Role of NAD binding and catalytic residues in the C‐terminal binding protein corepressor

Abstract

CtBP corepressor proteins potentiate the activity of many metazoan transcriptional repressors. These proteins are homologous to prokaryotic D-2-hydroxyacid dehydrogenases, possessing an NAD/NADH binding fold and conserved active site residues. When expressed in Drosophila, a catalytic site mutant retains biological activity, however, we find that an NAD binding mutant lacks biological activity. The NAD mutant, similar to a dimerization mutant, is expressed at low levels, indicating that binding of NAD/NADH may affect CtBP stability. These data support the idea that the ancestral dehydrogenase activity is not required for CtBP function, and NAD binding may play a regulatory, rather than catalytic, role.

Conserved Catalytic and C-Terminal Regulatory Domains of the C-Terminal Binding Protein Corepressor Fine-Tune the Transcriptional Response in Development

“…1). These mutations have been described for previous forms of CtBP (16,19,34). A previously described CtBP-NAD mutation affecting conserved glycines present in the NAD(H) binding Rossmann fold (GXGXXG) was not used in this study because of its strong destabilizing effect (19).…”

“…These mutations have been described for previous forms of CtBP (16,19,34). A previously described CtBP-NAD mutation affecting conserved glycines present in the NAD(H) binding Rossmann fold (GXGXXG) was not used in this study because of its strong destabilizing effect (19). A fourth gene, CtBP S , produces only the abundant short isoform, while CtBP L joins the alternatively spliced 3Ј exon directly to the main coding region to produce this isoform at levels similar to that of the more abundant endogenous CtBP S isoform.…”

“…Structurally, CtBP shares extensive homology with D-2-hydroxy acid dehydrogenases, including the conserved NAD(H) binding domain and putative catalytic site, and the protein has weak in vitro dehydrogenase activity (15). Although CtBP active site residues are highly conserved, it is not clear whether the dehydrogenase activity participates in CtBP-mediated transcriptional repression, because most cell-based assays have shown that this enzymatic activity is dispensable for repression (12,15,19,34). The NAD(H) binding activity, however, appears to be critical for CtBP repression activity (12,15,19,34).…”

“…Structural studies revealed that NAD(H) binding induces a conformational change in CtBP, which may explain the importance of this cofactor for the interaction of CtBP with cofactors and transcription factors (15,40). An NAD(H) binding mutant is still capable of forming dimers in vitro, but there is evidence to suggest that NAD(H) binding can further stimulate CtBP dimerization (2,19).…”

“…Although CtBP active site residues are highly conserved, it is not clear whether the dehydrogenase activity participates in CtBP-mediated transcriptional repression, because most cell-based assays have shown that this enzymatic activity is dispensable for repression (12,15,19,34). The NAD(H) binding activity, however, appears to be critical for CtBP repression activity (12,15,19,34). Structural studies revealed that NAD(H) binding induces a conformational change in CtBP, which may explain the importance of this cofactor for the interaction of CtBP with cofactors and transcription factors (15,40).…”