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 (Trp318) 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 Trp318 switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex. Background: C-terminal binding protein 1 (CtBP1) assembles into a tetrameric transcriptional co-repressor but how it directs gene expression is not clear. Results: CtBP1 requires NAD(H) for transition into multimers. Its biochemical activities are separable from transcriptional repression. Conclusion: Tryptophan 318 permits CtBP1 to first dimerize and then tetramerize after the binding of NAD(H). Significance: Clarification of how CtBP1 tetramerizes will permit development of CtBP inhibitors to target oncogenesis.