Structural Basis of Chemokine Sequestration by a Herpesvirus Decoy Receptor

Abstract

The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.

Human cytomegalovirus encodes a highly specific RANTES decoy receptor

“…4C Right), indicating that the 2:2 complex was formed preferentially. A 2:2 stoichiometry is consistent with earlier demonstrations that the gamma herpesvirus 68 M3 protein and vaccinia virus p35 chemokine-binding proteins form dimers (25,26).…”

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

“…Resonance assignments of both components in the complex were carried out with a series of standard 2D and 3D experiments by using 15 N-labeled, 13 C, 15 N-labeled, and 2 H, 13 C, 15 Nlabeled proteins. Essentially complete backbone and side-chain 1 H, 13 C, and 15 N chemical-shift assignments for bound MIP-1␤ and 1 H N , 13 C ␣ , 13 C ␤ , 13 CЈ, 15 N chemical-shift assignments for bound and free vCCI were obtained and are reported elsewhere (21,22). Deviation of chemical shift from random coil values allowed predictions of secondary structure for both protein components in the complex (23) and suggests that, in the complex, both proteins preserve the secondary structure elements that are present in their unliganded forms (7,19).…”

“…NOESY data were also obtained by using complexes composed of proteins with various isotopic labeling schemes to facilitate resolution of the spectra. Three-dimensional 15 N-NOESY of 1 H N , 15 N, 13 C-labeled vCCI in complex with unlabeled MIP-1␤ and 3D 13 C-NOESY and 15 N-NOESY of 13 C, 15 N-labeled MIP-1␤ in the presence of unlabeled vCCI were used for verification of chemicalshift assignments and to obtain intramolecular NOE constraints. These data also confirm that both proteins in the complex retain the same overall tertiary fold and secondary structures as in the unbound form.…”

“…Direct structural information of the binding interface was obtained by using a 4D 15 N, 13 C-edited NOESY experiment (24) that was measured on a 2 H, 1 H N , 15 N vCCI: 13 C, 14 N MIP-1␤ sample (see the supporting information). Because of the differential isotopic labeling of the proteins in the complex, this experiment shows resonances only for intermolecular contacts, providing distance information between the amide protons of vCCI and any nearby side-chain and backbone H C protons of MIP-1␤.…”

“…However, structure determinations of chemokines in complex with any biological molecule are rare and include only a chemokine:GAG complex (12), a chemokine:protein complex (13), and a chemokine:receptor fragment peptide complex (14).…”