Molecular Basis of Chemokine CXCL5-Glycosaminoglycan Interactions

Abstract

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.

Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin

“…It is likely that the absence of chemical shift changes is due to cancellation between contributions from direct and indirect interactions of similar magnitude but opposite sign. Lack of CSP of lysine residues involved in GAG binding has been previously observed in other chemokines [46,47,48,49]. Our docking model and CSP data collectively indicate that hydrophobic packing, guided by H-bonding and ionic interactions, mediates Site-I binding.…”

“…The binding mode and the nature of these interactions are similar to that observed for other CXCR2-activating chemokines CXCL1, CXCL5 and CXCL8 [37,39,49,58]. Binding is principally mediated by hydrophobic packing interactions that are conserved across the NAC family (Figure 1).…”

“…However, CXCL7 is unique, as it alone forms a weak dimer and also a tetramer at high concentrations, whereas other members form stronger dimers and no tetramers. Previous NMR studies have characterized binding interactions for native CXCL1, CXCL5 and CXCL8 dimers and engineered CXCL1 and CXCL8 monomers, as only dimers could be studied at concentrations used for NMR [37,39,46,48,49]. In this study, for the very first time, we have successfully characterized the binding interactions of an NAC monomer.…”

Perspective on computational simulations of glycosaminoglycans

“…Solvent interaction appears to be different for different sequences and this may play a role in protein recognition . In fact, a number of computational studies have predicted the importance of water molecules in GAG recognition of basic binding sites on proteins, which have been supported by experimental results . These and other studies suggest that intermolecular GAG–water interactions are important to gain insight into GAG recognition of proteins.…”

Glycosaminoglycan Interactions Fine-Tune Chemokine-Mediated Neutrophil Trafficking: Structural Insights and Molecular Mechanisms

“…Furthermore, these studies show not only basic residues that are conserved but also those that are unique to a given chemokine mediate binding. [30][31][32][33][34] Most interestingly, these chemokines show a diversity of GAG-binding surfaces, indicating chemokine-specific residues play an important role in dictating the binding interactions, and that location and distribution of both conserved and chemokine-specific residues in the context of three-dimensional structure determine binding geometry. Residues implicated in binding are highlighted, and those that are conserved (in at least five out of seven sequences) are in red and shaded in gray and are labeled from B1 to B8.…”