title: α2 and α3 helices of dystrophin R16 and R17 frame a microdomain in the α1 helix of dystrophin R17 for neuronal NOS binding.
abstract: Homologous spectrin-like repeats can mediate specific protein interaction. The underlying mechanism is poorly understood. Dystrophin contains 24 spectrin-like repeats. However, only repeats 16 and 17 (R16/17) are required for anchoring neuronal NOS (nNOS) to the sarcolemma. Through an adeno-associated virus-based in vivo binding assay, we found that membrane expression of correctly phased R16/17 was sufficient to recruit nNOS to the sarcolemma in mouse muscle. Utrophin R15/16 is homologous to dystrophin R16/17. Substitution of dystrophin R16/17 microdomains with the corresponding regions of utrophin R15/16 suggests that the nNOS binding site is located in a 10-residue fragment in dystrophin R17 α1 helix. Interestingly, swapping this microdomain back into utrophin did not convey the nNOS binding activity. To identify other structural features that are required for nNOS interaction, we replaced an individual α-helix of dystrophin R16/17 with an equivalent α-helix from another dystrophin repeat. In vitro study with yeast two-hybrid suggests that most α-helices of R16/17, except for the R17 α1 helix, were dispensable for nNOS interaction. Surprisingly, in vivo binding assay showed that α2 and α3 helices of both R16 and R17 were essential for nNOS binding in muscle. We concluded that a microdomain in the α1 helix of dystrophin R17 binds to nNOS in a way uniquely defined by two pairs of the flanking helices. Our results provide an explanation for how structurally similar spectrin-like repeats in dystrophin display selective interaction with nNOS. The results also open new therapeutic avenues to restore defective nNOS homeostasis in dystrophin-null Duchenne muscular dystrophy.
authors.affiliation: Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lai
authors.forename: Junling
authors.initials: J
authors.lastname: Zhao
authors.forename: Yongping
authors.initials: Y
authors.lastname: Yue
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Duan
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D018101:Mice, Inbred mdx; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013049:Spectrin / Q000235:genetics; D020798:Two-Hybrid System Techniques; D049411:Utrophin / Q000235:genetics / Q000378:metabolism
chemical_list: D016189:Dystrophin; D049411:Utrophin; C041393:spectrin-like proteins; D013049:Spectrin; D052248:Nitric Oxide Synthase Type I