title: Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17.
abstract: Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main β-sheet and its specific C-terminal β-finger.
authors.affiliation: From the Université de Rennes1, Campus Santé, 35043 Rennes, France, the Institut de Génétique et Développement de Rennes, IGDR, UMR CNRS 6290, Rennes, France, and.
authors.forename: Anne-Elisabeth
authors.initials: AE
authors.lastname: Molza
authors.affiliation: the Illinois Institute of Technology, Chicago, Illinois 60616.
authors.forename: Khushdeep
authors.initials: K
authors.lastname: Mangat
authors.affiliation: From the Université de Rennes1, Campus Santé, 35043 Rennes, France, the Institut de Génétique et Développement de Rennes, IGDR, UMR CNRS 6290, Rennes, France, and.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Le Rumeur
authors.affiliation: From the Université de Rennes1, Campus Santé, 35043 Rennes, France, the Institut de Génétique et Développement de Rennes, IGDR, UMR CNRS 6290, Rennes, France, and.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Hubert
authors.affiliation: the Illinois Institute of Technology, Chicago, Illinois 60616.
authors.forename: Nick
authors.initials: N
authors.lastname: Menhart
authors.affiliation: From the Université de Rennes1, Campus Santé, 35043 Rennes, France, the Institut de Génétique et Développement de Rennes, IGDR, UMR CNRS 6290, Rennes, France, and olivier.delalande@univ-rennes1.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Delalande
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D019921:Biotinylation; D016189:Dystrophin / Q000737:chemistry*; D049029:Dystrophin-Associated Proteins / Q000737:chemistry; D005091:Exons; D006801:Humans; D056004:Molecular Dynamics Simulation; D018482:Muscle, Skeletal / Q000201:enzymology; D016296:Mutagenesis; D009154:Mutation; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D014965:X-Rays
chemical_list: C484258:DMD protein, human; D016189:Dystrophin; D049029:Dystrophin-Associated Proteins; C091824:syntrophin; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I; D000409:Alanine
keywords: dystrophin; molecular dynamics; muscular dystrophy; nitric-oxide synthase; site-directed mutagenesis