title: Identification of the pharmacophore of the CC chemokine-binding proteins Evasin-1 and -4 using phage display.
abstract: To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.
authors.affiliation: Merck Serono Geneva Research Centre, 9 chemin des Mines, 1202 Geneva, Switzerland,; NovImmune SA, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland, and.
authors.forename: Pauline
authors.initials: P
authors.lastname: Bonvin
authors.affiliation: Merck Serono Geneva Research Centre, 9 chemin des Mines, 1202 Geneva, Switzerland.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Dunn
authors.affiliation: NovImmune SA, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland, and.
authors.forename: François
authors.initials: F
authors.lastname: Rousseau
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093-0684.
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Dyer
authors.affiliation: Merck Serono Geneva Research Centre, 9 chemin des Mines, 1202 Geneva, Switzerland.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shaw
authors.affiliation: Merck Serono Geneva Research Centre, 9 chemin des Mines, 1202 Geneva, Switzerland.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Power
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093-0684.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
authors.affiliation: Merck Serono Geneva Research Centre, 9 chemin des Mines, 1202 Geneva, Switzerland,; NovImmune SA, 14 chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland, and. Electronic address: amandapf@orange.fr.
authors.forename: Amanda E I
authors.initials: AEI
authors.lastname: Proudfoot
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D054405:Chemokine CCL3 / Q000737:chemistry; D018946:Chemokine CCL5 / Q000737:chemistry / Q000235:genetics; D054412:Chemokine CCL8 / Q000737:chemistry; D019742:Chemokines, CC / Q000737:chemistry*; D002633:Chemotaxis; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019707:Receptors, Chemokine / Q000737:chemistry*; D048508:Rhipicephalus sanguineus; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: C515429:CCL3 protein, human; C483024:CCL5 protein, human; C515453:CCL8 protein, human; D054405:Chemokine CCL3; D018946:Chemokine CCL5; D054412:Chemokine CCL8; D019742:Chemokines, CC; C523601:Evasin-1, Rhipicephalus sanguineus; D008024:Ligands; D019151:Peptide Library; D019707:Receptors, Chemokine; D000409:Alanine
keywords: Cell Migration; Chemokine; Chemokine Binding Protein; Chemotaxis; Epitope Mapping; Phage Display; Pharmacophore