Nicotinamide adenine dinucleotide-induced multimerization of the co-repressor CtBP1 relies on a switching tryptophan

J Biol Chem. 2013 Sep 27;288(39):27836-48. doi: 10.1074/jbc.M113.493569. Epub 2013 Aug 12.


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.

Keywords: C-terminal-binding Protein; Corepressor Transcription; Dehydrogenase; NAD; Protein-Protein Interactions; Transcription.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Amino Acid Motifs
  • Binding Sites
  • Cell Line, Tumor
  • Chromatography, Gel
  • Cross-Linking Reagents / pharmacology
  • DNA-Binding Proteins / chemistry*
  • Fluorescence Resonance Energy Transfer
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Mutagenesis
  • NAD / metabolism*
  • Nucleotides / chemistry
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Tryptophan / chemistry*
  • Two-Hybrid System Techniques


  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • Nucleotides
  • NAD
  • Tryptophan
  • Alcohol Oxidoreductases
  • C-terminal binding protein