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• W2016064695 abstract "Interleukin (IL-1)α and IL-1β are important mediators of inflammation. The binding of IL-1 to interleukin-1 receptor (IL-1R) type 1 is the initial step in IL-1 signal transduction and therefore is a tempting target for anti-inflammatory therapeutics. To advance our understanding of IL-1R1 binding interactions, we have determined the structure of the extracellular domains of IL-1R1 bound to a 21-amino acid IL-1 antagonist peptide at 3.0-Å resolution. The antagonist peptide binds to the domain 1/2 junction of the receptor, which is a conserved binding site for IL-1β and IL-1 receptor antagonist (IL-1ra). This co-crystal structure also reveals that considerable flexibility is present in IL-1R1 because the carboxyl-terminal domain of the receptor is rotated almost 170° relative to the first two domains of the receptor compared with the previously solved IL-1R1·ligand structures. The structure shows an unexpected binding mode for the peptide and may contribute to the design of smaller IL-1R antagonists. Interleukin (IL-1)α and IL-1β are important mediators of inflammation. The binding of IL-1 to interleukin-1 receptor (IL-1R) type 1 is the initial step in IL-1 signal transduction and therefore is a tempting target for anti-inflammatory therapeutics. To advance our understanding of IL-1R1 binding interactions, we have determined the structure of the extracellular domains of IL-1R1 bound to a 21-amino acid IL-1 antagonist peptide at 3.0-Å resolution. The antagonist peptide binds to the domain 1/2 junction of the receptor, which is a conserved binding site for IL-1β and IL-1 receptor antagonist (IL-1ra). This co-crystal structure also reveals that considerable flexibility is present in IL-1R1 because the carboxyl-terminal domain of the receptor is rotated almost 170° relative to the first two domains of the receptor compared with the previously solved IL-1R1·ligand structures. The structure shows an unexpected binding mode for the peptide and may contribute to the design of smaller IL-1R antagonists. interleukin-1 interleukin-1 receptor interleukin-1 receptor antagonist 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Interleukin (IL-1)1α and IL-1β are pro-inflammatory cytokines that are implicated in a variety of infectious responses as well as in rheumatoid arthritis and other inflammatory diseases (1Dinarello C. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). Increased IL-1 production has been observed in patients with several autoimmune disorders, ischemia, and various cancers, therefore implicating IL-1 as a potential mediator of many diseases. IL-1α and IL-1β signal by binding to interleukin-1 receptor (IL-1R) type 1. The receptor-ligand complex binds IL-1R accessory protein, and the resulting receptor heterodimer transduces a cellular signal (2Cullinan E.B. Kwee L. Nunes P.D.J.S. Ju G. McIntyre K.W. Chizzonite R.A. Labow M.A. J. Immunol. 1998; 161: 5614-5620PubMed Google Scholar). Recent studies have shown that IL-1R family members may be implicated in processes as diverse as IL-18 binding (3Hoshino K. Tsutsui H. Takeda K. Nakanishi K. Takeda Y. Akira S. J. Immunol. 1999; 162: 5014-5044Google Scholar) and neural development (4Born T.L. Smith D.E. Garka K.E. Renshaw B.R. Bertles J.S. Sims J.E. J. Biol. Chem. 2000; 275: 29946-29954Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Because of the highly inflammatory properties of IL-1, multiple control mechanisms exist to moderate the IL-1R response. These mechanisms include IL-1R2, soluble receptor fragments, and IL-1R antagonist (IL-1ra). IL-1R2 binds IL-1α and IL-1β, but does not signal, whereas proteolytically processed soluble versions of IL-1R1 and IL-1R2 bind the interleukin-1 molecules in circulation. IL-1ra is a naturally occurring antagonist that binds to IL-1R1 and that blocks the binding of IL-1α or IL-1β to IL-1R1. The IL-1R1·IL-1ra complex does not interact with IL-1R accessory protein, and so IL-1 signaling does not occur. IL-1ra shares ∼30% sequence identity with IL-1α and IL-1β (5Eisenberg S.P. Brewer M.T. Verderber E. Heimdal P.L. Brandhuber B.J. Thompson R.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5232-5236Crossref PubMed Scopus (221) Google Scholar) and has the same β-barrel tertiary structure (6Vigers G.P.A. Caffes P. Evans R.J. Thompson R.C. Eisenberg S.P. Brandhuber B.J. J. Biol. Chem. 1994; 269: 12874-12879Abstract Full Text PDF PubMed Google Scholar). It also has an extremely high (150 pm) affinity for IL-1R1 (7Dripps D.J. Brandhuber B.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1991; 266: 10331-10336Abstract Full Text PDF PubMed Google Scholar). Two recent studies describing IL-1ra-deficient mice further support the role of IL-1ra in inflammatory and autoimmune diseases (8Nicklin M.J.H. Hughes D.E. Barton J.L. Ure J.M. Duff G.W. J. Exp. Med. 2000; 191: 303-311Crossref PubMed Scopus (272) Google Scholar, 9Horai R. Saijo S. Tanioka H. Nakae S. Sudo K. Okahara A. Ikuse T. Asano M. Iwakura Y. J. Exp. Med. 2000; 191: 313-320Crossref PubMed Scopus (623) Google Scholar). IL-1ra is currently being investigated clinically as a treatment for rheumatoid arthritis. Ultimately, however, an orally deliverable IL-1 antagonist is desirable, which requires the discovery of a much lower molecular mass compound. Extensive searches for lower molecular mass IL-1R antagonists have been conducted; and in 1996, Yanofsky et al. (10Yanofsky S.D. Baldwin D.N. Butler J.H. Holden F.R. Jacobs J.W. Balasubramanian P. Chinn J.P. Cwirla S.E. Peters-Bhatt E. Whitehorn E.A. Tate E.H. Akeson A. Bowlin T.L. Dower W.J. Barrett R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7381-7386Crossref PubMed Scopus (105) Google Scholar) published the sequences of several IL-1R1 antagonist peptides identified from phage display libraries, including a 21-mer IL-1 antagonist peptide (referred to as AF10847). The peptide binds to IL-1R1 with an impressively tight IC50 of 2.6 nm and, like IL-1ra, appears to be a pure receptor antagonist. To determine how the peptide binds to IL-1R1, we have co-crystallized AF10847 with IL-1R1 and solved the structure at a resolution of 3.0 Å. The extracellular portion of IL-1R1 (expressed residues Asp4–Thr315 for full-length IL-1R1 or Asp4–Lys205 for two-domain IL-1R1) (11Sims J.E. Acres R.B. Grubin C.E. McMahan C.J. Wignall J.M. March C.J. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8946-8950Crossref PubMed Scopus (242) Google Scholar) was cloned into pVL1392 (Invitrogen) and expressed in baculovirus-infected Sf9 cells. Approximately 68 h after infection, the media were harvested, centrifuged, and loaded onto an IL-1ra/Affi-Gel 15 affinity column. The receptor was eluted at room temperature using a gradient from pH 5.0 to 2.5 containing 0.2 m sodium acetate, 0.2 m NaCl, 10% glycerol, and 0.25% CHAPS. The fractions were immediately neutralized with 1 m Tris (pH 9.0). The IL-1R1 fractions were pooled and dialyzed against 10 mm HEPES (pH 7.0), 100 mm NaCl, and 0.25% CHAPS. The protein was concentrated and loaded onto a Superdex 75 column (Amersham Pharmacia Biotech) equilibrated in the dialysis buffer. The monomer-containing fractions were pooled, concentrated, and stored frozen at −70 °C. IL-1R1 (3.3 mg/ml) and AF10847 (synthesized by standard solid-phase synthesis) were incubated for 2 h on ice at a 1:2 molar ratio, and crystals were grown by hanging-drop vapor diffusion at 20 °C. 25% polyethylene glycol 4000, 0.2 mammonium sulfate, and 100 mm sodium acetate (pH 4.5) were used as the precipitant. Crystals were cryoprotected with 90% precipitant and 10% ethylene glycol. All diffraction data were collected at 120 K on a Rigaku RU-H3R generator with an R-AxisII image plate detector (Molecular Structure Corp., The Woodlands, TX). The heavy atom derivative was prepared by soaking with 1 mmK2PtCl4 overnight at 4 °C. X-ray diffraction data were processed using HKL, Denzo, and Scalepack (12Otwinowski Z. Sawyer L. Isaacs N. Bailey S. Data Collection and Processing. Science and Engineering Research Council Daresbury Laboratory, Warrington, United Kingdom1993Google Scholar). Molecular replacement solutions (either for single or multiple domains) were generated by Amore (13Collaborative Computational Research Project, Number 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) and evaluated by Xplor (14Brunger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar), but no good solutions were found. Heavy atom data were therefore collected on a K2PtCl4 derivative. The heavy atom Patterson map was solved by hand, and Siras phases were calculated with Mlphare (13Collaborative Computational Research Project, Number 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). No solvent flattening was employed. The positions of the three receptor domains were found using Essens (15Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D. 1997; 53: 179-185Crossref PubMed Scopus (96) Google Scholar) to search the Siras map with polyalanine models of each domain individually. Refinement of the structure was carried out using Xplor, and the peptide was built using O (16Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). In view of the somewhat low resolution of the molecular replacement structure (3.0 Å), no water molecules were added to the structure. Changes in solvent-accessible area were computed using Xplor following the method of Lee and Richards (17Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5360) Google Scholar), making the assumption that the protein structures were unchanged upon ligand binding. Hydrogen bonds were assigned using Xplor to find nitrogen-oxygen pairs separated by <3.5 Å. Figures were prepared using Ribbons (18Carson M. Bugg C.E. J. Mol. Graphics. 1986; 4: 121-122Crossref Scopus (128) Google Scholar) or InsightII (Molecular Simulations Inc., San Diego, CA). Binding of AF10847 to both full-length extracellular IL-1R1 and two-domain IL-1R1 was evaluated using BIAcore 2000 (BIAcore, Inc., Piscataway, NJ) (19Schuck P. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 541-566Crossref PubMed Scopus (549) Google Scholar). IL-1R1 was immobilized on the sensor surface by amine coupling. Binding of the peptide was analyzed over a concentration range of 3–200 nm at a flow rate of 10 μl/min in 10 mm HEPES (pH 7.2), 150 mm NaCl, 1 mm EDTA, and 0.005% polysorbate 20 (Biacore). Association and dissociation rate constants were calculated using BIAevaluation software. Peptide AF10847 was co-crystallized with the extracellular domains of IL-1R1. The crystals were space group P6522 with unit cell dimensions a =b = 95.90 Å, c = 208.02 Å, α = β = 90°, and γ = 120°. X-ray diffraction data were collected to a resolution of 3.0 Å, and the data collection statistics are shown in Table I. We attempted to solve the complex structure by molecular replacement, starting from the structure of IL-1R1 in complex with either IL-1β or IL-1ra. Search models were constructed using either all atoms or a polyalanine trace for the whole molecule, for the first two domains, or for single domains. We searched for possible solutions with Amore and Xplor using multiple resolution ranges, but all attempts to find a convincing solution failed.Table ICrystallographic data and refinement statisticsData setNativeK2PtC4Resolution (Å)3.03.0No. of unique reflections11,89411,701No. of observations125,32067,989R sym10.310.8 (Last shell)(36.9)(38.8)Completeness 1-aI> = 2o-cutoff. No water molecules were added to the structure. Final bulk solvent density = 0.278 e/Å3; B-factor = 16.2 Å2. Number of residues built = 331.91.086.5 (Last shell)(82.3)(77.6)Rcullis_c0.91Rcullis_anom (30.0 to 6.5 Å)0.99Phasing power_c0.42Refinement statisticsRR free%% Polyalanine search model after rigid body fitting62.463.2 All atom model after rigid body fitting55.555.6 After Powell refinement39.650.0 With bulk solvent correction32.141.5 After simulated annealing27.140.0 Addition of peptide and simulated annealing22.333.21-a I> = 2o-cutoff. No water molecules were added to the structure. Final bulk solvent density = 0.278 e/Å3; B-factor = 16.2 Å2. Number of residues built = 331. Open table in a new tab We therefore decided to collect anomalous heavy atom data using a K2PtCl4 derivative that had worked well for the IL-1R1·IL-1β complex. The resulting Siras map was then searched in real space using Essens to locate each of the three domains of IL-1R1 individually. Even though the phasing power of the Siras derivative was very weak (Table I), the three domains of IL-1R1 were successfully located individually in the IL-1R1·peptide electron density map. At this point, it became quite obvious why the molecular replacement attempts had failed: a rotation of almost 170° had occurred between the third domain relative to the first two (compare Figs.1 and 2). After refinement of IL-1R1, the peptide structure was determined. In the final structure, the root mean square deviation from ideality is 0.0121 Å for bond lengths and 1.91° for bond angles. One residue of IL-1R1 (Glu203) lies in a disallowed region of the Ramachandran plot. This residue is at the sharpest part of the turn joining the second and third domains of IL-1R1. Previous mutagenesis and structural studies by our group (6Vigers G.P.A. Caffes P. Evans R.J. Thompson R.C. Eisenberg S.P. Brandhuber B.J. J. Biol. Chem. 1994; 269: 12874-12879Abstract Full Text PDF PubMed Google Scholar, 22Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and others (20Priestle J.P. Schaer H.P. Gruetter M.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9667-9671Crossref PubMed Scopus (161) Google Scholar, 21Labriola-Thompkins E. Chandran C. Kaffka K.L. Biondi D. Graves B.J. Hatada M. Madison V.S. Karas J. Kilian P.L. Ju G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11182-11186Crossref PubMed Scopus (73) Google Scholar) have shown that IL-1α and IL-1β have two binding sites for IL-1R1, whereas IL-1ra has only one binding site. We previously reported the structure of IL-1β complexed with IL-1R1 and showed that both binding sites on IL-1β bind to a single receptor (23Vigers G.P.A. Anderson L.J. Caffes P. Brandhuber B.J. Nature. 1997; 386: 190-194Crossref PubMed Scopus (233) Google Scholar). One site of IL-1β, referred to as Site A, binds at the junction of the first and second immunoglobulin-like domains of IL-1R1, whereas the second binding site of IL-1β (Site B) binds to the third domain of IL-1R1 (Fig. 1). This domain wraps around IL-1β and is connected to the first two domains by an 8-residue flexible linker. Schreuder et al. (24Schreuder H. Tardif C. Trump-Kallmeyer S. Soffientini A. Sarubbi E. Akeson A. Bowlin T. Yanofsky S. Barrett R.W. Nature. 1997; 386: 194-200Crossref PubMed Scopus (186) Google Scholar) have likewise solved the structure of IL-1ra bound to IL-1R1. Their structure shows that the single binding site of IL-1ra binds to Site A of IL-1R1 in a manner very similar to that of IL-1β. The third domain of IL-1R1, however, swings 20o away from IL-1ra by means of the flexible linker and makes very few contacts with IL-1ra (Fig. 1). These two x-ray structures confirmed the mutagenesis results and suggest that blocking either binding site of IL-1R1 with a small organic molecule or peptide might lead to a useful receptor antagonist. The structure of the IL-1R1·AF10847 complex is shown in Fig.2. IL-1R1 has three well defined immunoglobulin-like domains, as expected from the previously solved IL-1R1 complex structures. However, in this structure, the C-terminal domain is rotated ∼170° relative to its position in the previous complexes. This rotation means that Site B now points away from the ligand, rather than toward it. Meanwhile, the peptide binds in a manner entirely distinct from that of IL-1β and IL-1ra. Residues 6–13 form a short α-helix that binds at Site A of IL-1R1 in the domain 1/2 junction, whereas the N- and C-terminal residues of AF10847 form strands that interact with residues along the sides of the first two domains of the receptor. This complex is considerably more compact than the previous receptor complexes reported, with approximate dimensions of 60 × 52 × 35 Å. The three domains of IL-1R1 appear to be quite rigid. Comparing the x-ray structures of IL-1R1·AF10847 and IL-1R1·IL-1β, the domains are very similar, with root mean square deviations of 1.71, 1.92, and 0.937 Å for residues 8–98, 124–198, and 207–311 of IL-1R1, respectively. The largest changes seen in the structure of IL-1R1 are in residues 113–121 and 199–208. Residues 113–121 form a loop between strands b and c in the second domain of the receptor (23Vigers G.P.A. Anderson L.J. Caffes P. Brandhuber B.J. Nature. 1997; 386: 190-194Crossref PubMed Scopus (233) Google Scholar). Since Lys112 and Lys114 of IL-1R1 make hydrogen bonds with the peptide (TableII), it is likely that this change in conformation is due to the binding of the peptide to IL-1R1. Interestingly, Lys114 makes contacts with all three ligands solved to date, but the contacts are not the same in all three cases (Table II). Residues 199–208 form the flexible linker that joins the second and third immunoglobulin domains and clearly have to move to allow the third domain to take up its dramatically different orientation.Table IIListing of hydrogen bonds between IL-1R1 and the three ligands (inferred from Xplor)IL-1R1AF10847 peptideIL-1βIL-1raNo.ResidueAtomNo.ResidueAtomNo.ResidueAtomNo.ResidueAtom11GluOɛ27LysNζ26ArgNɛ12LysNζ25LeuO14IleN39AsnOδ14IleO34GlnNɛ39AsnNδ16ValN15GlnOɛ32GlnOɛ36GlnOɛ16ValO15GlnNɛ32GlnNɛ36GlnNɛ95LysNζ19LeuO95LysNζ20ProO108GlnOɛ19LeuN109AlaO15GlnNɛ32GlnNɛ36GlnNɛ110IleO33GlyN37Glyn112LysN14TrpO112LysNζ17TyrOH112LysO14TrpN114LysN12TyrO20GlnOɛ114LysNζ11ArgNH114LysNζ11ArgNH114LysO15GlnNɛ20GlnNɛ114LysO16TrpNɛ122GlyO15GlnNɛ20GlnNɛ124ValO13TyrOH127TyrN9SerOδ128GluOɛ127TyrOH10AsnOδ128AspOδ127TyrOH34TyrO129GluOɛ6TrpN128GluN127AlaN163ArgNɛ14GlnOɛ163ArgNH1GluOɛ18ValO204AsnNδ108AsnOδ234ThrOγ12TyrOH237LeuO4ArgNH238SerOγ11GlnOɛ250IleO93LysNζ252GluOɛ93LysN252GluOɛ94LysN259GluOɛ93LysNζ260AspGδ1AlaN261TyrN1AlaO261TyrOH93LysNζ263SerOγ4ArgNH271ArgNH150GluOɛ298LysNζ51GluOɛ299AsnO53ProN300ThrO54HisN300ThrOγ105GluOɛResidues in IL-1R1 that make identical bonds to all three ligands are shown in boldface, whereas those that make non-identical bonds to all three are shown in italics. Residues in IL-1β and IL-1ra that were identified by site-directed mutagenesis as being essential (see, for example, Ref. 22Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) are also shown in boldface. Open table in a new tab Residues in IL-1R1 that make identical bonds to all three ligands are shown in boldface, whereas those that make non-identical bonds to all three are shown in italics. Residues in IL-1β and IL-1ra that were identified by site-directed mutagenesis as being essential (see, for example, Ref. 22Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) are also shown in boldface. Comparing the IL-1R1·AF10847 complex with the previously solved IL-1R1·IL-1ra complex, the first domain of IL-1R1 has rotated 5° with respect to the second domain (the rotation is 2° in the IL-1β complex structure), whereas the third domain has rotated 168°. The C-terminal strand of the third domain of IL-1R1 now touches the N-terminal strand of the first domain. Residues 7–13 of IL-1R1 now form an antiparallel β-sheet with residues 216–220. The two strands have seven hydrogen bonds linking them, which presumably serve to stabilize the structure. This arrangement of the first and third domains of the receptor was unexpected. Despite numerous experimental attempts to determine whether this new IL-1R conformation is physiologically relevant, we have been unable to determine whether this arrangement occurs in the free receptor or is due to peptide binding, to the low pH used to grow the crystals, or to crystal packing forces. In view of the extensive hydrogen-bonding pattern observed between the amino and carboxyl termini of IL-1R1, it is tempting to speculate that this arrangement of the IL-1R1 domains may have physiological significance in preventing the binding of any of the natural IL-1R ligands and that this may be yet another mechanism to modulate IL-1 responses. Because the peptide binds to the same binding site as IL-1ra and IL-1β and because the three IL-1R1 domains are quite rigid, one might expect that the AF10847 peptide would have a structure similar to that of IL-1β or IL-1ra and make similar contacts. This, however, is not the case (Fig.3). The peptide forms an α-helix (residues 6–13) through the binding “groove” of the receptor, whereas β-strands in the protein ligands interact with this receptor binding site. The peptide also positions long strands along the sides of IL-1R1 (residues 1–5 and 14–21), which have no analogue in the natural IL-1 proteins. Most of the residues in these two strands probably do not directly interfere with the binding of IL-1, but instead provide binding energy to hold the α-helical section of the peptide in the groove of the receptor. We speculate that possibly one or both of the long “tails” of the peptide initially bind to IL-1R1, after which the α-helical segment can form and block subsequent IL-1 binding. All three ligands have approximately equal solvent-accessible areas buried at Site A of IL-1R1, with 1301, 1164, and 1009Å2 for AF10847, IL-1ra, and IL-1β, respectively. However, as shown in Table II and Figs. 3 and4, only 1 residue is closely conserved among AF10847, IL-1β, and IL-1ra. This residue (Gln15 in AF10847, Gln32 in IL-1β, and Gln36 in IL-1ra) makes multiple hydrogen bonds to IL-1R1 and was previously identified by mutagenesis studies as being essential for receptor binding (22Evans R.J. Bray J. Childs J.D. Vigers G.P.A. Brandhuber B.J. Skalicky J.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1995; 270: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, other “essential” residues such as Gln15 in IL-1ra (Gln20 in IL-1β) have no analogue in the peptide.Figure 4IL-1R1 binding site surfaces.Illustrated is the solvent-accessible surface of Site A of the receptor, showing all residues of the ligands with >30.0-Å2 change in solvent accessibility upon complex formation. Upper, AF10847 peptide; middle, IL-1β; lower, IL-1ra. The only conserved residue lies on the right-hand side of each panel and is marked with a gold asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This short peptide binds with remarkably high affinity to IL-1R1. As shown in Table III and Fig. 4, most of the residues in the peptide either make hydrogen bonds to IL-1R1 or bury a large amount of their surface area at the receptor-ligand interface. Only 6 of the 21 residues have no hydrogen bonds to the receptor and bury <40 Å2 at the receptor. Four of these (Glu7, Glu8, Ser9, and Ala11) are in the α-helix of the peptide and probably confer solubility to the peptide and keep the helix in register. Pro16 is not in the helix, but serves to redirect the peptide so that the long strand of residues 14–21 lies along the side of IL-1R1. Residues Thr2 and Thr5 seem less important since they lie in the N-terminal part of the peptide that shows fewer interactions with the receptor (Table II).Table IIIAF10847 peptide interactions with IL-1R1AF10847 sequenceBuried areaSide chain hydrogen bondBackbone hydrogen bondÅ21E402T3P744F73*5T276W188*7E8E9S38**10N24*11A1512Y57*13Y137*14W67*15Q113*16P3817Y109*18A4619L46*20P25*21L139Surface area buried per residue is listed. Main chain and side chain hydrogen bonds from the peptide to the receptor are denoted with an asterisk. Residues in the α-helical portion of the peptide are shown in boldface. Open table in a new tab Surface area buried per residue is listed. Main chain and side chain hydrogen bonds from the peptide to the receptor are denoted with an asterisk. Residues in the α-helical portion of the peptide are shown in boldface. Our structural results are consistent with the three high affinity peptides found by panning phage display libraries as described by Yanofsky et al. (10Yanofsky S.D. Baldwin D.N. Butler J.H. Holden F.R. Jacobs J.W. Balasubramanian P. Chinn J.P. Cwirla S.E. Peters-Bhatt E. Whitehorn E.A. Tate E.H. Akeson A. Bowlin T.L. Dower W.J. Barrett R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7381-7386Crossref PubMed Scopus (105) Google Scholar). These peptides showed considerable variability in the first 12 residues (the N-terminal strand and the α-helix) and a strict conservation of the last 9 residues (the C-terminal strand). Synthetically prepared peptides containing truncations of the amino terminus of AF10847 showed that removal of the first 3 residues did not significantly reduce the affinity of the peptide for the receptor, but removal of Phe4 from this peptide resulted in at least a 30-fold decrease in affinity. Approximately 70 Å2 of Phe4 are buried in the receptor complex, and one hydrogen bond is formed; but Phe4is probably also required to position Trp6 into a deep pocket of the receptor. The 9 C-terminal residues are highly conserved and form 10 of the 16 hydrogen bonds between the peptide and receptor. A further indication of the importance of this portion of the peptide is that AF10847 does not bind to murine IL-1R: 5 of the 10 hydrogen bonds formed between the C-terminal portion of the peptide and human IL-1R1 are made at Lys95 and Lys112. In murine IL-1R1, these residues are threonine and proline, which implies that the hydrogen-bonding pattern is unlikely to be maintained. Even though the crystal structure indicates that the third domain of the receptor touches the peptide, we do not believe that much binding energy is contributed by this interaction. In the x-ray structure, the only hydrogen bond with the third domain is from the OH of Tyr12, and only 115 Å2 are buried at the third domain. Comparison of the sequences of the three high affinity peptides of Yanofsky et al. (10Yanofsky S.D. Baldwin D.N. Butler J.H. Holden F.R. Jacobs J.W. Balasubramanian P. Chinn J.P. Cwirla S.E. Peters-Bhatt E. Whitehorn E.A. Tate E.H. Akeson A. Bowlin T.L. Dower W.J. Barrett R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7381-7386Crossref PubMed Scopus (105) Google Scholar) shows that either glycine or arginine at this position results in tight receptor binding. We have also determined the apparent association and dissociation rates of AF10847 by BIAcore measurements (data not shown) on both full-length IL-1R1 (Asp1–Thr315) and a truncated version of the receptor containing the first two domains of IL-1R1 (Asp1–Lys205). The association rates were 9.8 × 105 and 9.9 × 105m−1 s−1, respectively, whereas the dissociation rates were 5.8 × 10−3 and 6.0 × 10−3 s−1, respectively. Because both the association and dissociation rates are virtually identical, the interactions of the peptide with the third domain of the receptor appear to be kinetically negligible. IL-1R1 is remarkably pleiotropic in that it binds to three different naturally occurring 20-kDa protein ligands with high affinity, even though they share <30% homology. In addition, IL-1R also binds with nanomolar affinity to a 21-amino acid peptide that has a completely different secondary structure than any of the natural ligands. Comparisons of these structures show that both natural ligands and the peptide antagonist interact with the receptor in the junction area of domains 1 and 2, but that neither IL-1ra nor the peptide interacts with the second binding site located in the third receptor domain. The structure shows an unexpected binding mode for the peptide, with it being in a mainly extended conformation with a central short helical section. The interactions with the receptor are not well conserved between the ligands and therefore provide new insights into IL-1R1 interactions to aid in the design of smaller IL-1 antagonists." @default.
• W2016064695 created "2016-06-24" @default.
• W2016064695 creator A5015825916 @default.
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