The three-dimensional structure of the ligand-binding region of human E-selectin has been determined at 2.0 A resolution. The structure reveals limited contact between the two domains and a coordination of Ca2+ not predicted from other C-type lectins. Structure/function analysis indicates a defined region and specific amino-acid side chains that may be involved in ligand binding. These features of the E-selectin/ligand interaction have important implications for understanding the recruitment of leukocytes to sites of inflammation.

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....

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.