The Acidic Residues of the IκBα PEST Sequence are Responsible for “Stripping” NFκB from DNA

Nuclear factor kappa B (NFκB) transcription factors are responsible for the regulation of more than 150 target genes, their expression is induced by many classes of stimuli, and NFκBs play essential roles in the healthy regulation of cellular development and proliferation in inflammatory and immune responses. Diseases such as cancer, heart disease, Alzheimer's disease, and AIDS can be attributed to the aberrant regulation of NFκB. The transcriptional activity of NFκB is controlled by its inhibitors, the IκBs. IκBα, in particular, dynamically responds to extracellular stimuli releasing a burst of NFκB that enters the nucleus and activates hundreds of target genes. The transcriptional activation is short-lived, and our lab has been investigating the mechanism of post-induction repression. We previously showed that IκBα actively dissociates or "strips" NFκB from DNA. Analysis of the crystal structures of NFκB (RelA/p50) with DNA and with IκBα shows that the IκBα PEST sequence, which is rich in glutamate and aspartate residues, forms similar electrostatic contacts to NFκB as the DNA. Given this, we hypothesized that the IκBα PEST sequence electrostatically repels DNA from NFκB during the stripping process. Here we present fascinating results that show that the individual and collective, conservative mutation of these acidic residues to their amide counterparts does not affect the binding affinities of these mutants to NFκB; however, the mutant in which all five acidic residues are neutralized is incapable of stripping NFκB from DNA and instead forms a stable IκBα-NFκB-DNA ternary complex.