Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor

Abstract

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus E1A. The finding that CtBP-E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD+ and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD+ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD+. The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD+ affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD+. Our studies show a >100-fold higher affinity of NADH than NAD+, consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD+ required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.

Fig. 1.

Removal of bound NADH with pyruvate. Excitation of bacterially expressed purified CtBP at 340 nm results in an emission with a peak at 425 nm (middle trace). Saturating levels of NADH demonstrate fully bound CtBP (top trace). The addition of pyruvate abolished NADH fluorescence (bottom trace).

Fig. 2.

NAD⁺ binding site of CtBP, taken from Kumar et al. (17). Several contacts of CtBP residues and NAD⁺ are shown. The distance between W318 and the nicotinamide moiety of NAD⁺ is within the Förster distance observed for tryptophan to NADH energy transfer (21). This structure is deposited in the Protein Data Bank (accession number 1MX3; ref. 17).

Fig. 3.

FRET signal of CtBP. (a) The excitation of CtBP at 285 nm resulted in a typical tryptophan fluorescence emission with a peak at 340 nm (bold trace). The titration of NADH resulted in a decrease of tryptophan fluorescence at 340 nm and an increase in NADH fluorescence at 425 nm. (b) Single tryptophan mutants were generated by mutating two of the three tryptophan residues to phenylalanines. The emission scan of each single tryptophan mutant excited at 285 nm is shown. Each mutant has a typical emission scan with a peak at 340 nm. (c) The addition of 200 nM NADH resulted in a strong energy transfer observed only with W318 (bold trace).

Fig. 4.

Plot of free NADH versus ΔF. Data were fit with Eq. 2.

Fig. 5.

(a) Blue shift and enhanced fluorescence of bound NADH. Free NADH has a peak emission at 455 nm when excited at 340 nm (lower trace). Addition of CtBP results in a shift of the emission peak to 425 nm and an increase in the quantum yield (upper trace). (b) NAD⁺ displacement of NADH. The emission scan of CtBP bound with NADH excited at 340 nm (top trace). The titration of NAD⁺ resulted in a loss of NADH fluorescence. At saturating levels of NAD⁺, an emission scan typical of free NADH was observed. (c) Plot of NAD⁺ versus ΔF. Data were fit with Eq. 2. See Materials and Methods for calculation of K_d.