Interleukin-1 (IL-1) proteins, such as IL-1 beta, play a key role in immune and inflammatory responses. Interaction of these cytokines with the IL-1 receptor induces a variety of biological changes in neurologic, metabolic, hematologic, and endocrinologic systems. Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. The 153-residue protein binds to the receptor with an affinity similar to that of IL-1 beta but does not elicit any physiological responses. As a first step toward understanding IRAP's mode of action, we have used multidimensional, heteronuclear NMR spectroscopy to determine the antagonist's solution secondary structure and global fold. Using a combination of 3D 1H-15N NOESY-HMQC and TOCSY-HMQC and 3D 1H-15N-13C HNCA and HN(CO)CA experiments on uniformly 15N- or doubly 13C/15N-enriched IRAP, we have made resonance assignments for more than 90% of the main-chain atoms. Analysis of short- and long-range NOE's indicates that IRAP is predominantly beta-sheet, with the same overall topology as IL-1 beta but with different regions of the primary sequence comprising the beta-strands. Two short helical segments also were identified. The 14% sequence identity between IL-1 beta and IRAP increases to 25% when differences in the locations of secondary structure elements in the primary sequences are taken into account. Still, numerous differences in side chains, which ultimately play a major role in receptor interaction, exist.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. In contrast to IL-lb, IRAP binds to the IL-l receptor but does not elicit a physiological response. We have determined the solution structure of IRAP using NMR spectroscopy. While the overall topology of the two 153-residue proteins is quite similar, functionally critical differences exist concerning the residues of the linear amino acid sequence that constitute structurally homologous regions in the two proteins. Structurally homologous residues important for IL-l receptor binding are conserved between IRAP and IL-l/!?. By contrast, structurally homologous residues critical for receptor activation are not conserved between the two proteins.

We report the crystallization of samples of a recombinant preparation of human interleukin-I receptor antagonist protein (IRAP) and solution of the crystal structure by isomorphous replacement methods. Crystals were obtained by the hanging-drop vapordiffusion method at 277 K from solutions of PEG 4000 containing sodium chloride, dithiothreitol and PIPES [sodium piperazione-N,N'-bis(2-ethanesulfonate)] buffer at pH 7.0. Crystals appear within about a week and grow as truncated tetragonal bipyramids to 0.3-0.6 mm on an edge. X-ray diffraction data from these crystals specify space group P43212 and unit-cell dimensions of a=b= 72.35(26), c = 114.7(8),~ and Z = 16 (two molecules per asymmetric unit). Fresh crystals diffract to about 2.3 A resolution. The search for heavy-atom derivatives has produced two, potassium gold cyanide and trimethyl lead chloride, as same-site, single-site derivatives. Inspection of an electrondensity map at 4 A resolution calculated with these derivatives confirms that the IRAP molecule is a member of the interleukin-1 structural family.The interleukin-I (IL-1) proteins IL-la and IL-lfl are cytokines active in a variety of processes associated with the inflammatory response (Dinarello, 1992, and references therein). Cells that secrete IL-la and IL-lfl are able to influence the behavior of cells that have specific receptors for these cytokines. IL-la and IL-lfl, which are about 25% identical in amino-acid sequence, act by binding to the extracellular domains of IL-1 receptors and triggering *

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