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Query: UNIPROT:P10145 (IL-8)
 23,849 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The solution structure of the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) has been determined using NMR spectroscopy. Backbone and side-chain 1H and 15N assignments have been obtained using a combination of two-dimensional homonuclear and three-dimensional heteronuclear spectra. Regular elements of secondary structure have been identified on the basis of a qualitative interpretation of NOE data, J(NH-H alpha) coupling constants, and amide exchange rates. Three-dimensional structures were calculated from a total of 2146 experimental restraints using a combination of distance geometry and simulated annealing protocols. For the 13 best structures the average backbone (N, C alpha, C) atomic rmsd from the mean coordinates for residues 5-65 is 0.64 A (+/- 0.14 A) for the dimer and 0.50 A (+/- 0.08 A) for the individual monomers. Each monomer consists of a three-stranded antiparallel beta-sheet (residues 26-30, 38-43, 48-51) in a Greek key motif with a C-terminal helix (56-65) packed across the sheet, an arrangement similar to the monomeric structure of other members of this chemokine family (IL-8, PF4, MGSA/Gro alpha, and MIP-1 beta). Overall, the RANTES dimer resembles that previously reported for MIP-1 beta.
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PMID:The three-dimensional solution structure of RANTES. 754 19

In structure determination by X-ray crystallography and solution NMR spectroscopy, experimental data are collected as time and ensemble-averages. Thus, in principle, appropriate time and ensemble-averaged models should be used. Refinement of an ensemble of conformers rather than one single structure against the experimental NMR data could, however, result in overfitting the data because of the significantly increased number of parameters. To avoid overfitting, complete cross-validation, which provides an unbiased measure of the fit, has been applied to nuclear Overhauser effect derived distance refinement. Using two synthetic test cases, a correlation was demonstrated between the cross-validated measure to the fit (defined in terms of root-mean-square deviations from the distance restraints and number of violations) and the number of models that best reproduce the conformational variability in solution. A new method, based on a probability map, has been used to generate good representations of the resulting ensembles of structures. The method has also been applied to observed NMR data for two proteins, interleukin 4 and interleukin 8. For interleukin 4, cross-validation indicates that a single-conformer model gives the most accurate representation of the structure, whereas conventional measures of fit between the experimental data and those calculated from the model decrease when increasing the number of conformers, indicating overfitting. For interleukin 8, complete cross-validation predicts a twin-conformer model to be the most faithful representation of the experimental data. Two distinct conformations for the loop formed by residues 16 to 22 emerge from the family of twin-conformer structures. The putative alternate conformation of the loop is not observed in the crystal structure of interleukin 8. However, because of crystal packing contacts in this region this does not necessarily exclude the presence of the alternate conformation in solution. The twin-conformer model is supported by observed chemical exchange line broadening for the amide of His18 obtained by 15N relaxation studies. This region has also been implied to be involved in receptor binding.
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PMID:Conformational variability of solution nuclear magnetic resonance structures. 760 99

Interleukin-8 has been shown by X-ray crystallography and NMR to be a homodimer, suggesting that this is the form which binds to its receptor. Here we measure, for the first time, the monomer-dimer equilibrium of interleukin-8 using analytical ultracentrifugation and titration microcalorimetry and find that it dissociates readily to monomers with an equilibrium dissociation constant of 18 +/- 6 microM at 37 degrees C. The present findings suggest that the monomer is the form which binds to the receptor. Comparison of experimental and structure-based calculated thermodynamics of interleukin-8 dimerization argues for limited subunit conformational changes upon dissociation to monomer.
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PMID:Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations. 794 77

The solution conformation of rat cytokine-induced neutrophil chemoattractant (CINC/Gro), a small protein consisting of 72 amino acid residues with proinflammatory activities, and a member of the interleukin 8 family corresponding to a counterpart of human Gro, was investigated with homonuclear 2D and 3D NMR spectroscopy. At each phase of the structural analysis, the homonuclear 3D NOESY-HOHAHA and HOHAHA-NOESY spectra afforded valuable data, removing ambiguities intractable by conventional 2D NMR techniques. CINC/Gro exists as a dimer in solution and contains a triple stranded anti-parallel beta-sheet and C-terminal alpha-helix in the monomer structure, as observed in human IL-8, but non-trivial differences are also observed.
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PMID:The three dimensional structure of rat cytokine CINC/Gro in solution by homonuclear 3D NMR. 795 25

1H NMR studies were carried out on interleukin 8 (IL-8) analogs in order to probe the structural features that are essential for receptor binding and function. The analogs studied were the chemically synthesized IL-8 (1-72), a series of N-terminally truncated derivatives (4-72, 5-72, and 6-72), and derivatives with single amino acid substitutions (I10A, R6K, and H33A). Previous functional studies have shown that the N-terminal residues, especially the residues at positions 4-6, and the beta turn containing Cys-34, which is disulfide linked to Cys-7, are important for receptor affinity and functional activity [Clark-Lewis, I., Schumacher, C., Baggiolini, M., & Moser, B. (1991) J. Biol. Chem. 266, 23128-23134; Clark-Lewis, I., Dewald, B., Loetscher, M., Moser, B., & Baggiolini, M. (1994) J. Biol. Chem. (in press)]. The 6-72 and R6K analogs also showed properties of an antagonist. Analysis of the 1H NMR parameters such as chemical shifts, amide proton chemical shift temperature coefficients, and NOESY data indicates that the core structure is the same for all these proteins. Small differences were observed in some of the NMR properties for some of the residues in the N-terminal region and the turn containing Cys-34. Detailed analysis suggests that there is no correlation between these differences and observed function. Thus functional differences between the N-terminal analogs are a direct consequence of changes in receptor binding due to substitutions/deletions in the N-terminal sequence and not due to structural changes elsewhere.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:1H NMR studies of interleukin 8 analogs: characterization of the domains essential for function. 820 99

Interleukin-8 and its receptors are key mediators of immune and inflammatory responses. Heteronuclear NMR spectroscopy has been utilized to map the binding surface on interleukin-8 (IL-8) for an N-terminal fragment of the human Type-1 IL-8 receptor. A peptide corresponding to residues 1-40 of the IL-8 type 1 receptor (IL8-r1) was titrated into a sample of uniformly 15N-labeled IL-8. IL8-r1 binds to IL-8 with a dissociation constant of 170 +/- 50 microM assuming the peptide binds with a stoichiometry of one peptide per IL-8 monomer, exchanges rapidly (> 900 s-1) between free and bound states, and selectively perturbs the chemical environment of several IL-8 residues. The binding surface on IL-8 suggested by our results is comprised of residues in strand beta 3 of the beta-sheet (Glu48 to Cys50), the turn preceding beta 3 (Ser44), the C-terminal alpha-helix (Val61) and the irregular N-terminal loop region (Thr12, Lys15, Phe17, His18, Lys20 and Phe21). The IL-8 dimer appears to present two symmetrical binding surfaces for the IL8-r1 peptide, suggesting two receptor peptides may bind per dimer.
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PMID:Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 human interleukin-8 receptor. 830 64

A 72-amino-acid chimeric protein, Chi1, was constructed from the N-terminal part of interleukin 8, IL-8-(1-53), and the C-terminal part of melanoma growth stimulatory activity, MGSA-(54-72). Chi1 protein showed receptor-binding specificity and biological activity similar, but not identical to IL-8 and decidedly different from MGSA. The structure of Chi1 was determined in solution by two-dimensional NMR and molecular-dynamics calculations. The structure resembled the structures of MGSA and IL-8 closely, containing a triple-stranded beta-sheet in the IL-8 part and an amphipathic alpha-helix in the MGSA part. Chi1 formed dimers at millimolar concentrations via the first strand from the N-terminus, analogous to IL-8 and MGSA. In contrast to the latter molecules, however, the alpha-helix of Chi1 did not pack against the beta-sheet part, but was an independent structural element. This structural difference could be explained mainly by the modulation of hydrophobic interactions between the helix and the rest of the protein in Chi1 as compared to IL-8 and MGSA. It is concluded that tight helix packing is not required for receptor binding and biological activity of Chi1.
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PMID:Structure and activity of a chimeric interleukin-8-melanoma-growth-stimulatory-activity protein. 863 39

Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B. The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils. In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity. Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al. (1994) J. Mol. Biol. 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors. The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand. When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced. However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced. Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity. Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8. Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity.
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PMID:Exchanging interleukin-8 and melanoma growth-stimulating activity receptor binding specificities. 866 82

In an effort to determine which regions of IL-8 are involved in interactions with its receptors, eight peptides were designed to correspond to distinct exposed regions of the IL-8 monomer, using the proton NMR-derived structure of the dimer as a basis. The peptides were evaluated singularly, and as equimolar mixtures of two to six peptides, in an IL-8 receptor binding assay and found to have no binding interaction with either alpha or beta IL-8 receptor as single peptides or mixtures of two peptides. In contrast, one of these peptides having the sequence AVLPRSAKEL, which corresponds to the N-terminal 10 amino acid residues of the 77 amino acid form of IL-8, exhibited potent chemotactic activity in human neutrophils. These results indicate that there is no contiguous ligand that can be designed based on the NMR and X-ray determined structure of IL-8 and that there may be multiple receptors responsible for neutrophil activation and chemotaxis.
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PMID:Design and evaluation of small peptides mapping the exposed surface of IL-8. 874 Sep 72

IL-8 dimers have been observed in NMR and X-ray structures of the protein. We have engineered IL-8 monomers by mutations of residues throughout the dimer interface, which introduce hindrance determinants to dimerization. These IL-8 variants are shown by NMR to have wild-type monomer folding, but by ultracentrifugation to have a range of dimerization constants from microM to mM, as compared with a dimerization constant of about 10 microM for wild-type IL-8, under physiological salt and temperature conditions. The monomeric variants of IL-8 bind the erythrocyte chemokine receptor DARC, as well as the neutrophil IL-8 receptors CXCR1 and CXCR2 with affinities similar to that of wild-type IL-8. In addition, the monomeric variants were shown to have agonist activity, with similar potency to wild-type, in both Ca(2+)-flux assays on CXCR1 and CXCR2 transfected cells, and in chemotaxis assays on neutrophils. Thus, these variants confirm that monomeric IL-8 is functionally equivalent to wild-type in vitro assays. We have also investigated the effects of various solution conditions upon IL-8 dimer formation using analytical ultracentrifugation. At salt concentrations, temperatures, and pH conditions lower than physiological, the dimerization affinity of IL-8 is greatly enhanced. This suggests that, under some conditions, IL-8 dimer formation may occur at concentrations of IL-8 considerably lower than 10 microM, with consequences in vivo that are yet to be determined.
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PMID:Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation. 907 Apr 42


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