Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.

Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain.

The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. A single GalNAc residue was incorporated at each glycosylation site using standard Fmoc-chemistry to achieve the first total synthesis of a mucin-type glycoprotein. Using this approach quantities of homogeneous material were obtained for structural and functional analysis.

The crystal structure of the chemokine domain of fractalkine shows a novel quaternary arrangement.

Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX(3)C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX(3)CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 A resolution. The CDF monomers form a dimer through an intermolecular beta-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX(3)C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivo is not well understood.

Solution structure and dynamics of the CX3C chemokine domain of fractalkine and its interaction with an N-terminal fragment of CX3CR1.

Fractalkine, a novel CX3C chemokine, is unusual because of both its membrane-associated structure and its direct role in cell adhesion. We have solved the solution structure of the chemokine domain of fractalkine (residues 1-76) by heteronuclear NMR methods. The 20 lowest energy structures in the ensemble have an average backbone rmsd of 0.43 A, excluding the termini. In contrast to many other chemokines which form homodimers, fractalkine's chemokine module is monomeric. Comparison of the structure to CC and CXC chemokines reveals interesting differences which are likely to be relevant to receptor binding. These include a bulge formed by the CX3C motif, the relative orientation of the N-terminus and 30's loop (residues 30-38), and the conformation of the N-loop (residues 9-19). 15N backbone relaxation experiments indicate that these same regions of the protein are dynamic. We also titrated 15N-labeled protein with a peptide from the N-terminus of the receptor CX3CR1 and confirmed that this region of the receptor contacts the fractalkine chemokine domain. Interestingly, the binding site maps roughly to the regions of greatest flexibility and structural variability. Together, these data provide a first glimpse of how fractalkine interacts with its receptor and should help guide mutagenesis studies to further elucidate the molecular details of binding and signaling through CX3CR1.