Identification of the Pharmacophore of the CC Chemokine-binding Proteins Evasin-1 and -4 Using Phage Display

Pauline Bonvin,Steven M Dunn,François Rousseau,Douglas P Dyer,Jeffrey Shaw,Christine A Power,Tracy M Handel,Amanda E.I Proudfoot

doi:10.1074/jbc.m114.599233

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.

Highlights

The selectivity profiles of the closely related chemokine-binding proteins Evasin-1 and -4 differ
We identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8
We constructed an in silico model of the structure of Evasin-4 in complex with CCL3 based on the crystal structure of the Evasin-11⁄7CCL3 complex using Maestro software (Schrödinger)

Summary

Background

The selectivity profiles of the closely related chemokine-binding proteins Evasin-1 and -4 differ. The structure of the complex of Evasin-1 with CCL3 revealed two prominent ␲-␲ interactions between the chemokine and the CKBP, involving amino acids Phe-14 and Trp-89, respectively Mutation of these two residues did not completely abolish binding, which motivated the current investigation to: (i) define the precise binding interactions that confer strict chemokine selectivity to Evasin-1 and (ii) understand the interactions that enable many members of the CC chemokine class to bind Evasin-4. Phage display allows a direct link between the nucleotide sequence encoded by the phage(mid) and the phenotype of the displayed protein variant or protein variant mutant library and is a powerful approach for studying ligand binding without the need for individually expressing and chromatographically purifying large numbers of individual protein variants Using this technique, we report progress toward identifying sequence determinants that confer a broad yet selective binding profile of Evasin-4 for CC chemokines and confirm the importance of Phe-14 and Trp-89 for the interaction of Evasin-1 with CCL3. Despite having different binding hotspots, both Evasins block the key signaling domain of chemokines

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION