Molecular modeling-based evaluation of dual function of IκB∫ ankyrin repeat domain in toll-like receptor signaling

IκB∫ (inhibitor of NF-κB (nuclear factor κB) ∫) is a nuclear protein induced upon stimulation of toll-like receptors (TLRs) and interleukin-1 receptor. Induced IκB∫, especially its C-terminal ankyrin repeat domain (ARD), interacts with NF-κB in the nucleus, where it regulates the transcriptional activity of target genes. Recent studies have shown that human ARD of IκB∫ binds with p50/p65 heterodimer and inhibits the transcription of NF-κB regulated genes, whereas mouse ARD of IκB∫ binds with p50/p50 homodimer and exhibits transcriptional activation activity. Since human and mouse IκB∫ ARD are identical, it is unclear how IκB∫ can be a positive and negative regulator of NF-κB-mediated transcription. Therefore, we generated a structural model of IκB∫ ARD and constructed a detailed molecular dynamics (MD) simulation of IκB∫ in explicit solvent to investigate ARD flexibility. In addition, we used molecular docking to screen for potential sites of interaction between IκB∫ and the p50/p65 heterodimer and IκB∫ and the p50/p50 homodimer. The docking experiments revealed that the binding of IκB∫ ankyrin repeats with the p50/p65 N-terminal DNA binding domain prevents NF-κB-mediated transcriptional activation. Furthermore, the IκB∫-p50 homodimer complex, which lacks Pro, Glu (and Asp), Ser and Thr (PEST motif), facilitated gene expression. These two different binding schemes of IκB∫ may be responsible for its opposite function, which is consistent with the currently available biochemical data. Moreover, our data implicate structurally highly flexible ARD residues as the prime contributors to this dual function.