The salt dependence of DNA recognition by NF-κB p50: A detailed kinetic analysis of the effects on affinity and specificity

Abstract

The binding kinetics of NF-κB p50 to the Ig-κB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmon resonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k(a) for both sequences was in the order of 107 M-1 S-1 whilst the k(d) values varied 600-fold in a sequence-dependent manner between 10-1 and 10-4 S-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K(D) value for the Ig-κB sequence was 16 pM, whilst the K(D) for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.