Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor

Abstract

Poxviruses express a family of secreted proteins that bind with high affinity to chemokines and antagonize the interaction with their cognate G protein-coupled receptors (GPCRs). These viral inhibitors are novel in structure and, unlike cellular chemokine receptors, are able to specifically interact with most, if not all, CC-chemokines. We therefore sought to define the structural features of CC-chemokines that facilitate this broad-spectrum interaction. Here, we identify the residues present on human monocyte chemoattractant protein-1 (MCP-1) that are required for highaffinity interaction with the vaccinia virus 35-kDa CC-chemokine binding protein (VV-35kDa). Not only do these residues correspond to those required for interaction with the cognate receptor CCR2b but they are also conserved among many CC-chemokines. Thus, the results provide a structural basis for the ability of VV-35kDa to promiscuously recognize CC-chemokines and block binding to their receptors.CCR2b ͉ chemokine binding protein ͉ monocyte chemoattractant protein-1 ͉ mutagenesis ͉ surface plasmon resonance

Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1β

“…In the current structure, the 48th position is approached closely by the side chains of vCCI Tyr-80 and Arg-89, so substitution of Lys-48 by Ala in MIP-1␤ could alleviate steric crowding and poor electrostatic effects of the WT protein. In support of this, the analogous mutation in MCP-1 Lys-49-Ala actually showed an increase in affinity for vCCI (27,28).…”

“…An amino acid with a shorter or a polar side chain would not be able to make these contacts and would therefore lack this component of binding affinity. Indeed, detailed mutagenesis studies of the CC chemokine MCP-1 show an Ϸ10-fold reduction in affinity when analogous residue Tyr-13 is replaced by Ala (27,28). In all human chemokines that bind tightly to vCCI, this position is an aromatic or a large hydrophobic amino acid (26), again demonstrating the importance of this residue in both the function and inhibition of CC chemokines.…”

“…3). Although this interaction is not shielded from water, mutagenesis experiments on MCP-1 indicate its significance, showing a Ͼ20-fold reduction in affinity when Arg-18 is substituted with Ala (27,28). Additionally, sequence analysis of human chemokines that bind vCCI shows a nearly universal positive charge at this position (Fig.…”

“…An extensive binding study with vaccinia vCCI using Ͼ80 chemokines from several organisms revealed 26 CC chemokines that bind with high affinity, including 13 human chemokines, such as MCP-1 (monocyte chemoattractant protein 1), MIP-1␣, MIP-1␤, and RANTES (26). To probe the chemokine residues that are important in vCCI binding, two previous studies using vCCI and variants of human CC chemokine MCP-1 suggested that the MCP-1 monomeric subunit binds vCCI through an interface that is partially overlapping with the site that MCP-1 uses to bind its natural receptors, with residues Tyr-13, Arg-18, and Arg-24 found to be particularly important (27,28). The present work displays the structural underpinning of the high affinity between poxvirus vCCI and CC chemokines.…”

“…The arrangement of negative charges and the confor- mational plasticity in this region of vCCI allow favorable interactions to positively charged residues in the chemokine and have minimal hindrance to large hydrophobic residues. A negatively charged chemokine residue at this position would be expected to be disfavored by the highly acidic loop, which explains the observation that an Arg-24-Glu MCP-1 mutant has no detectable binding to vCCI by surface plasmon resonance (28). Regarding vCCI interactions with those chemokines having a neutral rather than a basic residue at this 24th position, the high flexibility of the acidic vCCI loop region surrounding MIP-1␤ residue Phe-24 makes it difficult to pinpoint specific contacts, so it is possible that Phe-24 itself does not contribute favorably to binding vCCI.…”