Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo.
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.
Lau
Elaine K
EK
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Paavola
Chad D
CD
Johnson
Zoë
Z
Gaudry
Jean-Philippe
JP
Geretti
Elena
E
Borlat
Frédéric
F
Kungl
Andreas J
AJ
Proudfoot
Amanda E
AE
Handel
Tracy M
TM
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
D018932:Chemokine CCL2; D003432:Cross-Linking Reagents; D000939:Epitopes; D006025:Glycosaminoglycans; D008024:Ligands; D009844:Oligosaccharides; D011509:Proteoglycans; D006493:Heparin; D008239:Lysine; D000409:Alanine