Chemokine CXCL7 Monomer, Homodimer, and Heterodimer: Structural Insights, CXCR2 Receptor Function, and Glycosaminoglycan Interactions

Platelet-derived chemokine CXCL7 (NAP-2) plays a critical role in mediating the crosstalk between platelets and neutrophils for initiating repair during vascular injury. CXCL7 function is coupled to CXCR2 receptor activation and interactions with sulfated glycosaminoglycans (GAG) that regulate receptor activity. CXCL7 exists as monomers and dimers and there is also evidence that it could form heterodimers. Currently, nothing is known regarding the structural features of the monomer, dimer, and heterodimer, and the molecular basis by which these various forms mediate receptor and GAG interactions. We have addressed this missing knowledge as a part of this doctoral thesis. Structural characterization of each of the different CXCL7 forms was challenging because multiple species (say monomer and dimer) always coexist, and structural studies demand that only a single species is present for unambiguous characterization. Using a combination of solution conditions and concentrations, NMR spectroscopy, modeling, protein engineering, and cellular assays, the structural features of the monomer, homodimer, and heterodimer, their receptor activity, and GAG interactions have been successfully characterized. Our findings indicated that the receptor activity and binding interactions were similar for all the variants, with the CXCR2 N-domain binding a hydrophobic groove along the CXCL7 N-loop. However, the GAG binding properties of the monomer, homodimer, and heterodimer vary, and most interestingly, dimer is favored in the GAG bound form. This was an unexpected finding as the dimer is the minor species in solution. Further, several of the residues involved in GAG binding are also involved in receptor interactions, indicating that the GAG-bound monomer, homodimer, and heterodimer cannot activate the receptor. We conclude that both homodimers and heterodimers play an important role in mediating CXCL7 function via their interactions with GAG and propose that the GAG-bound dimers regulate the steepness and duration of concentration gradients, which in turn regulates the levels of free monomer available for CXCR2 activation and neutrophil recruitment. Further, this work 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 for a variety of chemokine pairs.