Solution Structures of Glycosaminoglycan-bound CXCL8 Complexes Determined by Small-Angle X-Ray Scattering (SAXS).

Abstract

Glycosaminoglycans (GAGs) play diverse and fundamental roles in physiology by regulating the function of large classes of proteins. Despite their importance, knowledge of how GAGs are organized in protein-bound complexes remains limited. This can be attributed to the linear structure, conformational flexibility, and high negative charge of GAGs, all of which disfavor structure determination by crystallography or NMR spectroscopy. A hybrid approach based on GAG-binding-induced changes in NMR protein chemical shifts, computational docking, and molecular dynamics simulations has proven to be valuable in providing structural models. However, these approaches can identify multiple plausible GAG geometries, making it difficult to determine whether the observed geometries reflect intrinsic plasticity or limitations of the NMR data and docking methods. In the case of chemokine CXCL8, two GAG-binding modes have been proposed, one within a monomer and the other across the dimer interface. Here, we determined low-resolution solution structures of heparin and chondroitin sulfate octasaccharides bound to the CXCL8 dimer using small-angle X-ray scattering (SAXS). SAXS analyses show that both heparin and chondroitin sulfate bind to a surface within a monomer and are incompatible with binding across the dimer. NMR paramagnetic relaxation enhancement measurements for heparin-bound CXCL8 dimer and monomer complexes show that heparin engages a similar surface within the monomer in both complexes, consistent with the SAXS models. Together, these studies establish how GAGs are organized in the CXCL8-bound complex and highlight the value of complementary low-resolution structural methods for characterizing GAG-protein complexes.