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.