Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, β₃ strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function.

Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play non-redundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer, and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin–KC and heparin–MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn, β3-strand, and C-terminal helix form a binding surface within a monomer, and that both conserved and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play non-overlapping roles in resolution of inflammation.

Chemokines promote directional cell migration through binding to G-protein-coupled receptors, and as such are involved in a large array of developmental, homeostatic and pathological processes. They also interact with heparan sulfate (HS), the functional consequences of which depend on the respective location of the receptor- and the HS-binding sites, a detail that remains elusive for most chemokines. Here, to set up a biochemical framework to investigate how HS can regulate CXCL13 activity, we solved the solution structure of CXCL13. We showed that it comprises an unusually long and disordered C-terminal domain, appended to a classical chemokine-like structure. Using three independent experimental approaches, we found that it displays a unique association mode to HS, involving two clusters located in the α-helix and the C-terminal domain. Computational approaches were used to analyse the HS sequences preferentially recognized by the protein and gain atomic-level understanding of the CXCL13 dimerization induced upon HS binding. Starting with four sets of 254 HS tetrasaccharides, we identified 25 sequences that bind to CXCL13 monomer, among which a single one bound to CXCL13 dimer with high consistency. Importantly, we found that CXCL13 can be functionally presented to its receptor in a HS-bound form, suggesting that it can promote adhesion-dependent cell migration. Consistently, we designed CXCL13 mutations that preclude interaction with HS without affecting CXCR5-dependent cell signalling, opening the possibility to unambiguously demonstrate the role of HS in the biological function of this chemokine.