Exclusivity and Compensation in NFκB Dimer Distributions and IκB Inhibition

The NF kappa B transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by I kappa B alpha, I kappa B beta, and I kappa B epsilon. These NF kappa B family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NF kappa B dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NF kappa B dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report binding affinities much stronger than those that had been previously reported by non-equilibrium gel shift and analytical ultracentrifugation assays. The results reveal a wide range of NF kappa B dimer affinities and a strong preference of each I kappa B for a small subset of NF kappa B dimers. Once the preferred I kappa B is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NF kappa B transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.