A MODEL OF GAG:MIP-2:CXCR2 INTERFACES AND ITS FUNCTIONAL EFFECTS

Chemokines orchestrate leukocyte recruitment to sites of injury or infection. Some chemokines, particularly CXCL12, have roles during embryonic development (1). Chemotaxis is a complicated process that can be simplified into three events. First, chemokines are involved in transcytosis through the endothelium from the site of cellular secretion (2–5). Second, chemokines bind and activate specific G-protein coupled chemokine receptors (GPCRs) to initiate integrin-mediated adhesion and transmigration (6, 7). Finally, neutrophils respond to a chemokine gradient through the same GPCR to migrate through the matrix to the site of chemokine release (8). The interactions of glycosaminoglycans (GAGs) and chemokines regulate these individual processes (9, 10). The chemokine superfamily is classified into four families based on the presence or absence of intervening amino acids between two conserved cysteines near the N-terminus that form disulfides with other cysteines in the protein sequence. Chemokines from three (CXC, CC, CX3C) of the four families essentially have the same monomeric structure, but tend to differ in their dimeric structure. The chemokine in the fourth family (XC) lacks one of the two cysteines, and forms two interconverting structures, a canonical chemokine monomer and a monomer with four β-strands lacking a C-terminal α-helix (11). Chemokines activate their GPCRs in a family-dependent manner. While there may be a number of chemokines within a family that activate the same receptor, chemokines from different families never activate the same receptor. Structures of chemokines from all four families have been solved with most having a monomeric or dimeric structure, but higher oligomers up to a decamer have been determined (12, 13). The dimeric chemokines from each family are sufficiently different to explain family specificity, but most functional studies indicate that the activating chemokine is a monomer (14). There is also some evidence chemokine receptors can be activated by monomers and dimers, but monomers and dimers have different receptor affinities and activating properties, with the monomer having a higher binding affinity and eliciting most biological effects (15–17). More recently, structures of linear forms of oligomeric chemokines have been determined and proposed to have important roles in chemotaxis (13, 18–21). In contrast to the many structures available for chemokines, only one chemokine receptor (CXCR4) structure is available. CXCR4 was co-crystallized with two small molecule antagonists and is a dimer in five different space groups (22). The interactions of chemokines with proteoglycans and glycosaminoglycans (GAGs) regulate biological activity (9, 10) but the mechanisms that lead to regulation are still unclear. GAGs enhance dimerization or oligomerization of chemokines (23–25) and may present chemokines to their receptors (4). It has also been suggested that GAG-mediated linear (as opposed to globular) oligomerization is important for presentation to receptors under flow conditions in the endothelium (19, 26). GAG-mediated oligomerization of chemokines (23–25) has also been proposed to mediate a chemokine gradient within the matrix that is necessary for chemotaxis (8). The functional effects of GAG-deficient mutant chemokines have been investigated in vitro and in vivo but the results are confounding (24, 27–29). The in vitro activities vary depending on chemokines. Some GAG-deficient mutants are as active as WT (30, 31), whereas GAG-deficient mutants of other chemokines are approximately 1000-fold less active (24). The in vivo activities also vary from increased cell recruitment (e.g., for CXCL8) to no activity at all (e.g., CCL2, CCL4, and CCL5) (30, 32) but, generally, there is no correspondence to the in vitro effects. In the present study we determined the X-ray structure of MIP-2 to resolve whether the four monomers in the asymmetric (33) make linear oligomers recently reported for CCL4(34), CCL5 (21), CXCL10 (18, 26) and CXCL12 (13), speculated to be important for the function of chemokines (19). We were also interested in the interface between MIP-2 and murine CXCR2 (mCXCR2), and the structural and functional aspects of regulation by GAGs. A model of mCXCR2 was created based on the structure of human CXCR4 (22) and the MIP-2 X-ray structure to analyze the potential binding surface of MIP-2 and CXCR2. We also used NMR to identify MIP-2 residues that interact with a heparin disaccharide and developed a model of the GAG:MIP-2:CXCR2 complex. The GAG-binding residues identified by NMR were mutated to alanine and tested for in vitro chemotaxis and neutrophil recruitment to the mouse peritoneum and lung. We observe differences of GAG-deficient mutants between in vitro chemotaxis and the in vivo results, as well as differences of GAG-deficient mutants in in vivo neutrophil recruitment to the peritoneum and lung. This study was compared to neutrophil recruitment from CXCL8 GAG-deficient mutants to the murine peritoneum and lung (30, 31). This comparison of two chemokines in the ELR subfamily leads us to conclude GAG regulation is tissue- and chemokine-dependent and could differ dramatically in its effects, suggesting markedly different mechanisms.