title: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555 and.
authors.forename: Krishna Mohan
authors.identifier: 0000-0002-3675-6294
authors.initials: KM
authors.lastname: Sepuru
authors.affiliation: the Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery and Development, Virginia Commonwealth University, Richmond, Virginia 23219.
authors.forename: Balaji
authors.identifier: 0000-0003-3892-6178
authors.initials: B
authors.lastname: Nagarajan
authors.affiliation: the Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery and Development, Virginia Commonwealth University, Richmond, Virginia 23219.
authors.forename: Umesh R
authors.identifier: 0000-0002-1976-6597
authors.initials: UR
authors.lastname: Desai
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555 and krrajara@utmb.edu.
authors.forename: Krishna
authors.identifier: 0000-0002-0077-700X
authors.initials: K
authors.lastname: Rajarathnam
mesh_terms: D054360:Chemokine CXCL1 / Q000737:chemistry / Q000378:metabolism; D054365:Chemokine CXCL5 / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: C475829:CXCL1 protein, human; C486263:CXCL5 protein, human; C508606:CXCL8 protein, human; D054360:Chemokine CXCL1; D054365:Chemokine CXCL5; D016209:Interleukin-8; D009844:Oligosaccharides; D006493:Heparin
keywords: NMR; glycobiology; heparin; isothermal titration calorimetry (ITC); molecular dynamics