title: Molecular basis of glycosaminoglycan heparin binding to the chemokine CXCL1 dimer.
abstract: Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (Kd = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of "active" chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.
authors.affiliation: From the Department of Biochemistry and Molecular Biology,; Sealy Center for Structural Biology and Molecular Biophysics, and.
authors.forename: Krishna Mohan
authors.initials: KM
authors.lastname: Poluri
authors.affiliation: From the Department of Biochemistry and Molecular Biology,; Sealy Center for Structural Biology and Molecular Biophysics, and.
authors.forename: Prem Raj B
authors.initials: PRB
authors.lastname: Joseph
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Kirti V
authors.initials: KV
authors.lastname: Sawant
authors.affiliation: From the Department of Biochemistry and Molecular Biology,; Sealy Center for Structural Biology and Molecular Biophysics, and; Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555. Electronic address: krrajara@utmb.edu.
authors.forename: Krishna
authors.initials: K
authors.lastname: Rajarathnam
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D054360:Chemokine CXCL1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018922:HL-60 Cells; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability
chemical_list: D054360:Chemokine CXCL1; C475895:Cxcl1 protein, mouse; D006493:Heparin
keywords: Chemokines; Glycobiology; Glycosaminoglycan; Heparin; NMR; Neutrophil