Heterologous quaternary structure of CXCL12 and its relationship to the CC chemokine family

Abstract

X-ray crystallographic studies reveal that CXCL12 is able to form multiple dimer types, a traditional CXC dimer and a 'CC-like' form. Phylogenetic analysis of all known human chemokines demonstrates CXCL12 is more closely related to the CC chemokine class than other CXC chemokines. These observations indicate that CXCL12 contains genomic and structural elements characteristic of both CXC and CC chemokines.Chemokines are members of a superfamily of proteins involved in the migration of cells to the proper anatomical position during embryonic development or in response to infection or stress during an immune response. There are two major (CC and CXC) and two minor (CX3C and XC) families based on the sequence around the first conserved cysteine. The topology of all structures is essentially identical with a flexible N-terminal region of 3-8 amino acids, a 10-20 residue N-terminal loop, a short 3{sub 10}-helix, three {beta}-strands, and a {alpha}-helix. The major consequence of the subtle difference between the families occurs at the oligomeric level. Monomers of the CC, CXC, and CX3C families form dimers in a family-specific manner. The XCL1 chemokine is a monomer that can interconvert between two folded states. All chemokines activate GPCRs according to family-specificity, however there are a fewmore » examples of chemokines crossing the family boundary to function as antagonists. A two-stage mechanism for chemokine activation of GPCRs has been proposed. The N-terminal region of the receptor interacts with the chemokine, followed by receptor activation by the chemokine N-terminal region. Monomeric chemokines have been demonstrated to be the active form for receptor function. There are numerous examples of both chemokines and their receptors forming dimers. While family-specific dimerization may be an attractive explanation for why specific chemokines only activate GPCRs within their own family, the role of dimers in the function of chemokines has not been resolved. Given that CXCL12 is in the CXC family, the CXC dimer is considered the physiologic dimer in all previous studies based on crystallographic evidence. NMR and mutational studies agree with the CXC dimer form in solution. The CXC form of the dimer is seen in recent structures of CXCL12 bound to a heparin disaccharide and several CXCR4 peptides. In one case, crystals of the CXC-type dimer were soaked in a heparin disaccharide solution to determine the interactions between this dimer and bound disaccharide. In another case, in order to overcome NMR chemical shift line broadening when CXCR4 peptides are added, a 'locked' dimer was constructed by introducing a cysteine mutant that linked subunits as a CXC dimer through an inter-subunit disulfide bond. The solution structures of the locked CXC dimer with CXCR4 peptides were determined. The locked CXC dimer retained Ca{sup 2+} mobilization yet lost chemotaxis activity, presumably because the monomer is the active form. In addition to existing as a monomer and CXC dimer, CXCL12 is now demonstrated to have the capacity to form CC type dimers in the presence of a CXCR4 peptide.« less