Mapping Receptor Binding Sites in Interleukin (IL)-1 Receptor Antagonist and IL-1β by Site-directed Mutagenesis
1995; Elsevier BV; Volume: 270; Issue: 19 Linguagem: Inglês
10.1074/jbc.270.19.11477
ISSN1083-351X
AutoresR J Evans, Jeff Bray, John D. Childs, Guy Vigers, Barbara J. Brandhuber, Jack J. Skalicky, Robert C. Thompson, Stephen P. Eisenberg,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoInterleukin-1 receptor antagonist (IL-1ra), an IL-1 family member, binds with high affinity to the type I IL-1 receptor (IL-1RI), blocking IL-1 binding but not inducing an IL-1-like response. Extensive site-directed mutagenesis has been used to identify residues in IL-1ra and IL-1β involved in binding to IL-1RI. These analyses have revealed the presence of two discrete receptor binding sites on IL-1β. Only one of these sites is present on IL-1ra, consisting of residues Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147. Interestingly, the absent second site is at the location of the major structural difference between IL-1ra and IL-1β, which are otherwise structurally similar. The two receptor binding sites on IL-1β are also present on IL-1α. Thus, it appears that the two IL-1 agonist molecules have two sites for IL-1RI binding, and the homologous antagonist molecule, IL-1ra, has only one. Based on these observations, a hypothesis is presented to account for the difference in activity between the agonist and antagonist proteins. It is proposed that the presence of the two receptor binding sites may be necessary for agonist activity. Interleukin-1 receptor antagonist (IL-1ra), an IL-1 family member, binds with high affinity to the type I IL-1 receptor (IL-1RI), blocking IL-1 binding but not inducing an IL-1-like response. Extensive site-directed mutagenesis has been used to identify residues in IL-1ra and IL-1β involved in binding to IL-1RI. These analyses have revealed the presence of two discrete receptor binding sites on IL-1β. Only one of these sites is present on IL-1ra, consisting of residues Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147. Interestingly, the absent second site is at the location of the major structural difference between IL-1ra and IL-1β, which are otherwise structurally similar. The two receptor binding sites on IL-1β are also present on IL-1α. Thus, it appears that the two IL-1 agonist molecules have two sites for IL-1RI binding, and the homologous antagonist molecule, IL-1ra, has only one. Based on these observations, a hypothesis is presented to account for the difference in activity between the agonist and antagonist proteins. It is proposed that the presence of the two receptor binding sites may be necessary for agonist activity. The IL-1 1The abbreviations used are: IL-1, interleukin-1; IL-1ra, interleukin-1 receptor antagonist; IL-1RI, 80-kDa type 1 IL-1 receptor. family of proteins are related by sequence similarity, gene organization, and three-dimensional structure (Eisenberg et al., 1990Eisenberg S.P. Evans R.J. Arend W.P. Verderber E. Brewer M.T. Hannum C.H. Thompson R.C. Nature. 1990; 343: 341-346Crossref PubMed Scopus (927) Google Scholar, Eisenberg et al., 1991Eisenberg S.P. Brewer M.T. Verderber E. Heimdal P. Brandhuber B.J. Thompson R.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5232-5236Crossref PubMed Scopus (221) Google Scholar; Carter et al., 1990Carter D.B. Deibel Jr., M.R. Dunn C.J. Tomich C-S. C Laborde A.L. Slightom J.L. Berger A.E. Bienkowski M.J. Sun F.F. McEwan R.N. Harris P.K.W. Yem A.W. Waszak G.A. Chosay J.G. Sieu L.C. Hardee M.M. Zurcher-Neely H.A. Reardon I.M. Heinrickson K.L. Truesdell S.E. Shelly J.A. Eessalu T.E. Taylor B.M. Tracey D.E. Nature. 1990; 344: 633-638Crossref PubMed Scopus (537) Google Scholar. The three proteins, IL-la, IL-1β, and IL-1ra, all exhibit a β-trefoil topology characterized by six β-strands forming a tapered β-barrel, which is closed at the wide end by another six β-strands (Murzin et al., 1992Murzin A.G. Lesk A.M. Chothia C. J. Mol. Biol. 1992; 223: 531-543Crossref PubMed Scopus (307) Google Scholar; Vigers et al., 1994Vigers 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. The two agonist proteins, IL-1α and IL-1β, have similar biological activities, mediated through their high affinity interaction with the type 1 IL-1 receptor (IL-1RI) (Sims et al., 1993Sims J.E. Gayle M.A. Slack J.L. Alderson M.R. Bird T.A. Giri J.G. Colotta F. Re F. Mantovani A. Shanebeck K. Grabstein K.H. Dower S.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6155-6159Crossref PubMed Scopus (548) Google Scholar. These two proteins are believed to play an important role in causing both local and systemic inflammatory responses (Dinarello, 1991Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar). The third family member, IL-1ra, also binds with high affinity to IL-1RI but does not elicit a biological response (Hannum et al., 1990Hannum C.H. Wilcox C.J. Arend W.P. Joslin F.G. Dripps D.J. Heimdal P.L. Armes L.G. Sommer A. Eisenberg S.P. Thompson R.C. Nature. 1990; 343: 336-340Crossref PubMed Scopus (956) Google Scholar; Dripps et al., 1991Dripps D.J. Brandhuber B.J. Thompson R.C. Eisenberg S.P. J. Biol. Chem. 1991; 266: 10331-10336Abstract Full Text PDF PubMed Google Scholar. IL-1ra competitively inhibits the binding of IL-1α and IL-1β and thus acts as a specific inhibitor of IL-1 activity. Exogenously administered human recombinant IL-1ra can significantly reduce the severity of inflammation in many animal models of inflammatory disease, thus implicating IL-1 as an important mediator of inflammation in these models (Dinarello and Thompson, 1991Dinarello C.A. Thompson R.C. Immunol. Today. 1991; 12: 404-410Abstract Full Text PDF PubMed Scopus (680) Google Scholar). Endogenous IL-1ra plays an important role in reducing the severity of the inflammatory response by IL-1, since administration of neutralizing antibodies to IL-1ra causes an increase in severity and a prolongation of intestinal inflammation (Ferretti et al., 1994Ferretti M. Casini-Raggi V. Pizarro T.T. Eisenberg S.P. Nast C.C. Cominelli F. J. Clin. Invest. 1994; 94: 449-453Crossref PubMed Scopus (147) Google Scholar. Several studies utilizing site-directed mutagenesis have led to the identification of residues in IL-1a and IL-18 that are important for either receptor binding or receptor-mediated biological activity (MacDonald et al., 1986MacDonald H.R. Wmgfield P. Schmeissner U. Shaw A. Clore G.M. Gronenborn A.M. FEBS Lett. 1986; 209: 295-298Crossref PubMed Scopus (47) Google Scholar; Gehrke et al., 1990Gehrke L. Jobling S.A. Paik L.S.K. McDonald B. Rosenwasser L.J. Auron P.E. J. Biol. Chem. 1990; 265: 5922-5925Abstract Full Text PDF PubMed Google Scholar; Yamayoshi et al., 1990Yamayoshi M. Ohue M. Kawashima H. Kotani H. Iida M. Kawata S. Yamada M. Lymphokine Res. 1990; 9: 405-413PubMed Google Scholar; Ju et al., 1991Ju G. Labriola-Tompkins E. Campen C.A. Benjamin W.R. Karas J. Plocinski J. Biondi D. Kaffka K.L. Kilian P.L. Eisenberg S.P. Evans R.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2658-2662Crossref PubMed Scopus (74) Google Scholar; Labriola-Tompkins et al., 1991Labriola-Tompkins E. Chandran C. Kaflka K.L. Biondi D. Graves B.J. Hatada M. Madison V.S. Karas J. Kilian P.L. Ju G. Proc. Natl. Acad. Sci. V. S. A. 1991; 88: 11182-11186Crossref PubMed Scopus (73) Google Scholar, Labriola-Tompkins et al., 1993Labriola-Tompkins E. Chandran C. Varnell T.A. Madison V.S. Ju G. Protein Eng. 1993; 6: 535-539Crossref PubMed Scopus (21) Google Scholar; Grütter et al., 1994Grütter M.G. van Oostrum J. Priestle J.P. Edelmann E. Joss U. Feige U. Vosbeck K. Schmitz A. Protein Eng. 1994; 7: 663-671Crossref PubMed Scopus (30) Google Scholar; Gayle et al., 1993Gayle III, R.B. Poindexter K. Cosman D. Dower S.K. Gillis S. Hopp T. Jerzy R. Kronheim S. Lum V. Lewis A. Goodgame M.M. March C.J. Smith D.L. Srinivasan S. J. Biol. Chem. 1993; 268: 22105-22111Abstract Full Text PDF PubMed Google Scholar. An analysis of these results suggests there are two main sites on the surface of the IL-1 agonist molecules that are involved in receptor binding. One site (site A), located on the side of the 8-barrel structure, was originally identified by mutagenesis of IL-18 residue histidine 30 (His-30). Additional residues in this region have more recently been identified in both IL-1α and IL-1β. A second site (site B), approximately 20–25 À from the first site, is located at the open end of the 8-barrel and includes several charged residues, e.g. Arg-4, Lys-93, Lys-103, and Glu-105 of IL-1β. We report here the results of site-directed mutagenesis studies to identify residues on IL-1ra important for IL-1RI binding. Our results indicate that the IL-1ra residues homologous to receptor binding site A of IL-1a and IL-18 also play a role in IL-1RI binding of IL-1ra but that residues of IL-1ra homologous to receptor binding site B on IL-1α and IL-1β do not interact with the receptor. In addition, we believe that site A is the only receptor binding site on IL-1ra because we have mutagenized or deleted almost every residue on the surface of the molecule (a total of 103 residues) and found that only the residues of site A affect receptor binding. We have also performed site-directed mutagenesis of IL-18 and found that site A on IL-18 is larger than previously thought. Site-directed mutagenesis of IL-1ra and IL-1β genes was performed using a Bio-Rad Mutagene kit. Muteins were expressed in E. coli and purified to near homogeneity from the soluble cell lysate as previously described (Eisenberg et al., 1990Eisenberg S.P. Evans R.J. Arend W.P. Verderber E. Brewer M.T. Hannum C.H. Thompson R.C. Nature. 1990; 343: 341-346Crossref PubMed Scopus (927) Google Scholar. Protein concentrations were determined by BCA protein assay (Pierce), absorbance at 280 nm, and relative intensity of bands on a Coomassie-stained polyacrylamide gel. Receptor binding assays were done as described (Hannum et al., 1990Hannum C.H. Wilcox C.J. Arend W.P. Joslin F.G. Dripps D.J. Heimdal P.L. Armes L.G. Sommer A. Eisenberg S.P. Thompson R.C. Nature. 1990; 343: 336-340Crossref PubMed Scopus (956) Google Scholar. Briefly, a standard amount of 35S-labeled IL-1ra at a concentration approximately equal to its Kd (150 pM) was incubated with mouse EL4 thymoma (ATCC, TIB181, ~5000 receptors per cell) or a Chinese hamster ovary cell line (kindly provided by R. Chizzonite) expressing the human type 1 IL-1 receptor (~30,000 receptors per cell) for 4 h at 4 °C with varying concentrations of cold competitor. The cells were harvested through a Millipore millititer plate filter system, and radioactivity retained on the filter was counted on an Ambis radioanalytical imaging system. Percent wild type activity was defined as IC50 (wild type)/IC50 (mutein). The Kd for the wild type IL-1ra (as competitor) was estimated using a simplification of the Cheng-Prusoff relationship (Kd = IC50/2, Cheng and Prusoff", 1973Cheng Y. Prusoff" W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12330) Google Scholar and ranged from 150 to 400 pM, consistent with values previously reported. In each assay, a wild type control was included. All muteins were assayed a minimum of two times with the standard error between assays generally ≤25%. One-dimensional 1H-labeled NMR spectra were collected for IL-1ra and several of its muteins. The spectra were recorded on a VXR500 spectrometer housed at the University of Colorado-Boulder. The 1.5 mM IL-1ra solutions (90% H2O, 10% D2O) were prepared in 10 mM phosphate (pH = 6.0), 100 mM NaCl, and 0.1 mM EDTA. Spectra were recorded at temperatures of 25, 30, and 35 °C. Each spectrum was collected with 8192 complex points over a spectral width of 7000 Hz, and a total of 64 transients were signal averaged for each free induction decay. All data were processed on a Sun 4260 using the NMR processing software FELIX (Hare Research). The spectra of IL-1ra wild type and mutants were plotted to allow for comparison of the backbone (Hn and Ha) and methyl proton chemical shifts and intensities. Since only the C-α coordinates of IL-1α have been deposited in the Brookhaven Data base, we modeled possible side chain positions for comparison to IL-1β and IL-1ra. Initial coordinates were assigned at random for missing atoms and then refined using simulated annealing in X-PLOR (Brünger et al., 1987Brünger A. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar. Three different starting positions gave similar final conformations in all areas of interest. 2G. Vigers, unpublished data. Using site-directed mutagenesis, we replaced 93 of the 152 residues of IL-1ra and made two deletion mutants of 3 and 10 amino acids at the amino terminus. Initial single replacements were to glycine or alanine or, in a few instances, to an amino acid of the opposite charge. Muteins were assayed by competition with 35S-labeled IL-1ra for binding to mouse EL4 cells or to Chinese hamster ovary cells expressing recombinant full-length human IL-1RI. From this initial round of mutagenesis, 54 of the muteins were assayed on EL4 cells, 33 were assayed on Chinese hamster ovary cells, and the remaining 6 were assayed on both cell lines. For the vast majority of muteins, including the two deletion mutants, competition was not significantly different from that of the wild type protein (Fig. 1). The muteins that had less than 35% of wild type activity were investigated further. Five residues in IL-1ra were initially found to be sensitive to alanine or glycine substitution. Four of them, Trp-16, Gln-20, Tyr-34, and Tyr-147, were sensitive to substitution by other residues as well (Table I). At each of these residues, certain changes reduced receptor binding by 100-fold or more. These four residues form a contiguous patch on the IL-1ra surface, and two of them, Trp-16 and Tyr-34, correspond to residues Arg-11 and His-30, which have been previously identified as important in site A of IL-1β. One of the four residues, Tyr-147, exhibited interesting receptor binding properties in that Y147G bound with lower affinity than wild type IL-1ra to human IL-1RI (34%) but with higher affinity to the mouse receptor (252%).Table IReceptor binding activity of IL-1ra muteinsIL-1ra muteinWild type activity%W16G12 ± 3 (4)W16K<1,<1W16M5,7W16Q9,18W16R21 ± 2 (3)W16Y53,85Q20A6,8Q20Y<1,<1Q20D<1,<1,<1Q20K<1,<1Q20M18,22Q20N43,46Y34G21,23Y34K<1,<1Y34D7,11Y34H80,84Y34W88,98Y34M86,100Q36F<1,<1Y147G24,44Y147K<1,<1Y147T3,5Y147H41,69Y147M63, 69 Open table in a new tab The fifth residue that exhibited ≤35% of wild type activity in the initial screen, D74G, exhibited ~30% of wild type IL-1ra binding to the mouse IL-1RI but ~50% binding to the human receptor. Since muteins D74A, D74F, D74C, and D74W all exhibit normal IL-1RI binding activity to the human receptor (data not shown), we believe that this residue is not fundamentally important for IL-1ra binding to IL-1RI. One additional residue, Gln-36, was investigated further (despite the fact that Q36G exhibited normal binding on EL4 cells) due to the importance of the homologous residue on IL-1β, Gln-32 (see below and Discussion). We found Q36F had only 1% of wild type activity (Table I), suggesting that this residue is also important for receptor binding. Gln-36 is adjacent to the four important residues discussed above. The reduced affinity of muteins with changes at the five residues that make up the important binding site in IL-1ra (Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147) suggests these residues contribute to the interaction of IL-1ra with IL-1RI. However, it is also possible that the mutations may disrupt the tertiary structure of the muteins, which causes a reduction in receptor affinity. To address this issue, we performed high resolution one-dimensional NMR on wild type IL-1ra and muteins Y34G, Q20A, and W16G. No significant differences were observed in the spectra of these four molecules, indicating there were no major differences among their structures. In addition, all muteins eluted at approximately the same salt concentration from an ion exchange column, suggesting that these molecules all have a similar conformation and overall charge. Seven IL-1β residues (Arg-4, Leu-6, Phe-46, Ile-56, Lys-93, Lys-103, and Glu-105) in the vicinity of the open end of the β-barrel have previously been shown to be important for binding to IL-1RI (Labriola-Tompkins et al., 1991Labriola-Tompkins E. Chandran C. Kaflka K.L. Biondi D. Graves B.J. Hatada M. Madison V.S. Karas J. Kilian P.L. Ju G. Proc. Natl. Acad. Sci. V. S. A. 1991; 88: 11182-11186Crossref PubMed Scopus (73) Google Scholar. Four of these seven are charged residues. Based on previously published sequence alignments (Stockman et al., 1992Stockman B.J. Scahill T.A. Roy M. Ulrich E.L. Strakalaitis N.A. Brunner D.P. Yem A.W. Deibel Jr., M.R. Biochemistry. 1992; 31: 5237-5244Crossref PubMed Scopus (35) Google Scholar, these charges are conserved in IL-1ra and have been suggested to be important for receptor binding. Thus, the homologous region of IL-1ra (Pro-50, Glu-52, His-54, Ser-89, Glu-90, Asn-91, Arg-92, Lys-93, Gln-94, Asp-95, Lys-96, Arg-102, Ser-103, Asp-104, Ser-105) was extensively mutagenized (FIG. 1, FIG. 2, see also Vigers et al., 1994Vigers 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) to investigate whether it had a similar function in the binding of IL-1ra to IL-1RI. Interestingly, none of the changes in this region of IL-1ra affected activity in the competitive receptor binding assay (Fig. 1). An additional residue near the carboxyl terminus of these proteins, IL-1α Asp-151, IL-1β Asp-145, and IL-1ra Lys-145, has been characterized as important for biological activity but not for IL-1RI affinity. Of particular interest is the human IL-1ra mutein, K145D that has been shown to exhibit partial agonist activity on mouse cells but exhibits normal affinity for both the mouse and human receptors (Yamayoshi et al., 1990Yamayoshi M. Ohue M. Kawashima H. Kotani H. Iida M. Kawata S. Yamada M. Lymphokine Res. 1990; 9: 405-413PubMed Google Scholar; Ju et al., 1991Ju G. Labriola-Tompkins E. Campen C.A. Benjamin W.R. Karas J. Plocinski J. Biondi D. Kaffka K.L. Kilian P.L. Eisenberg S.P. Evans R.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2658-2662Crossref PubMed Scopus (74) Google Scholar. This is consistent with our observation that replacing Lys-145 of IL-1ra with glycine has no affect on receptor binding activity (Fig. 1). We performed site-directed mutagenesis on IL-1β to reevaluate the role of the known site A residues and to examine other IL-1β residues in the vicinity of this site. In addition, we wished to analyze residues in or near site B of IL-1β. We find that three residues in the region of site A appear to be important for binding IL-1β to IL-1RI (Table II). Two residues, Gln-15 and His-30 of IL-1β, align with IL-1ra residues Gln-20 and Tyr-34, which were identified as important from the mutagenesis of that protein. The third residue, Gln-32, corresponds to Gln-36 of IL-1ra, which was not originally identified by the scanning mutagenesis shown in Fig. 1. However, further mutagenesis of this residue in IL-1ra did suggest its importance in receptor binding (Table I).Table IIReceptor binding activity of IL-1β muteinsIL-1β muteinWild type activity%R11GaSite A residues.45R11AaSite A residues.55Q15GaSite A residues.<1Q15HaSite A residues.130H30GaSite A residues.<1H30EaSite A residues.2H30AaSite A residues.10H30SaSite A residues.10Q32GaSite A residues.<1156GbSite B residues.10K92GbSite B residues.8K93GbSite B residues.<1K93MbSite B residues. 53 kJ/mol. Thus there is reasonably close agreement between these numbers, considering the broad assumptions made in the calculations. A simple way of defining the regions of a protein important for receptor binding is through the use of in vitro site-directed mutagenesis. This method has been used over the past few years by several groups to identify residues involved in the high affinity binding of IL-1α and IL-1β to IL-1RI. These studies have led to the conclusion that two distinct receptor binding regions (site A and site B) are present on both IL-1α and IL-1β. Site A, located on the side of the β-barrel structure, was originally identified by the mutagenesis of IL-1β residues His-30 (MacDonald et al., 1986MacDonald H.R. Wmgfield P. Schmeissner U. Shaw A. Clore G.M. Gronenborn A.M. FEBS Lett. 1986; 209: 295-298Crossref PubMed Scopus (47) Google Scholar and Arg-11 (Gehrke et al., 1990Gehrke L. Jobling S.A. Paik L.S.K. McDonald B. Rosenwasser L.J. Auron P.E. J. Biol. Chem. 1990; 265: 5922-5925Abstract Full Text PDF PubMed Google Scholar. Recently, additional residues in this region have been identified on IL-1α (Gayle et al., 1993Gayle III, R.B. Poindexter K. Cosman D. Dower S.K. Gillis S. Hopp T. Jerzy R. Kronheim S. Lum V. Lewis A. Goodgame M.M. March C.J. Smith D.L. Srinivasan S. J. Biol. Chem. 1993; 268: 22105-22111Abstract Full Text PDF PubMed Google Scholar and on IL-1β (Grütter et al., 1994Grütter M.G. van Oostrum J. Priestle J.P. Edelmann E. Joss U. Feige U. Vosbeck K. Schmitz A. Protein Eng. 1994; 7: 663-671Crossref PubMed Scopus (30) Google Scholar. We have extensively mutagenized IL-1ra and identified only five residues, Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147, that are important for binding to the IL-1RI. These five residues, when changed to glycine, were also found to be important for binding to recombinant soluble human IL-1RI using surface plasmon resonance technology on a BIAcore instrument. Glycine substitutions at other positions had no significant affect on binding to human IL-1RI either on the surface of cells or to the purified receptor on the BIAcore. 3D. J. Dripps and J. Jordan, unpublished data. All of these important amino acids map to site A, which is conformationally conserved among the IL-1 family members. The chemical nature of the IL-1ra site A side chains is critical for high affinity binding to IL-1RI, as shown by the effect of amino acid substitution on binding. In addition to identifying substitutions that lead to a significant loss in binding, we have found some substitutions that have little or no affect on binding. In this regard, we show that replacing the aromatic amino acids Trp-16 and Tyr-34 in IL-1ra with other aromatic amino acids has no significant affect on binding. It is also interesting to note that Tyr-34 of IL-1ra can be replaced with histidine, which is the corresponding residue in IL-1β, with no loss in binding, and Gln-20 of IL-1ra can be replaced with asparagine, which is the corresponding IL-1α residue, with only a moderate loss in binding. In addition, Gln-20 and Gln-36 of IL-1ra correspond to Gln-15 and Gln-32 of IL-1β, based on the three-dimensional structures of these molecules. In light of the chemical similarity of site A among these proteins and the effect of mutagenesis of residues comprising this site, it appears that this region is both structurally and functionally homologous among the three IL-1 family members. The second receptor binding region (site B) is located at the open end of the β-barrel and includes IL-1β residues Arg-4, Lys-6, Phe-46, Ile-56, Lys-92, Lys-93, Lys-103, and Glu-105 and IL-la residues Arg-16, Ile-18, Asp-64, Asp-65, Ile-68, Lys-100, Asn-108, Trp-113, Gly-17, and Gln-136 (Labriola-Tompkins et al., 1991Labriola-Tompkins E. Chandran C. Kaflka K.L. Biondi D. Graves B.J. Hatada M. Madison V.S. Karas J. Kilian P.L. Ju G. Proc. Natl. Acad. Sci. V. S. A. 1991; 88: 11182-11186Crossref PubMed Scopus (73) Google Scholar, Labriola-Tompkins et al., 1993Labriola-Tompkins E. Chandran C. Varnell T.A. Madison V.S. Ju G. Protein Eng. 1993; 6: 535-539Crossref PubMed Scopus (21) Google Scholar. The area in IL-1ra that is structurally analogous to site B of IL-1β is not involved in binding to IL-1RI. A careful comparison of IL-1β and IL-1ra in this region revealed very significant structural differences between the two molecules (Vigers et al., 1994Vigers 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. Thus, the difference in function in this region between the agonists and the antagonist is consistent with this being the region exhibiting the greatest difference in structure between these two classes of protein. Through the extensive use of site-directed mutagenesis, we have demonstrated that the IL-1 antagonist is missing one of the two receptor binding regions identified on the two IL-1 agonists. This finding suggests a model for receptor activation in which IL-1α and IL-1β, by contacting two sites on IL-1RI, causes activation, but IL-1ra, in making contacts with the receptor at only one of the two receptor binding sites, is unable to induce an IL-1-like signal (Fig. 4). An alternative model, in which the IL-1 agonists act by dimerizing two IL-1 receptors molecules, as in the case of human growth hormone (Wells et al., 1993Wells J.A. Cunningham B.C. Fuh G. Lowman H.B. Bass S.H. Mulkerrin M.G. Ultsch M. deVos A.M. Recent Prog. Harm. Res. 1993; 48: 253-275Crossref PubMed Google Scholar, or by forming a heterodimer of IL-1R1 and the recently identified IL-1 receptor accessory protein (Greenfeder et al., 1994Greenfeder S. Chizzonite R. Ju G. J. Cell. Biochem. 1994; 18: 217Google Scholar is also possible and is consistent with the observation that IL-1ra binds with a substantially higher affinity to the soluble form of IL-1RI than either IL-1α or IL-1β (Svenson et al., 1993Svenson M. Hansen M.B. Heegaard P. Abell K. Bendtzen K. Cytokine. 1993; 5: 427-435Crossref PubMed Scopus (75) Google Scholar. However, dimerizing two IL-1RI molecules seems unlikely because attempts to demonstrate the involvement of aggregation of IL-1RI by either IL-1α or IL-1β in receptor activation have been unsuccessful (Slack et al., 1993Slack J. McMahan C.J. Waugh S. Schooley K. Spriggs M.K. Sims J.E. Dower S.K. J. Biol. Chem. 1993; 268: 2513-2524Abstract Full Text PDF PubMed Google Scholar. In addition, binding experiments with IL-1β muteins and soluble IL-1RI on the BIAcore indicate that site A and site B residues both interact with the purified IL-1RI. 4P. Caffes and R. J. Evans, unpublished data. IL-1ra binds to IL-1RI with the same affinity as the IL-1 agonists but uses only one binding site instead of two. Since site A of IL-1ra is compact and generates high affinity binding with the IL-1RI, it may act as a template for rational design for a small molecule IL-1 antagonist. Such a small molecule is likely to contain pharmacophoric elements that match sidechain atoms of the site A residues. Additional structure-function studies are being done to further discern how these side chain atoms of IL-1ra contact residues in IL-1RI. We thank Richard Chizzonite for kindly providing the cell line expressing the human IL-1RI, Marlyn Troetsch for expert technical assistance, and Hiko Kohno for useful critique of the manuscript.
Referência(s)