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Circulating neutrophils, rapidly recruited in response to microbial infection, form the first line in host defense. Humans express ~50 chemokines, of which a subset of seven chemokines, characterized by the conserved “Glu-Leu-Arg” motif, mediate neutrophil recruitment. Neutrophil-activating chemokines (NACs) share similar structures, exist as monomers and dimers, activate the CXCR2 receptor on neutrophils, and interact with tissue glycosaminoglycans (GAGs). Considering cellular assays have shown that NACs have similar CXCR2 activity, the question has been and remains, why do humans express so many NACs? In this review, we make the case that NACs are not redundant and that distinct GAG interactions determine chemokine-specific in vivo functions. Structural studies have shown that the GAG-binding interactions of NACs are distinctly different, and that conserved and specific residues in the context of structure determine geometries that could not have been predicted from sequences alone. Animal studies indicate recruitment profiles of monomers and dimers are distinctly different, monomer–dimer equilibrium regulates recruitment, and that recruitment profiles vary between chemokines and between tissues, providing evidence that GAG interactions orchestrate neutrophil recruitment. We propose in vivo GAG interactions impact several chemokine properties including gradients and lifetime, and that these interactions fine-tune and define the functional response of each chemokine that can vary between different cell and tissue types for successful resolution of inflammation.

Glycosaminoglycans (GAGs) bind a large array of proteins and mediate fundamental and diverse roles in human physiology. Ion pair interactions between protein lysines/arginines and GAG sulfates/carboxylates mediate binding. Neutrophil-activating chemokines (NAC) are GAG-binding proteins, and their sequences reveal high selectivity for lysines over arginines indicating they are functionally not equivalent. NAC binding to GAGs impacts gradient formation, receptor functions, and endothelial activation, which together regulate different components of neutrophil migration. We characterized the consequence of mutating lysine to arginine in NAC CXCL8, a well-characterized GAG-binding protein. We chose three lysines — two highly conserved lysines (K20 and K64) and a CXCL8-specific lysine (K67). Interestingly, the double K64R/K20R and K64R/K67R mutants are highly impaired in recruiting neutrophils in a mouse model. Further, both the mutants bind GAG heparin with higher affinity but show similar receptor activity. NMR and MD studies indicate that the structures are essentially identical to the WT, but the mutations alter the network of intramolecular ion pair interactions. These observations collectively indicate that the reduced in vivo recruitment is due to altered GAG interactions, higher GAG binding affinity can be detrimental, and specificity of lysines fine-tunes in vivo GAG interactions and function.

Structural plasticity plays a major role in determining differential binding of CXCL8 monomer and dimer to glycosaminoglycans (GAGs) and that dimer is the high-affinity GAG ligand. We propose that these properties play important roles in orchestrating in vivo chemokine-mediated neutrophil function., Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8–GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer–GAG interactions and function.

Chemokines mediate diverse fundamental biological processes, including combating infection. Multiple chemokines are expressed at the site of infection; thus chemokine synergy by heterodimer formation may play a role in determining function. Chemokine function involves interactions with G-protein-coupled receptors and sulfated glycosaminoglycans (GAG). However, very little is known regarding heterodimer structural features and receptor and GAG interactions. Solution nuclear magnetic resonance (NMR) and molecular dynamics characterization of platelet-derived chemokine CXCL7 heterodimerization with chemokines CXCL1, CXCL4, and CXCL8 indicated that packing interactions promote CXCL7-CXCL1 and CXCL7-CXCL4 heterodimers, and electrostatic repulsive interactions disfavor the CXCL7-CXCL8 heterodimer. As characterizing the native heterodimer is challenging due to interference from monomers and homodimers, we engineered a "trapped" disulfide-linked CXCL7-CXCL1 heterodimer. NMR and modeling studies indicated that GAG heparin binding to the heterodimer is distinctly different from the CXCL7 monomer and that the GAG-bound heterodimer is unlikely to bind the receptor. Interestingly, the trapped heterodimer was highly active in a Ca2+ release assay. These data collectively suggest that GAG interactions play a prominent role in determining heterodimer function in vivo. Further, this study provides proof-of-concept that the disulfide trapping strategy can serve as a valuable tool for characterizing the structural and functional features of a chemokine heterodimer.

BRCT(BRCA1) plays a major role in DNA repair pathway, and does so by recognizing the conserved sequence pSXXF in its target proteins. Remarkably, tetrapeptides containing pSXXF motif bind with high specificity and micromolar affinity. Here, we have characterized the binding interactions of pSXXF tetrapeptides using NMR spectroscopy and calorimetry. We show that BRCT is dynamic and becomes structured on binding, that pSer and Phe residues dictate overall binding, and that the binding affinities of the tetrapeptides are intimately linked to structural and dynamic changes both in the BRCT(BRCA1) and tetrapeptides. These results provide critical insights for designing high-affinity BRCT(BRCA1) inhibitors.

Chemokine CXCL8 plays a pivotal role in host immune response by recruiting neutrophils to the infection site. CXCL8 exists as monomers and dimers, and mediates recruitment by interacting with glycosaminoglycans (GAGs) and activating CXCR1 and CXCR2 receptors. How CXCL8 monomer and dimer interactions with both receptors and GAGs mediate trafficking is poorly understood. In particular, both haptotactic (mediated by GAG-bound chemokine) and chemotactic (mediated by soluble chemokine) gradients have been implicated, and whether it is the free or the GAG-bound CXCL8 monomer and/or dimer that activates the receptor remains unknown. Using solution NMR spectroscopy, we have now characterized the binding of heparin-bound CXCL8 monomer and dimer to CXCR1 and CXCR2 receptor N-domains. Our data provide compelling evidence that heparin-bound monomers and dimers are unable to bind either of the receptors. Cellular assays also indicate that heparin-bound CXCL8 is impaired for receptor activity. Considering dimer binds GAGs with higher affinity, dimers will exist predominantly in the GAG-bound form and the monomer in the free form. We conclude that GAG interactions determine the levels of free CXCL8, and that it is the free, and not GAG-bound, CXCL8 that activates the receptors and mediates recruitment of blood neutrophils to the infected tissue.

Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. Mutations in the GP motif caused various differences from native-like function to complete loss of activity that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity.

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K(i)1 μm). Further, CC-CXCL8 failed to mobilize Ca(2+) in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca(2+) in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.

The Gag polyproteins of gammaretroviruses contain a conserved p12 domain between MA and CA that plays critical roles in virus assembly, reverse transcription and nuclear integration. Here we show using nuclear magnetic resonance, that p12 is unstructured in a Moloney murine leukemia virus (MMLV) Gag fragment that includes the N-terminal domain of CA (p12-CA(N)). Furthermore, no long range interactions were observed between the domains, as has been previously predicted. Flexibility appears to be a common feature of Gag "late" domains required for virus release during budding. Residues near the N-terminus of CA(N) that form a beta-hairpin in the mature CA protein are unfolded in p12-CA(N), consistent with proposals that hairpin formation helps trigger capsid assembly.