title: Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo.
abstract: In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Elaine K
authors.initials: EK
authors.lastname: Lau
authors.forename: Chad D
authors.initials: CD
authors.lastname: Paavola
authors.forename: Zoë
authors.initials: Z
authors.lastname: Johnson
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Gaudry
authors.forename: Elena
authors.initials: E
authors.lastname: Geretti
authors.forename: Frédéric
authors.initials: F
authors.lastname: Borlat
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Kungl
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Proudfoot
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002499:Centrifugation, Density Gradient; D018932:Chemokine CCL2 / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D005260:Female; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D010537:Peritoneum / Q000166:cytology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
chemical_list: D018932:Chemokine CCL2; D003432:Cross-Linking Reagents; D000939:Epitopes; D006025:Glycosaminoglycans; D008024:Ligands; D009844:Oligosaccharides; D011509:Proteoglycans; D006493:Heparin; D008239:Lysine; D000409:Alanine