Selectivity and antagonism of chemokine receptors

Abstract

The chemokine superfamily can be subdivided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonist. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1 beta, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

Identification of Receptor Binding and Activation Determinants in the N-terminal and N-loop Regions of the CC Chemokine Eotaxin

“…The receptor binding and activation data are summarized in Table I. DISCUSSION Previous chemokine mutational studies, and their interpretation according to the prevailing two-step model for chemokine receptor activation, have suggested a separation of function between the N-loop and N-terminal regions of chemokines (21,(23)(24)(25). The N-loop is proposed to be important for providing the initial binding energy, whereas the N-terminus is suggested to be required for triggering receptor activation subsequent to binding.…”

“…Extensive mutational studies of several chemokines have led to a proposed "two-step" model for receptor interaction in which the N-loop of the chemokine initially binds to the receptor then the N-terminal region of the chemokine docks with the receptor to induce a conformational change, resulting in receptor activation (21,(23)(24)(25). Recent papers describing the binding of receptor-derived peptides to a groove located between the Nloop and ␤2-␤3 hairpin of their cognate chemokines (interleukin-8, fractalkine, eotaxin, and eotaxin-2) have reinforced the importance of the N-loop region for receptor binding (22, 26 -28).…”

Identification of Residues of CXCR4 Critical for Human Immunodeficiency Virus Coreceptor and Chemokine Receptor Activities

“…The ECL2 mutants (except D182G) bear an NH 2 -terminal Myc tag (45), which does not affect CXCR4 function. 2 The COOH-terminal intracellular domain of CXCR4 (residues 310 -354) was deleted in mutants ⌬2-9, ⌬4 -36, and N11Q. This deletion has no influence on HIV-1 infection, or on SDF-1 binding and signaling, but abolishes ligand-induced endocytosis (27).…”

Design and Receptor Interactions of Obligate Dimeric Mutant of Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

“…2A), with their O␥ atoms separated by only 3.1 Å. Thr 10 is unlikely to be specifically important for MCP-1 structure or function, as simultaneous mutation of Val 9 and Thr 10 to Ala in previous experiments did not affect binding or signaling properties (23,24). Therefore, we reasoned that substitution of Thr 10 by Cys might enable the formation of a disulfide bond across the dimerization interface without compromising wild type structure.…”