ABSTRACT
Using site-directed mutagenesis, we have determined the location and composition of the binding sites in human IL-1 alpha and IL-1 beta for the Type I IL-1 receptor (IL-1R). The binding site in each ligand is a discontinuous epitope made up of at least seven amino acids whose side chains are exposed on a contiguous region of the protein surface. Although human IL-1 alpha and IL-1 beta have similar affinities and cross-compete for binding to the human Type I IL-1R, the binding site residues are not identical in the two ligands. In addition, the residues in the binding site of each ligand contribute differently to binding of the human versus the mouse IL-1R. The structure of the IL-1 binding site has implications for the rational design of IL-1 antagonists.
Subject(s)
Drug Design , Interleukin-1/chemistry , Mutagenesis, Site-Directed , Receptors, Interleukin-1/metabolism , Animals , Binding Sites , Binding, Competitive , Humans , Interleukin-1/genetics , Mice , Receptors, Interleukin-1/chemistry , Recombinant Proteins/metabolism , Species Specificity , Structure-Activity RelationshipABSTRACT
Using oligonucleotide-directed mutagenesis, the binding site on human interleukin-1 alpha (IL-1 alpha) for the human type I IL-1 receptor (IL-1R) has been analyzed. Substitution of seven amino acids (Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resulted in a significant loss of binding to the receptor. Based on crystallographic information, the side chains of these residues are clustered in one region of IL-1 alpha and exposed on the surface of the protein. Five of the residues in the IL-1 alpha binding site align with the binding residues previously determined in human IL-1 beta, demonstrating that the type I IL-1R recognizes homologous regions in both ligands. Unexpectedly, only three of the aligned residues are identical between IL-1 alpha and IL-1 beta. These observations suggest that the composition of contact residues in the binding site is unique for each ligand-receptor complex in the IL-1 system.
Subject(s)
Interleukin-1/genetics , Interleukin-1/metabolism , Receptors, Interleukin-1/metabolism , Animals , Binding Sites/genetics , Binding, Competitive , CHO Cells , Cricetinae , DNA Mutational Analysis , Epitopes/genetics , Humans , Interleukin-1/analogs & derivatives , Mice , Mutagenesis, Site-Directed , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Human interleukin 1 beta (IL-1 beta) exerts its diverse biological effects by binding to specific receptors on target cells. Two types of IL-1 receptor (IL-1R) have been identified: the type I IL-1R (p80) and the type II IL-1R (p68). Using site-specific mutagenesis, we have identified the binding site on IL-1 beta for the murine type I IL-1R. Analogs of the IL-1 beta protein containing defined amino acid substitutions were produced and tested for competitive binding to the two IL-1Rs. Substitutions of the amino acids at seven positions resulted in analogs that had greater than or equal to 100-fold reductions in competitive binding to the type I IL-1R, while maintaining substantial binding to the type II IL-1R. These seven amino acids (Arg-4, Leu-6, Phe-46, Ile-56, Lys-93, Lys-103, and Glu-105) are clustered in the IL-1 beta molecule, forming a discontinuous binding site. The side chains of all seven residues are exposed on the surface of IL-1 beta. The cumulative binding energies contributed by each of the residues predict a binding affinity that is consistent with the observed Kd of the wild-type protein for the type I IL-1R.
Subject(s)
Interleukin-1/metabolism , Receptors, Immunologic/metabolism , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Humans , Interleukin-1/chemistry , Interleukin-1/genetics , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Interleukin-1 , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , X-Ray DiffractionABSTRACT
Interleukin 1 (IL-1) receptor antagonist (IL-1ra) is a naturally occurring protein that binds to the IL-1 receptor present on T cells, fibroblasts, and other cell types and acts to block IL-1-induced responses. IL-1ra is a pure antagonist and has no agonist activity in in vitro or in vivo systems. By site-specific mutagenesis, an analog of IL-1ra was created that contained a substitution of a single amino acid, Lys-145----Asp. This analog, IL-1ra K145D, exhibited partial agonist activity in the D10.G4.1 cell proliferation assay. The newly acquired agonist activity could not be neutralized by antisera to IL-1 alpha or IL-1 beta, but it could be blocked by a monoclonal antibody to the T-cell IL-1 receptor. The analog also showed agonist activity as assayed by increased prostaglandin E2 synthesis from CHO cells expressing recombinant mouse IL-1 receptor. These results with IL-1ra K145D demonstrate the importance of the region surrounding the corresponding Asp-145 residue in IL-1 beta for triggering the biological response to IL-1.
