Nucleotides; D009243:NAD; D014364:Tryptophan; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein
C-terminal-binding Protein; Corepressor Transcription; Dehydrogenase; NAD; Protein-Protein Interactions; Transcription