Proton NMR Assignments and Solution Conformation of RANTES, a Chemokine of the C-C Type

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH 3 NCH 3 ). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca 2؉ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50 -100 M. Similarly, MGSA showed a monomer-dimer equilibrium with a K d of ϳ5 M. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers.Recruitment and accumulation of circulating leukocytes at the site of inflammation are mediated in part by a family of small molecular weight proteins called chemokines (1-3). They have four conserved cysteines and are called either CXC or CC chemokines, depending on whether the first two cysteines are separated by one amino acid (CXC) or are adjacent (CC). Functionally, the CXC chemokines predominantly attract neutrophils, whereas CC chemokines attract monocytes, lymphocytes, and eosinophils.Three well studied proteins of the CXC chemokine family are interleukin-8 (IL-8) 1

Section: Resultsmentioning

confidence: 99%

Neutrophil-activating Peptide-2 and Melanoma Growth-stimulatory Activity Are Functional as Monomers for Neutrophil Activation

Rajarathnam

Kay

Dewald

et al. 1997

“…Above micromolar concentrations many form homodimers, whereas at nanomolar concentrations the monomeric form predominates in solution. High resolution structures of IL-8 (31), MGSA/GRO (32), MCP-1 (33,34), RAN-TES (35,36), and MIP-1␤ (37) have also revealed a striking correlation between chemokine class and mode of dimerization (38), suggesting that dimerization may play an important role in chemokine function. A key question, however, is whether dimerization is required for receptor binding and activation or whether it plays a more subtle role in protein stability, regu-lation, surface presentation and retention, formation of the chemotactic gradient, or other processes unrelated to chemotaxis.…”

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confidence: 99%

Monomeric Monocyte Chemoattractant Protein-1 (MCP-1) Binds and Activates the MCP-1 Receptor CCR2B

Paavola¹,

Hemmerich²,

Grünberger³

et al. 1998

184

223

To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr 13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, Chemokines are small secreted proteins that function as intercellular messengers to control migration and activation of specific subsets of leukocytes (1-3). This process is mediated by the interaction of chemokines with seven transmembrane Gprotein-coupled receptors on the surface of target cells. Interest in these proteins was first stimulated by the observation of elevated levels in a number of inflammatory diseases (4, 5) including rheumatoid arthritis (6, 7), arteriosclerosis (8, 9), and asthma (10). Although it is not clear whether excessive production is the cause or consequence of these diseases, the demonstration that neutralizing antibodies (11-13) reduced symptoms in a number of animal models generated optimism that receptor antagonists may have therapeutic benefit (14). Recently, it has been shown that certain chemokine receptors also serve as obligate coreceptors for entry of the human immunodeficiency virus into CD4ϩ cells (15)(16)(17) and that viral replication can be inhibited by the ligands of the coreceptors (18 -21). Thus a wide range of clinically important diseases are associated with chemokines and their receptors, motivating many studies to understand the molecular details of chemokine function.Chemokines have been classified into two major families based on their pattern of cysteine residues, their chromosomal location, and their cell specificities (22). ␣-Chemokines such as IL-8 1 have a conserved CXC cysteine motif and act predominantly on neutrophils, whereas ␤-chemokines have a CC signature and attract monocytes and T-cells. The recently discovered chemokines lymphotactin (23) and fractalkine/neurotactin (24,25) are characterized by C and CX 3 C motifs, respectively, and chemoattract T-cells and NK cells. Mutagenesis studies, particularly of ␣-and ␤-chemokines, have provided some insight into the structural determinants of receptor binding and the specificity of these proteins, but many details have yet to emerge.Considerable effort has been devoted to characterizing the stochiometry of chemokine-receptor complexes because most chemokines oligomerize to an extent tha...

“…Eotaxin has been implicated as the predominant chemokine in the recruitment of eosinophils and the eosinophil-mediated tissue damage associated with asthma. Thus eotaxin is a possible target for anti-asthmatic drugs.The three-dimensional structures of RANTES and MCP-3 have been solved (12)(13)(14). Both of these proteins bind CCR3 among other receptors, so there is presently no structure for a chemokine that is selective in its binding to CCR3.…”

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confidence: 99%

“…The three-dimensional structures of RANTES and MCP-3 have been solved (12)(13)(14). Both of these proteins bind CCR3 among other receptors, so there is presently no structure for a chemokine that is selective in its binding to CCR3.…”

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confidence: 99%

Solution Structure of Eotaxin, a Chemokine That Selectively Recruits Eosinophils in Allergic Inflammation

Crump¹,

Rajarathnam²,

Kim³

et al. 1998

102

The solution structure of the CCR3-specific chemokine, eotaxin, has been determined by NMR spectroscopy. The quaternary structure of eotaxin was investigated by ultracentrifugation and NMR, and it was found to be in equilibrium between monomer and dimer under a wide range of conditions. At pH < 5 and low ionic strength, eotaxin was found to be predominantly a monomer. The three-dimensional structure of the eotaxin monomer solved at pH 5.0 revealed that it has a typical chemokine fold, which includes a 3-stranded ␤-sheet and an overlying ␣-helix. Except for the N-terminal residues (residues 1-8), the core of the protein is well defined. The eotaxin structure is compared with the chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1); eotaxin binds only CC chemokine receptor CCR3, whereas RANTES binds many receptors including CCR3, and MCP-1 binds a distinct receptor, CCR2. The RMSD of the eotaxin ensemble of structures with the RANTES average minimized monomeric subunit is 5.52 ؎ 0.87 Å over all backbone atoms and 1.14 ؎ 0.09 Å over backbone atoms of residues 11-28 and 34 -65. The most important difference between the structures is in the N-terminal residues that are unstructured in eotaxin but structured in RANTES and MCP-1. Several residues in the loop region of RANTES show similar packing in eotaxin (residues 11-17). As the N-terminal and loop regions have been shown to be critical for receptor binding and signaling, this structure will be useful for determining the basis for CCR3 selectivity of the eotaxin.Eotaxin (1, 2) is a member of the chemoattractant cytokine (chemokine) family of proteins that mediates the migration and accumulation of eosinophils at sites of parasitic infection and allergic response (3-5). The chemokines play a pivotal role in the control of leukocyte chemotaxis and represent attractive targets for the blockade of inflammatory disease progression. Eotaxin belongs to the CC chemokine family which is distinguished by the presence of two adjacent cysteines close to the N terminus (6). The CC chemokines activate a wide range of leukocytes including eosinophils, basophils, monocytes, dendritic cells, and T lymphocytes. Chemokine activities are mediated through the binding and activation of seven transmembrane helix G-coupled receptors (6), and eight distinct functional CC chemokine receptors have been identified (6 -9). Their broad leukocyte distribution coupled with a lack of CC chemokine specificity means there is generally considerable redundancy among the CC chemokine family. However, the cloning and characterization of eotaxin and the related protein eotaxin-2 (10) and its receptor CCR3 1 (11) has revealed its unique and most important feature, a high selectivity for eosinophils (1, 2). Eotaxin has been implicated as the predominant chemokine in the recruitment of eosinophils and the eosinophil-mediated tissue damage associated with asthma. Thus eotaxin is a possible target for anti-asthmatic drugs.The three-di...