Alteration of NF-κB p50 DNA Binding Kinetics by S-Nitrosylation

Abstract

Nitric oxide (NO) regulates a wide variety of cellular functions, in part, by formation of S–NO bonds at critical active site thiol groups within proteins, including transcription factors. Previous studies have qualitatively demonstrated that S-nitrosothiol formation can alter transcription factor binding to the DNA recognition site. To more precisely define the effect of S-nitrosylation on transcription factor binding, the equilibrium binding constant was derived for S-nitrosylated NF-κB p50 (S-NO-p50) in a cell free system utilizing gel shift assays. Binding of NF-κB p50 subjected to the nitrosylation conditions in the absence of NaNO2(C-p50-2) was not different from that of wild type NF-κB (C-p50-1). The extent of S-NO-p50 binding to its DNA target sequence was significantly decreased in comparison to that noted with C-p50-1 and C-p50-2. The binding constant was derived for each of the NF-κB variants: C-p50-1 = 1.01 × 1010M−1; C-p50-2 = 0.92 × 1010M−1; and S-NO-p50 = 0.28 × 1010M−1. These data indicate that S-nitrosylation of p50 decreases its affinity for the target DNA sequence by four-fold.