Molecular modeling‐based evaluation of dual function of IκBζ ankyrin repeat domain in toll‐like receptor signaling

Abstract

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

Structure-Function Relationship of Cytoplasmic and Nuclear IκB Proteins: An In Silico Analysis

“…In IκBζ modelling, we deleted a 28 residue insertion, which was located between α1 and α2 of the fourth ANK repeat. The reason for deletion of this insertion region has been previously described [21]. Nevertheless, we also observed a 20 amino acid residues insertion in IκBNS corresponding to IκBζ.…”

“…Since only the C-terminal dimerization region remained, we have hypothesized that this region might play a crucial role in the binding of IκB proteins. Our hypothesis is bolstered by previous data on the prediction of the Bcl3-p50/p50 and IκBζ-p50/p50 complexes [4], [21]. Additionally, cytoplasmic IκBε proteins mask the NLS of the p50/p65 complex, thereby preventing its translocation into the nucleus, and this is in agreement with previously solved cytoplasmic IκB proteins [13], [14], [16].…”

“…These interactions are similar to those observed between IκBζ and the p50/p50 complex [21]. Additionally, ANK7 interacts with the N-terminal region of p50 subunit B.…”

“…However, these two residues did not participate in our currently predicted docking poses. Previous studies on IκBζ have shown a similar kind of insertion, but no functional significance was observed regarding binding with its partners [21]. Unlike nuclear protein, cytoplasmic IκBβ contains a unique insertion between ANK3-ANK4, and this region is important for masking the NLS of p65 subunit B [16].…”

Similar Structures but Different Roles – An Updated Perspective on TLR Structures

“…Although the structures are similar, the binding specificities of these proteins remain unknown. The modeling studies have identified that variation in charged surfaces among the IκB proteins and also differences in the flexible residual position might be the chief factor for the IκB protein binding specificities (Manavalan et al, 2010, 2011). …”