Global Stabilization of NFκB Upon IκBα Binding

Binding of IκBα to NFκB constitutes the primary and most robust mechanism of regulation in the NFκB signalling pathway. This phenomenon is facilitated by the sequestering of p50/p65 NFκB dimers in the cytosol via IκBα's masking of the p65 NLS. Interestingly, IκBα is one of the genes upregulated by NFκB binding to its promoter upon translocation into the nucleus; IκBα then facilitates dissocation of NFκB from DNA thereby creating a negative feedback loop to turn off the NFκB response. NFκB dimers consist of two domains, a dimerization domain and an N-terminal domain (NTD) which are responsible for dimerization/IκBα binding and DNA binding respectively. Since there are no native contacts between IκBα and the NTDs and further, constructs lacking the NTDs bind IκBα with comparable KD values, it has long been believed that the NTDs are not implicated in IκBα binding. I performed hydrogen-deuterium exchange mass spectrometry to analyze changes in amide exchange in NFκB when IκBα binds. The results show dramatic stabilization of the dimerization of NFκBs upon IκBα binding. In addition, surprisingly, we observe significant stabilization in the NTDs as well. This stabilization could potentially indicate a conformational change that would explain how IκBα binding may facilitate dissociation of NFκB from DNA.