Exclusivity and Compensation in NFkB Dimer distributions and IkB inhibition

The NF k B transcription factor family members RelA, p50, and cRel form homo and heterodimers that are inhibited by I k Bα, I k Bβ, and I k Bε. These NF k 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 k B dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NF k B dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report much stronger binding affinities than had been previously reported by non-equilibrium gel-shift and analytical ultracentrifugation assays. The results reveal a wide range of NF k B dimer affinities and a strong preference of each I k B for a small subset of NF k B dimers. Once the preferred I k B is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NF k B transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.