Identification of the discontinuous binding site in human interleukin 1 beta for the type I interleukin 1 receptor.

Abstract

Human interleukin 113 (IL-113) exerts its diverse biological effects by binding to specific receptors on target cells. Two ypes 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-11 for the murine type I IL-1R. Analogs of the IL-1fL 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 .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, are clustered in the IL-13 molecule, forming a discontinuous binding site. The side chains of all seven residues are exposed on the surface of IL-14. 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.Human interleukins la and 1,B (IL-la and IL-1*3) are polypeptide hormones that share limited identity in amino acid sequence but are functionally similar and exhibit a multiplicity of biological activities. Their diverse biological effects indicate that IL-1 is one of the major mediators in the regulation of immunologic and inflammatory responses (1-3). Both IL-la and IL-1p8 bind to the same receptors on different cell types with high affinity (4-6). Two types of IL-1 receptor (IL-lR) have been identified. The IL-lR that is present on T cells is an 80-kDa glycoprotein that has been molecularly cloned from mouse and human cells (7,8 MATERIALS AND METHODSBacterial Strains and Plasmids. The Escherichia coli expression plasmid for human IL-1f, pEV1-IL-lbeta, has been described (27). Oligonucleotide-directed mutagenesis was performed as described (27). For each mutation, a specific restriction site was included in the synthetic oligonucleotide to monitor the incorporation of the mutated sequence at the correct location in the IL-1p8 coding region. Mutations were confirmed by nucleotide sequence analysis as described (28).Preparation of IL-1(3 Analog Proteins. Crude extracts. Bacterial cells containing the wild-type or mutated IL-1p3 expression plasmids were grown at 300C to early logarithmic phase (OD6w = 0.5) in M9 medium. The cultures were then induced for protein synthesis by transfer to 420C for --2 h, until OD600 = 1.0 ± 0.1. Samples of the cultures were then pelleted by centrifugation at 12,000 x g for 20 min at 40C. Extracts were made from the cell pellets by vigorous Vortex mixing and heating at 1000C in Laemmli sample buffer (29). The extracted proteins were analyzed by SDS/PAGE to assess the size and levels of expression of the IL-1.3 analog proteins, relative to that of wild-type IL-1,3 induced in parallel cultures.Purified proteins. Pellets of E. coli cells induced for 8 synthesis were disrupted by sonication and then adjusted to pH 4 with 6 M HCL....

Molecular Cloning and Phylogenetic Analysis of Inflammatory Cytokines of the Ferret (Mustela putorius furo)

“…Ferrets have several amino acids that, from the discontinuous binding site for the type I IL-1 receptor, maintain the conformation of IL-1β and have carboxyl-terminal endpoints for active protein (TDF) that are conserved in the same positions, as seen in previous reports [10,14,16,19,20]. In ferret IL-1β, comparison of each residue suggests that these conserved amino acids have the same function as those of human IL-1β.…”

“…These amino acids form a discontinuous binding site for the type I IL-1 receptor in humans [16]. These amino acids were conserved in ferret were found at the same positions in other mammals, except for the mouse (Fig.…”

KdPT, a Tripeptide Derivative of α-Melanocyte–Stimulating Hormone, Suppresses IL-1β–Mediated Cytokine Expression and Signaling in Human Sebocytes

“…We thus turned to previously reported sitedirected mutation studies to investigate if any Lys, Pro, or Thr residues on IL-1b had been shown to be important or critical for receptor binding. Interestingly, Lys 209 of IL-1b was found to be absolutely essential for activity, with mutants showing ,1% of activity (37,38 , which is itself located on the third Ig-like C2 domain of IL-1RI. This loop on the receptor is rich in acidic residues; in particular, there are 3 Asp and 1 Glu residues in a continuous sequence (residues 251-254).…”

Insertion of a Structural Domain of Interleukin (IL)-1β Confers Agonist Activity to the IL-1 Receptor Antagonist

“…Extensive mutagenesis studies of both IL-1␣ and IL-1␤ have identified the amino acid residues important for binding of these ligands to the Type I IL-1R (21)(22)(23)(24)(25)(26). In these studies, all of the residues involved in binding to the Type I IL-1R, although not contiguous, are located on one face of the IL-1 structure (the open end of the ␤-barrel).…”