Double phosphorylation-induced structural changes in the signal-receiving domain of IκBα in complex with NF-κB.

Activation of the transcription factor NF-κB requires degradation of its physiological inhibitor IκBα in order to allow nuclear translocation of NF-κB. NF-κB activity links inflammation and carcinogenesis and makes its signaling pathway an important target for therapeutic intervention. The signal-receiving N-terminal domain (SRD) of the NF-κB inhibitor IκBα harbors the sites of post-translational modifications (Ser32 and 36) directed by the IκB kinase (IKK) complex. The SRD was originally recognized to be highly disordered, but was recently shown to possess stable secondary structural elements. Identifying and characterizing the structural effects that arise from phosphorylation may explain how phosphorylation regulates the IκBα-NF-κB protein complex. Therefore, the effect of post-translational mono- and double-phosphorylation of the serine residues of the SRD was analyzed. The structural modifications of the IκBα-NF-κB protein-protein complex due to mono-phosphorylation of either Ser32 or Ser36 amino acid residues or simultaneous phosphorylation were investigated by means of molecular dynamics simulations. Mono-phosphorylation at either Ser32 or Ser36 was not sufficient to induce significant structural changes in the secondary structure of the SRD of IκBα. Double-phosphorylation yielded an increase in distance between the Cα atoms of these serine residues, indicative of a structural change. Only this two-fold phosphorylation induced the extended, more stabilized conformation of the degron motif which renders it accessible by the E3 ligase. In summary, these results provide insight into the conformational changes induced in IκBα proteins upon phosphorylation that are vital to their signaling dynamics and enable us to propose a model for the phosphorylation of the SRD. Proteins 2016; 85:17-29. © 2016 Wiley Periodicals, Inc.