title: Unique properties of human β-defensin 6 (hBD6) and glycosaminoglycan complex: sandwich-like dimerization and competition with the chemokine receptor 2 (CCR2) binding site.
abstract: Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human β-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the β2 and β3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and β2-β3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a "sandwich-like" model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding.
authors: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas and Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-902, Brazil; Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-902, Brazil. Electronic address: vpaula@cnrmn.bioqmed.ufr.br.
authors: Viviane S
authors: VS
authors: De Paula
authors: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-902, Brazil.
authors: Vitor H
authors: VH
authors: Pomin
authors: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas and Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-902, Brazil; Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-902, Brazil. Electronic address: valente@cnrmn.bioqmed.ufrj.br.
authors: Ana Paula
authors: AP
authors: Valente
mesh_terms: D001665:Binding Sites; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D062105:Molecular Docking Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D054390:Receptors, CCR2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D023083:beta-Defensins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C515334:CCR2 protein, human; C465592:DEFB106A protein, human; D006025:Glycosaminoglycans; D054390:Receptors, CCR2; D023083:beta-Defensins; C020341:glucosaminoglycans
keywords: Chemokine Receptor; Docking; Glycosaminoglycan; Nuclear Magnetic Resonance (NMR); Protein Dynamic; defensin