title: Phosphate modulates receptor sulfotyrosine recognition by the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2).
abstract: Tyrosine sulfation is a widespread post-translational modification that mediates the interactions of secreted and membrane-associated proteins in such varied biological processes as peptide hormone action, adhesion, blood coagulation, complement activation and regulation of leukocyte trafficking. Due to the heterogeneous nature of tyrosine sulfation, detailed biochemical and biophysical studies of tyrosine sulfation rely on homogenous, synthetic sulfopeptides. Here we describe the synthesis of a fluorescent sulfopeptide (FL-R2D) derived from the chemokine receptor CCR2 and the application of FL-R2D in direct and competitive fluorescence anisotropy assays that enable the efficient measurement of binding affinities between sulfopeptides and their binding proteins. Using these assays, we have found that the binding of the chemokine monocyte chemoattractant protein-1 (MCP-1) to sulfated peptides derived from the chemokine receptor CCR2 is highly dependent on the assay buffer. In particular, phosphate buffer at close to physiological concentrations competes with the receptor sulfopeptide by binding to the sulfopeptide binding pocket on the chemokine surface. Thus, physiological phosphate may modulate the receptor binding selectivity of chemokines.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia. martin.stone@monash.edu.
authors.forename: Justin P
authors.initials: JP
authors.lastname: Ludeman
authors.forename: Mahdieh
authors.initials: M
authors.lastname: Nazari-Robati
authors.forename: Brendan L
authors.initials: BL
authors.lastname: Wilkinson
authors.forename: Cheng
authors.initials: C
authors.lastname: Huang
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Payne
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stone
mesh_terms: D001665:Binding Sites; D018932:Chemokine CCL2 / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D010710:Phosphates; C005662:tyrosine O-sulfate; D014443:Tyrosine