Glycan-mediated functional assembly of IL-1RI: structural insights into completion of the current description for immune response.

Interleukin 1 Receptor type I (IL-1RI) is a multi-domain transmembrane receptor that triggers the inflammatory response. Understanding its detailed mechanism of action is crucial for treating immune disorders. IL-1RI is activated upon formation of its functional assembly that occurs by binding of the IL-1 cytokine and the accessory protein (Il-1RAcP) to it. X-ray crystallography, small-Angle X-ray Scattering and molecular dynamics simulation studies showed that IL-1RI adopts two types of 'compact' and 'extended' conformational states in its dynamical pattern. Furthermore, glycosylation has shown to play a critical role in its activation process. Here, classical and accelerated atomistic molecular dynamics were carried out to examine the role of full glycosylation of IL-1RI and IL-1RAcP in arrangement of the functional assembly. Simulations showed that the 'compact' and 'extended' IL-1RI form two types of 'cytokine-inaccessible-non-signaling' and 'cytokine-accessible-signaling' assemblies with the IL-1RacP, respectively that are both abiding in the presence of glycans. Suggesting that the cytokine binding to IL-1RI is not required for the formation of IL-1RI-IL-1RAcP complex and the 'compact' complex could act as a down-regulatory mechanism. The 'extended' complex is maintained by formation of several persistent hydrogen bonds between the IL-1RI-IL-1RAcP inter-connected glycans. Taken together, it was shown that full glycosylation regulates formation of the IL-1RI functional assembly and play critical role in cytokine biding and triggering the IL-1RI involved downstream pathways in the cell.Communicated by Ramaswamy H. Sarma.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

GAG positioning on IL-1RI; A mechanism regulated by dual effect of glycosylation.

IL-1RI is the signaling receptor for the IL-1 family of cytokines that are involved in establishment of the innate and acquired immune systems. Glycosylated extracellular (EC) domain of the IL-1RI binds to agonist such as IL-1ß or antagonist ligands and the accessory protein to form the functional signaling complex. Dynamics and ligand binding of the IL-1RI is influenced by presence of the glycosaminoglycans (GAGs) of the EC matrix. Here a combination of molecular dockings and molecular dynamics simulations of the unglycosylated, partially N-glycosylated and fully N-glycosylated IL-1RI EC domain in the apo, GAG-bound and IL-1ß-bound states were carried out to explain the co-occurring dynamical effect of receptor's glycosylation and GAGs. It was shown that the IL-1RI adopts two types of "extended" and "locked" conformations in its dynamical pattern, and glycosylation maintains the receptor in the latter form. Maintaining the receptor in the locked conformation disfavors IL-1ß binding by burying its two binding site on the IL-1RI EC domain. Glycosylation disfavors GAG binding to the extended IL-1RI EC domain by sterically limiting the GAGs degrees of freedom in targeting its binding site, while it favors GAG binding to the locked IL-1RI by favorable packing interactions.

Identification of critical regions within the TIR domain of IL-1 receptor type I.

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC' helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.

Identification of potential small molecule allosteric modulator sites on IL-1R1 ectodomain using accelerated conformational sampling method.

The interleukin-1 receptor (IL-1R) is the founding member of the interleukin 1 receptor family which activates innate immune response by its binding to cytokines. Reports showed dysregulation of cytokine production leads to aberrant immune cells activation which contributes to auto-inflammatory disorders and diseases. Current therapeutic strategies focus on utilizing antibodies or chimeric cytokine biologics. The large protein-protein interaction interface between cytokine receptor and cytokine poses a challenge in identifying binding sites for small molecule inhibitor development. Based on the significant conformational change of IL-1R type 1 (IL-1R1) ectodomain upon binding to different ligands observed in crystal structures, we hypothesized that transient small molecule binding sites may exist when IL-1R1 undergoes conformational transition and thus suitable for inhibitor development. Here, we employed accelerated molecular dynamics (MD) simulation to efficiently sample conformational space of IL-1R1 ectodomain. Representative IL-1R1 ectodomain conformations determined from the hierarchy cluster analysis were analyzed by the SiteMap program which leads to identify small molecule binding sites at the protein-protein interaction interface and allosteric modulator locations. The cosolvent mapping analysis using phenol as the probe molecule further confirms the allosteric modulator site as a binding hotspot. Eight highest ranked fragment molecules identified from in silico screening at the modulator site were evaluated by MD simulations. Four of them restricted the IL-1R1 dynamical motion to inactive conformational space. The strategy from this study, subject to in vitro experimental validation, can be useful to identify small molecule compounds targeting the allosteric modulator sites of IL-1R and prevent IL-1R from binding to cytokine by trapping IL-1R in inactive conformations.

The structural pathway of interleukin 1 (IL-1) initiated signaling reveals mechanisms of oncogenic mutations and SNPs in inflammation and cancer.

Interleukin-1 (IL-1) is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance) the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor). We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. In silico mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP) mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.

A communication network within the cytoplasmic domain of toll-like receptors has remained conserved during evolution.

Toll/IL-1R (TIR) domain, that is, the cytoplasmic domain, in toll-like receptors (TLRs) from different species showed high sequence conservation in stretches spread across the surface as well as the core of the domain. To probe the structure-function significance of these residues, especially those coming from the core of TIR domains, we analyzed molecular dynamics trajectories of sequence similarity based models of human TIR domains. This study brought forth that N-terminal of the TIR domain simultaneously interacts with the flanking residues of the BB loop and central ß-sheets. At the same time, residues of the central ß-strands form favorable contacts with the DD loop and C-terminal, thus forming a two-way circuit between the N- and C-termini. In this work, the array of intradomain interactions is termed as communication network. Importantly, the "hubs" of this communication network were found to be conserved in all human TLRs. Earlier mutagenesis-function correlation work brought forth that certain mutations in the "core" of the TIR domain of TLR4 (e.g. in IFI767-769AAA and L815A) led to almost complete abrogation of signaling and reasoning for this dramatic loss-of-function has remained unclear, since these sites are not surface exposed. Using MD studies, we show here that this communication network gets disrupted in mutants of human TLR4 which were earlier reported to be functionally compromised. Extension of MD studies to heterodimer of TLR1/2 suggested that this evolutionarily conserved communication network senses the interactions formed upon dimerization and relays it to surfaces which are not involved in direct interdomain contacts.

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn led to antibodies with improved potencies. For this particular analysis, we also found that VH-VL interface positions provided a source of structural diversity for those derived from the ribosome display selections. This greater diversity is a likely consequence of the presence of a larger pool of recombinants in the ribosome display system, or the evolutionary capacity of ribosome display, but may also reflect differential selection of antibodies in the two systems.

Design of a superior cytokine antagonist for topical ophthalmic use.

IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1ß and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other ß-trefoil family proteins in the IL-1 and FGF families.

One target-two different binding modes: structural insights into gevokizumab and canakinumab interactions to interleukin-1ß.

Interleukin-1ß (IL-1ß) is a key orchestrator in inflammatory and several immune responses. IL-1ß exerts its effects through interleukin-1 receptor type I (IL-1RI) and interleukin-1 receptor accessory protein (IL-1RAcP), which together form a heterotrimeric signaling-competent complex. Canakinumab and gevokizumab are highly specific IL-1ß monoclonal antibodies. Canakinumab is known to neutralize IL-1ß by competing for binding to IL-1R and therefore blocking signaling by the antigen:antibody complex. Gevokizumab is claimed to be a regulatory therapeutic antibody that modulates IL-1ß bioactivity by reducing the affinity for its IL-1RI:IL-1RAcP signaling complex. How IL-1ß signaling is affected by both canakinumab and gevokizumab was not yet experimentally determined. We have analyzed the crystal structures of canakinumab and gevokizumab antibody binding fragment (Fab) as well as of their binary complexes with IL-1ß. Furthermore, we characterized the epitopes on IL-1ß employed by the antibodies by NMR epitope mapping studies. The direct comparison of NMR and X-ray data shows that the epitope defined by the crystal structure encompasses predominantly those residues whose NMR resonances are severely perturbed upon complex formation. The antigen:Fab co-structures confirm the previously identified key contact residues on IL-1ß and provide insight into the mechanisms leading to their distinct modulation of IL-1ß signaling. A significant steric overlap of the binding interfaces of IL-1R and canakinumab on IL-1ß causes competitive inhibition of the association of IL-1ß and its receptor. In contrast, gevokizumab occupies an allosteric site on IL-1ß and complex formation results in a minor reduction of binding affinity to IL-1RI. This suggests two different mechanisms of IL-1ß pathway attenuation.

Structure of the activating IL-1 receptor signaling complex.

Interleukin-1 (IL-1)-family cytokines are mediators of innate and adaptive immunity. They exert proinflammatory effects by binding a primary receptor that recruits a receptor accessory protein to form a signaling-competent heterotrimeric complex. Here we present the crystal structure of IL-1ß bound to its primary receptor IL-1RI and its receptor accessory protein IL-1RAcP, providing insight into how IL-1-type cytokines initiate signaling and revealing an evolutionary relationship with the fibroblast growth factor receptor family.

Distinct control of MyD88 adapter-dependent and Akt kinase-regulated responses by the interleukin (IL)-1RI co-receptor, TILRR.

Inflammatory responses are controlled through members of the interleukin-1 receptor (IL-1R)/Toll-like receptor superfamily. Our earlier work demonstrates that the IL-1 receptor type 1 (IL-1RI) co-receptor, Toll-like and IL-1 receptor regulator (TILRR), amplifies IL-1 activation of NF-κB and inflammatory genes. Here we show that TILRR similarly promotes IL-1-induced anti-apoptotic signals and reduces caspase-3 activity. Further, the TILRR-induced effects on cell survival and inflammatory responses are controlled through distinct parts of the IL-1RI regulatory Toll IL-1 receptor (TIR) domain. Alanine-scanning mutagenesis identified a functional TILRR mutant (R425A), which blocked increases in cell survival and upstream activation of Akt but had no effect on amplification of MyD88-dependent inflammatory responses. A second mutant (D448A) blocked TILRR potentiation of MyD88-dependent signals and inflammatory activation but had no impact on cell survival. Secondary structure predictions suggested that the mutations induce distinct alterations in the α-helical structure of the TILRR core protein. The results indicate a role for TILRR in selective amplification of NF-κB responses through IL-1RI and suggest that the specificity is determined by changes in receptor conformation and adapter protein recruitment.

Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency.

A hydrophobic interaction chromatography method was developed to analyze recombinant soluble Interleukin 1 receptor type II (sIL-1R type II) drug substance and assess the stability of the drug under accelerated degradation studies. HIC resolved the degraded molecules into three peaks. A combination of several analytical techniques, including cyanogen bromide cleavage, reversed-phase chromatography, mass spectrometry, and N-terminal sequencing, were used to identify the origins of these peaks. We found that accelerated degradation resulted from three different events, deamidation and isomerization at asparagine 317 (Asn317), C-terminal cleavage, and aggregation. The iso-aspartate 317 (iso-Asp317)-containing species were shown to elute in HIC peak I and the Asp317-containing species in HIC peak II, respectively. Deamidation-isomerization to iso-Asp317, but not deamidation to Asp317, resulted in altered retention time on HIC companied by loss of potency, presumably by introducing a significant conformational change. CNBr C-terminal analysis showed that the inactive HIC peak I consisted of sIL-1R type II with "large" C-terminal truncations of 13 or 14 amino acids, whereas the active HIC peak II contained C-terminally full length and "small" C-terminal clips of two amino acids. Molecular modeling indicates that the short loop D317-S320, in the third domain of IL-1R type II, has a crucial impact on the stability of the molecule.

[An investigation of polymorphism in IL-1R1 gene in Chinese population and the functional prediction of a variation in the transmembrane region of IL-1R1].

OBJECTIVE: To identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of human interleukin-1 receptor type I (IL-1R1) gene and to assess their potential effect on the function of IL-1R1. METHODS: The 5' flank region, exons, parts of the introns, as well as 3' flank region of IL-1R1 gene were sequenced to identify and characterize the SNPs in Chinese population. Effects of the SNP on the structure and function of IL-1R1 were analyzed by computational methods. RESULTS: Sixteen SNPs were identified through a 9643 bp sequencing of IL-1R1 gene. Among them, four were in 5' flank region, four in intron region, one in coding region, and seven in 3' untranslated region. A novel SNP in Chinese population was involved in a structural change in IL-1R1, which may influence the signal transduction of IL-1R1. CONCLUSION: The SNP in the IL-1R1 gene might influence its function as an important receptor of IL-1 family.