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.
Highlights
Nitric-oxide synthase (NOS) function in skeletal muscles is due to its interaction with dystrophin
Repeats R16 and R17 of the central domain of dystrophin, two spectrin-like repeats theoretically organized in a coiled-coil fold of three ␣-helices A, B, and C for R16 and AЈ, BЈ, and CЈ for R17, were shown to constitute an neuronal NOS (nNOS) interaction site that is distant from the dystrophin C-terminal region that associates with syntrophin (17–19)
Alanine-scanning mutagenesis of charged residues using 32 variants of dystrophin R16 –17 highlighted multiple regions where mutagenesis modified the binding affinity, indicating that these regions are involved in the interaction with nNOS
Summary
Nitric-oxide synthase (NOS) function in skeletal muscles is due to its interaction with dystrophin. 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. Repeats R16 and R17 of the central domain of dystrophin, two spectrin-like repeats theoretically organized in a coiled-coil fold of three ␣-helices A, B, and C for R16 and AЈ, BЈ, and CЈ for R17, were shown to constitute an nNOS interaction site that is distant from the dystrophin C-terminal region that associates with syntrophin (17–19) This explains why the presence of syntrophin at the sarcolemma through its association with dystrophin did not always maintain nNOS localization as in BMD cases (11). Analysis of the sequence similarities of the site to its modifications in several BMD deletions provides clues about the nNOS sarcolemma localization in these pathologies
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