Structural and biochemical analyses of the nuclear IκBζ protein in complex with the NF-κB p50 homodimer.

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.

The NF-κB regulator IκBß exhibits different molecular interactivity and phosphorylation status from IκBα in an IKK2-catalysed reaction.

Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (IκBα) by the Inhibitor of kappaB kinase (IKK) that triggers IκBα degradation. Although inhibitor of kappaB beta (IκBß) is structurally similar to IκBα, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of IκBß with the kinase-active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of IκBα. A mass spectrometry analysis revealed that IκBß phosphorylation sites are distributed in its C-terminal region, whereas IκBα phosphorylation sites are located in the N-terminal region. Furthermore, IKK2 phosphorylation sites in IκBß are found in a region distinct from typical degradation signals, such as phosphodegron and proline/glutamic acid/serine/threonine-rich sequence (PEST) motifs. Mutation of the IκBß phosphorylation sites enhances its resistance to homeostatic proteasomal degradation. These findings contribute a novel concept in NF-κB/IKK signalling research.

Statistical Framework for Uncertainty Quantification in Computational Molecular Modeling.

As computational modeling, simulation, and predictions are becoming integral parts of biomedical pipelines, it behooves us to emphasize the reliability of the computational protocol. For any reported quantity of interest (QOI), one must also compute and report a measure of the uncertainty or error associated with the QOI. This is especially important in molecular modeling, since in most practical applications the inputs to the computational protocol are often noisy, incomplete, or low-resolution. Unfortunately, currently available modeling tools do not account for uncertainties and their effect on the final QOIs with sufficient rigor. We have developed a statistical framework that expresses the uncertainty of the QOI as the probability that the reported value deviates from the true value by more than some user-defined threshold. First, we provide a theoretical approach where this probability can be bounded using Azuma-Hoeffding like inequalities. Second, we approximate this probability empirically by sampling the space of uncertainties of the input and provide applications of our framework to bound uncertainties of several QOIs commonly used in molecular modeling. Finally, we also present several visualization techniques to effectively and quantitavely visualize the uncertainties: in the input, final QOIs, and also intermediate states.

Prediction of the presence of a seventh ankyrin repeat in IκBε from homology modeling combined with hydrogen-deuterium exchange mass spectrometry (HDX-MS).

The ankyrin repeat (AR) structure is a common protein-protein interaction motif and ankyrin repeat proteins comprise a vast family across a large array of different taxa. Natural AR proteins adopt a conserved fold comprised of several repeats with the N- and C-terminal repeats generally being of more divergent sequences. Obtaining experimental crystal structures for natural ankyrin repeat domains (ARD) can be difficult and often requires complexation with a binding partner. Homology modeling is an attractive method for creating a model of AR proteins due to the highly conserved fold; however, modeling the divergent N- and C-terminal "capping" repeats remains a challenge. We show here that amide hydrogen/deuterium exchange mass spectrometry (HDX-MS), which reports on the presence of secondary structural elements and "foldedness," can aid in the refinement and selection of AR protein homology models when multiple templates are identified with variations between them localizing to these terminal repeats. We report a homology model for the AR protein IκBε from three different templates and use HDX-MS to establish the presence of a seventh AR at the C-terminus identified by only one of the three templates used for modeling.

Mechanism of vaccinia viral protein B14-mediated inhibition of IκB kinase ß activation.

Activation of IκB kinase ß (IKKß) is a central event in the NF-κB-mediated canonical pro-inflammatory pathway. Numerous studies have reported that oligomerization-mediated trans autophosphorylation of IKKß is indispensable for its phosphorylation, leading to its activation and IKKß-mediated phosphorylation of substrates such as IκB proteins. Moreover, IKKß's interaction with the NF-κB essential modifier (NEMO) is necessary for IKKß activation. Interestingly, some viruses encode virulence factors that target IKKß to inhibit NF-κB-mediated antiviral immune responses. One of these factors is the vaccinia viral protein B14, which directly interacts with and inhibits IKKß. Here we mapped the interaction interface on the B14 and IKKß proteins. We observed that B14 binds to the junction of the kinase domain (KD) and scaffold and dimerization domain (SDD) of IKKß. Molecular docking analyses identified key interface residues in both IKKß and B14 that were further confirmed by mutational studies to promote binding of the two proteins. During trans autophosphorylation of protein kinases in the IKK complex, the activation segments of neighboring kinases need to transiently interact with each other's active sites, and we found that the B14-IKKß interaction sterically hinders direct contact between the kinase domains of IKKß in the IKK complex, containing IKKß, IKKα, and NEMO in human cells. We conclude that binding of B14 to IKKß prevents IKKß trans autophosphorylation and activation, thereby inhibiting NF-κB signaling. Our study provides critical structural and mechanistic information for the design of potential therapeutic agents to target IKKß activation for the management of inflammatory disorders.

Molecular characterization of an inhibitor of NF-κB in the scallop Argopecten purpuratus: First insights into its role on antimicrobial peptide regulation in a mollusk.

Inhibitors of nuclear factor kappa B (IκBs) are major control components of the Rel/NF-κB signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates. The activation of the Rel/NF-κB signaling pathway depends largely in the degradation of IκB proteins and thus, IκBs are a main target for the identification of genes whose expression is controlled by Rel/NF-κB pathway. In order to identify such regulation in bivalve mollusks, the cDNA sequence encoding an IκB protein was characterized in the scallop Argopecten purpuratus, ApIκB. The cDNA sequence of ApIκB is comprised of 1480 nucleotides with a 1086 bp open reading frame encoding for 362 amino acids. Bioinformatics analysis showed that ApIκB displays the conserved features of IκB proteins. The deduced amino acid sequence consists of a 39.7 kDa protein, which has an N-terminal degradation motif, six ankyrin repeats and a C-terminal phosphorylation site motif. Phylogenetic analysis revealed a high degree of identity between ApIκB and other IκBs from mollusks, but also to arthropod cactus proteins and vertebrate IκBs. Tissue expression analysis indicated that ApIκB is expressed in all examined tissues and it is upregulated in circulating hemocytes from scallops challenged with the pathogenic Gram-negative bacterium Vibrio splendidus. After inhibiting ApIκB gene expression using the RNA interference technology, the gene expression of the antimicrobial peptide big defensin was upregulated in hemocytes from non-challenged scallops. Results suggest that ApIκB may control the expression of antimicrobial effectors such as big defensin via a putative Rel/NF-κB signaling pathway. This first evidence will help to deepen the knowledge of the Rel/NF-κB conserved pathway in scallops.

Molecular stripping in the NF-κB/IκB/DNA genetic regulatory network.

Genetic switches based on the [Formula: see text] system are master regulators of an array of cellular responses. Recent kinetic experiments have shown that [Formula: see text] can actively remove NF-κB bound to its genetic sites via a process called "molecular stripping." This allows the [Formula: see text] switch to function under kinetic control rather than the thermodynamic control contemplated in the traditional models of gene switches. Using molecular dynamics simulations of coarse-grained predictive energy landscape models for the constituent proteins by themselves and interacting with the DNA we explore the functional motions of the transcription factor [Formula: see text] and its various binary and ternary complexes with DNA and the inhibitor IκB. These studies show that the function of the [Formula: see text] genetic switch is realized via an allosteric mechanism. Molecular stripping occurs through the activation of a domain twist mode by the binding of [Formula: see text] that occurs through conformational selection. Free energy calculations for DNA binding show that the binding of [Formula: see text] not only results in a significant decrease of the affinity of the transcription factor for the DNA but also kinetically speeds DNA release. Projections of the free energy onto various reaction coordinates reveal the structural details of the stripping pathways.

The nonstructural protein 11 of porcine reproductive and respiratory syndrome virus inhibits NF-κB signaling by means of its deubiquitinating activity.

Since its emergence in the late 1980s, porcine reproductive and respiratory syndrome (PRRS) has been devastating the swine industry worldwide. The causative agent is an Arterivirus, referred to as PRRS virus (PRRSV). The pathogenic mechanisms of PRRS are poorly understood, but are believed to correlate with the ability of PRRSV to inhibit immune responses of the host. However, precisely how the virus is capable of doing so remains obscure. In this study, we showed that PRRSV infection led to reduced ubiquitination of cellular proteins. Screening all of the 12 nonstructural proteins (Nsps) encoded by PRRSV revealed that, apart from the Nsp2 which contains the deubiqintinating (DUB) ovarian tumor (OTU) domain, Nsp11, which encodes a unique and conserved endoribonuclease (NendoU) throughout the Nidovirus order, also possesses DUB activity. In vivo assay demonstrated that Nsp11 specifically removed lysine 48 (K48)-linked polyubiquitin chains and the conserved sites C112, H144, D173, K180, and Y219 were critical for its DUB activity. Remarkably, DUB activity was responsible for the capacity of Nsp11 to inhibit nuclear factor κB (NF-κB) activation. Mutations abrogating the DUB activity of Nsp11 toward K48-linked polyubiquitin chains of IκBα nullified the suppressive effect on NF-κB. Our data add Nsp11 to the list of DUBs encoded by PRRSV and uncover a novel mechanism by which PRRSV cripples host innate immune responses.

CgIκB3, the third novel inhibitor of NF-kappa B (IκB) protein, is involved in the immune defense of the Pacific oyster, Crassostrea gigas.

Inhibitor of NF-κB (IκB), the important regulator of NF-κB/Rel signaling pathway, plays the crucial role in immune response of both vertebrates and invertebrates. Here, a novel homologue of IκB was cloned from Crassostrea gigas, and designated as CgIκB3. The complete CgIκB3 cDNA was 1282 bp in length, including a 942 bp open reading frame (ORF), a 51 bp 5' UTR and a 289 bp 3' UTR. The ORF encodes a putative protein of 313 amino acids with a predicted molecular weight of approximately 34.7 kDa. Sequence analysis reveals that CgIκB3 contains a conserved degradation motif but with only five ankyrin repeats. Neither a PEST domain nor a C-terminal casein kinase II phosphorylation site was identified through either alignment or bioinformatic prediction. Phylogenetic analysis suggested that CgIκB3 shares common ancestor with CgIκB1 rather CgIκB2, and theoretically it may originate from one duplication event prior to divergence of CgIκB1 and CgIκB2. Tissue expression analyses demonstrated that CgIκB3 mRNA is the most abundant in gills and heart. The expression following PAMP infection showed that CgIκB3 was significantly up-regulated in a similar pattern when challenged with LPS, HKLM or HKVA, respectively. Moreover, similar to CgIκB1 and CgIκB2, CgIκB3 can also inhibit Rel dependent NF-κB activation in HEK293 cells in a dose-dependent manner. In summary, these findings suggest that CgIκB3 can be as the functional inhibitor of NF-κB/Rel and involved in the host defense of C. gigas. The discovery of the third IκB emphasizes the complexity and importance of the regulation on NF-κB activation.

GRK6 phosphorylates IκBα at Ser(32)/Ser(36) and enhances TNF-α-induced inflammation.

G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine kinases that phosphorylate agonist-activated G protein-coupled receptors (GPCRs). It has recently been reported that GRKs regulate GPCR-independent signaling through the phosphorylation of intracellular proteins. To date, several intracellular substrates for GRK2 and GRK5 have been reported. However, those for GRK6 are poorly understood. Here we identified IκBα, a negative regulator of NF-κB signaling, as a substrate for GRK6. GRK6 directly phosphorylated IκBα at Ser(32)/Ser(36), and the kinase activity of GRK6 was required for the promotion of NF-κB signaling after TNF-α stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-α stimulation. In addition, we developed a bioluminescence resonance energy transfer (BRET) probe to monitor GRK6 activity. Using this probe, we revealed that the conformational change of GRK6 was induced by TNF-α. In summary, our study demonstrates that TNF-α induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of IκBα.

A cytoplasmic NF-κB interacting long noncoding RNA blocks IκB phosphorylation and suppresses breast cancer metastasis.

NF-κB is a critical link between inflammation and cancer, but whether long non-coding RNAs (lncRNAs) regulate its activation remains unknown. Here, we identify an NF-KappaB Interacting LncRNA (NKILA), which is upregulated by NF-κB, binds to NF-κB/IκB, and directly masks phosphorylation motifs of IκB, thereby inhibiting IKK-induced IκB phosphorylation and NF-κB activation. Unlike DNA that is dissociated from NF-κB by IκB, NKILA interacts with NF-κB/IκB to form a stable complex. Importantly, NKILA is essential to prevent over-activation of NF-κB pathway in inflammation-stimulated breast epithelial cells. Furthermore, low NKILA expression is associated with breast cancer metastasis and poor patient prognosis. Therefore, lncRNAs can directly interact with functional domains of signaling proteins, serving as a class of NF-κB modulators to suppress cancer metastasis.

p100/IκBδ sequesters and inhibits NF-κB through kappaBsome formation.

Degradation of I kappaB (κB) inhibitors is critical to activation of dimeric transcription factors of the NF-κB family. There are two types of IκB inhibitors: the prototypical IκBs (IκBα, IκBß, and IκBε), which form low-molecular-weight (MW) IκB:NF-κB complexes that are highly stable, and the precursor IκBs (p105/IκBγ and p100/IκBδ), which form high-MW assemblies, thereby suppressing the activity of nearly half the cellular NF-κB [Savinova OV, Hoffmann A, Ghosh G (2009) Mol Cell 34(5):591-602]. The identity of these larger assemblies and their distinct roles in NF-κB inhibition are unknown. Using the X-ray crystal structure of the C-terminal domain of p100/IκBδ and functional analysis of structure-guided mutants, we show that p100/IκBδ forms high-MW (IκBδ)4:(NF-κB)4 complexes, referred to as kappaBsomes. These IκBδ-centric "kappaBsomes" are distinct from the 2:2 complexes formed by IκBγ. The stability of the IκBδ tetramer is enhanced upon association with NF-κB, and hence the high-MW assembly is essential for NF-κB inhibition. Furthermore, weakening of the IκBδ tetramer impairs both its association with NF-κB subunits and stimulus-dependent processing into p52. The unique ability of p100/IκBδ to stably interact with all NF-κB subunits by forming kappaBsomes demonstrates its importance in sequestering NF-κB subunits and releasing them as dictated by specific stimuli for developmental programs.

Predicted disorder-to-order transition mutations in IκBα disrupt function.

IκBα inhibits the transcription factor, NFκB, by forming a very tightly bound complex in which the ankyrin repeat domain (ARD) of IκBα interacts primarily with the dimerization domain of NFκB. The first four ankyrin repeats (ARs) of the IκBα ARD are well-folded, but the AR5-6 region is intrinsically disordered according to amide H/D exchange and protein folding/unfolding experiments. We previously showed that mutations towards the consensus sequence for stable ankyrin repeats resulted in a "prefolded" mutant. To investigate whether the consensus mutations were solely able to order the AR5-6 region, we used a predictor of protein disordered regions PONDR VL-XT to select mutations that would alter the intrinsic disorder towards a more ordered structure (D → O mutants). The algorithm predicted two mutations, E282W and P261F, neither of which correspond to the consensus sequence for ankyrin repeats. Amide exchange and CD were used to assess ordering. Although only the E282W was predicted to be more ordered by CD and amide exchange, stopped-flow fluorescence studies showed that both of the D → O mutants were less efficient at dissociating NFκB from DNA.

MiR-196a promotes pancreatic cancer progression by targeting nuclear factor kappa-B-inhibitor alpha.

Aberrant expression of miR-196a has been frequently reported in different cancers including pancreatic cancer. However, its function in pancreatic cancer has not been fully elucidated. Here, we investigated the expression pattern and the biological role of miR-196a in pancreatic cancer cell lines, as well as its interaction with a metastasis-related gene, nuclear factor-kappa-B-inhibitor alpha (NFKBIA). We demonstrated that miR-196a was up-regulated in human pancreatic cancer cell lines compared with immortalized pancreatic ductal epithelial cells by means of microRNAs microarray and qRT-PCR. Furthermore, down-regulation of miR-196a in PANC-1 suppressed its proliferation and migration with an increase in G0/G1 transition and decreased expression of Cyclin D1 and CDK4/6. Meanwhile, an increased expression in E-cadherin and decreased expression in N-cadherin and Vimentin were also observed. We identified a novel miR-196a target, NFKBIA, and down-regulation of miR-196a enhanced the expression of NFKBIA protein. Luciferase assay confirmed that NFKBIA was a direct and specific target of miR-196a. Silencing NFKBIA in PANC-1 cells enhanced its proliferation and migration. Taken together, our findings indicate that miR-196a is highly expressed in pancreatic cancer cell lines, and may play a crucial role in pancreatic cancer proliferation and migration, possibly through its downstream target, NFKBIA. Thus, miR-196a may serve as a potential therapeutic target for pancreatic cancer.

Ribosomal protein S3 interacts with the NF-κB inhibitor IκBα.

Ribosomal protein S3 (RPS3) is part of nuclear, transcriptionally active and cytoplasmic inhibitory complexes containing NF-κB variant p65. We show that in resting HEK293 cells, RPS3 interacts with NF-κB inhibitor IκBα. In contrast, efficient co-precipitation of p65 with RPS3 was only achieved in the presence of ectopic IκBα. In addition, a strong in vitro interaction was observed between RPS3 and IκBα, while binding between RPS3 and p65 was very weak. Furthermore, IκBα facilitated the reconstitution of p65 and RPS3 into one complex in vitro. Our results suggest that IκBα sequesters not only p65 but also RPS3 in the cytoplasm. This would ensure maintenance of an RPS3 pool for the NF-κB pathway as well as equimolar release of RPS3 and p65 upon stimulation.

Molecular cloning and expression of IRAK-4, IL-17 and I-κB genes in Haliotis rufescens challenged with Vibrio anguillarum.

The candidate genes interleukin-1 receptor associated kinase 4 (IRAK-4), Interleukin 17 (IL-17) and Inhibitor of NF-κB (I-κB) were cloned and evaluated in Californian abalone (Haliotis rufescens) hemocytes in response to Vibrio anguillarum. Molecular characterization evidenced that HrI-κB has a full cDNA sequence of 3027 bp with an encoding region of 401 amino acids (aa), HrIRAK-4 comprised 1969 bp that encoded for 516 aa, and Hr-IL17 had a full sequence of 806 bp encoding for 165 aa. qPCR analysis showed the higher constitutive expression level of Hr-IL17 in hemocytes; meanwhile Hr-IκB and Hr-IRAK4 gene expression levels were higher in gills and mantle. The assessment of gene expression in hemocytes after infection with V. anguillarum evidences the immune responses of Hr-IκB, Hr-IRAK4, and Hr-IL17 and their relationships through the NF-κB signaling pathway.

A disorder-induced domino-like destabilization mechanism governs the folding and functional dynamics of the repeat protein IκBα.

The stability of the repeat protein IκBα, a transcriptional inhibitor in mammalian cells, is critical in the functioning of the NF-κB signaling module implicated in an array of cellular processes, including cell growth, disease, immunity and apoptosis. Structurally, IκBα is complex, with both ordered and disordered regions, thus posing a challenge to the available computational protocols to model its conformational behavior. Here, we introduce a simple procedure to model disorder in systems that undergo binding-induced folding that involves modulation of the contact map guided by equilibrium experimental observables in combination with an Ising-like Wako-Saitô-Muñoz-Eaton model. This one-step procedure alone is able to reproduce a variety of experimental observables, including ensemble thermodynamics (scanning calorimetry, pre-transitions, m-values) and kinetics (roll-over in chevron plot, intermediates and their identity), and is consistent with hydrogen-deuterium exchange measurements. We further capture the intricate distance-dynamics between the domains as measured by single-molecule FRET by combining the model predictions with simple polymer physics arguments. Our results reveal a unique mechanism at work in IκBα folding, wherein disorder in one domain initiates a domino-like effect partially destabilizing neighboring domains, thus highlighting the effect of symmetry-breaking at the level of primary sequences. The offshoot is a multi-state and a dynamic conformational landscape that is populated by increasingly partially folded ensembles upon destabilization. Our results provide, in a straightforward fashion, a rationale to the promiscuous binding and short intracellular half-life of IκBα evolutionarily engineered into it through repeats with variable stabilities and expand the functional repertoire of disordered regions in proteins.

Identification and characterization of a novel IκB-ε-like gene from lamprey (Lampetra japonica) with a role in immune response.

Nuclear factor of kappa B (NF-κB) is a stimuli-activated transcription factor, regulates the expression of a diverse array of genes. Inhibitor of kappa B-epsilon (IκB-ε) is an inhibitor of NF-κB, which retains NF-κB in an inactive state in the cytoplasm. Lampreys (Lampetra japonica) belong to the lowest class of vertebrates with little information about its IκBs. We have identified a cDNA sequence IκB-ε-like in the lamprey and the deduced amino acid sequence of IκB-ε-like. It contains a conserved DSGxxS motif and six consecutive ankyrin repeats, which are necessary for signal-induced degradation of the molecule. Phylogenetic analysis indicated it had high sequence homology with IκB-εs from other vertebrates. FACS analysis showed that IκB-ε-like located in cytoplasm of leukocytes. The degradation of IκB-ε-like could be observed in leukocytes of L. japonica stimulated with lipopolysaccharide. These results indicate that IκB-ε proteins are conserved across vertebrates and the NF-κB-like signaling pathway may exist in the oldest agnatha.

Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain.

Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1-4), at ARs 2 and 5 (AR 2-5), and at ARs 3 and 6 (AR 3-6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 "loses grip". AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.

Evolutionary, structural and functional interplay of the IκB family members.

A primary level of control for nuclear factor kappa B (NF-κB) is effected through its interactions with the inhibitor protein, inhibitor of kappa B (IκB). Several lines of evidence confirm the existence of multiple forms of IκB that appear to regulate NF-κB by distinct mechanisms. Therefore, we performed a comprehensive bioinformatics analysis to understand the evolutionary history and intrinsic functional diversity of IκB family members. Phylogenetic relationships were constructed to trace the evolution of the IκB family genes. Our phylogenetic analysis revealed 10 IκB subfamily members that clustered into 5 major clades. Since the ankyrin (ANK) domain appears to be more ancient than the Rel homology domain (RHD), our phylogenetic analysis suggests that some undefined ancestral set of ANK repeats acquired an RHD before any duplication and was later duplicated and then diverged into the different IκB subfamilies. Functional analysis identified several functionally divergent sites in the ANK repeat domains (ARDs) and revealed that this region has undergone strong purifying selection, suggesting its functional importance in IκB genes. Structural analysis showed that the major variations in the number of ANK repeats and high conformational changes in the finger loop ARD region contribute to the differing binding partner specificities, thereby leading to distinct IκB functions. In summary, our study has provided useful information about the phylogeny and structural and functional divergence of the IκB family. Additionally, we identified a number of amino acid sites that contribute to the predicted functional divergence of these proteins.