The NF-kB TFs are related structurally by a ~300 residue homologous peptide sequence (a majority of their structure), the Rel homology region (RHR), and by their shared activity as homo- and heterodimers whose integrative diversity contributes to extensively variable DNA-binding capacity (Mosialos & Gilmore, 1993). Furthermore, NF-kB TFs are functionally related by interactions with IkB proteins that inhibit NF-kB nuclear localization and DNA-binding, relegating the resulting IkB-NF-kB complexes to the cytoplasm. One of the more common complexes is that formed between IkBb and the p65 homodimer. The ligand-dependent ubiquitination and phosphorylation of the former within the cytoplasm leads to its degradation and the release of the p65 homodimer to the nucleus where it takes effect (Fig. 1). Though, as Malek et al. (2003) show, hypophosphorylated IkBb-p65 homodimer complex may also migrate as a whole into the nucleus and bind DNA in a stable complex. Phosphorylation and acetylation of the latter also contributes to its DNA-binding capacity.

Figure 1. Annotated pathway of induction of IkBb degradation and release of p65 homodimer to localize to the nucleus and effect transcriptional change. Red oval represents IkBb. Blue circles represent p65 TF subunits. Green circle represents phosphate group(s).

The IkBb inhibitor is comprised of 6 , each consisting of two antiparallel a-helices linked by a sharp loop. In turn, each repeat is followed on either end by a short B-hairpin turn whose 180-degree turn retains the parallel orientation of the repeat domain. Hydrophobic stacking between the helices of the ankyrin repeats stabilizes the extended structure of IkBb. Each p65 subunit has a bipartite structure consisting of an N-terminal RHR and a C-terminal transactivation domain (TAD) separated by a nuclear localization signal (NLS) . The RHR is folded into two immunoglobulin-like domains that contribute to DNA-binding specificity while the TAD contributes to dimerization stability (*Chen et al., 1998) through a selection of hydrophobic and polar contacts: Cys 197, Asn 200, Phe 213, Leu 215, His 245, Val 248, Ala 249 and Val 251 form a at the dimer interface. Arg 198, Cys 216, Asp 217, Asp 243, His 245 and Arg 246 make .

Linkage between IkBb and the p65 homodimer is mediated by interactions between the first two ankyrin repeats of IkBb and the NLS domain of p65 subunit A, as well as contacts made between the last three ankyrin repeats of IkBb and the dimerization domain of the p65 subunits . Within the NLS domain, sandwiched stacking between Phe73 and Phe76 of IkBb and Phe318 (Phe318 not pictured) of p65 subunit A provides particularly stability among other (as well as salt bridge and polar contacts). 'Tis important to note that the minimal extended surface contact between IkBb and the p65 homodimer and the resultant reliance on individual single and sparse interactions is largely due to the bent structure of the ankyrin repeat domain and its limited shared surface area with the planar dimerization surface of the two p65 subunits. Nonetheless, in total, ~4000 Å2 of the surface area of the IkBb/NF-kB p65 homodimer complex - roughly half of its total surface area is buried (Malek et al., 2003).

III. Signaling

Signaling is essential in regulating the activity of both the IkBb/NF-kB p65 homodimer complex as a whole, as well as the p65 homodimer during its functioning as a transcription factor after release form the IkBb inhibitor. The most immediately effective regulatory step in control of the complex is the signal for degradation of the IkBb inhibitor and the subsequent release of the p65 homodimer to localize to the nucleus. This step is primarily controlled by phosphorylation of selective residues such as Ser32 and Ser36 and polyubiquitination of Lys9 within a signal response region of IkBb (overlapping the NLS therein as well) (Fig. 2). Phosphorylation of all IkB proteins occurs via three pathways involving kinase cascades. The canonical pathway is that by which pro-inflammatory cytokines recruit adaptor proteins to the cytoplasmic membrane where they, in turn, recruit IkB-kinase (IKK) complex to phosphorylate IkB. IkB is then ubiquitinated and degraded by protease activity, releasing the attached NF-kB dimer whose NLSs are now available to signal its travel to the nucleus where it can bind DNA and act in conjugation with other TFs to activate targeted transcription (Viatour et al., 2005). The release of the dimer is facilitated by the fact that IkB, as aforementioned, makes minimal and localized contact with the p65 homodimer, so degradation of IkBb affects release faster than would a protein with weaker but more extensive hydrophobic surface contacts.

Chemical modification of the p65 homodimer affects its later interactions at transcription sites. For example, phosphorylation of on either p65 subunit has the potential to change the dimer’s interactions with cofactors, variably determining the gene set it can coactivate. Acetylation of the p65 subunits also affects their activational capacity, specifically, acetylation of and Lys310 (Lys310 not pictured). Acetylation of Lys221 enhances p65 homodimer DNA binding, potentially by impairing interactions with IkBb. Acetylation of Lys310 is required for effective transcriptional activation, though not for DNA binding, potentially indicative of it being required for binding to cofactors (Chen et al., 2004).

Figure 2. Annotated peptide sequence of IkBb (residues 1-49) containing the signal response region (residues 5-42). Polyubiquination target lysine and phosphorylation target serines are all marked in red.

IV. DNA Binding

V References

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