Thermodynamics Reveal that Helix Four in the NLS of NF-κB p65 Anchors IκBα, Forming a Very Stable Complex

Abstract

IκBα is an ankyrin repeat protein that inhibits NF-κB transcriptional activity by sequestering NF-κB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IκBα ankyrin repeat domain to NF-κB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IκBα and NF-κB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K D,obs of approximately 40 pM at 37 °C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-κB(p50/p65) heterodimer and p65 homodimer bound IκBα with almost indistinguishable thermodynamics, except that the NF-κB p65 homodimer was characterized by a more favorable Δ H obs relative to the NF-κB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (Δ C P,obs), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.