α2 and α3 helices of dystrophin R16 and R17 frame a microdomain in the α1 helix of dystrophin R17 for neuronal NOS binding

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 mousemuscle. 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 alpha 1 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 alpha-helix of dystrophin R16/17 with an equivalent alpha-helix from another dystrophin repeat. In vitro study with yeast two-hybrid suggests that most alpha-helices of R16/17, except for the R17 alpha 1 helix, were dispensable for nNOS interaction. Surprisingly, in vivo binding assay showed that alpha 2 and alpha 3 helices of both R16 and R17 were essential for nNOS binding in muscle. We concluded that a microdomain in the alpha 1 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.