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Molecular dissection of dystrophin identifies the docking site for nNOS

Scott Q. Harper1 Department of Pediatrics, Ohio State University College of Medicine and Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH 43205

t current count, we human dystrophin links the internal A Normal Dystrophin beings have 20,687 - R R R R NT 1 166177 24 CR CT to the . These early A coding genes encoded within nNOS biochemical data led to the logical con- our DNA (1). The unfortunate clusion that dystrophin primarily functions NT R R R R CR CT reality is that we know very little about 1 1516 22 to stabilize muscle membranes during the functions of most of these . Homologous Utrophin repeats 15-16 do not bind nNOS contraction, thereby providing mechanical Because new scientific discovery is built B Micro-Dystrophin support to the cell. Within this context, upon the foundation of prior work, it is R R R R NT 1 161724 CR the 24 STRs of the dystrophin central rod perhaps obvious that knowledge gaps like nNOS were often considered as a single unit, these can have compounding effects to R1 R16 R17 R24 linked together to form a shock absorber fi slow further scienti c advancement. Indeed, V α helix 3 VI α helix 3 α helix 3 or a spacer that separated the business H1 IV VII XIV α helix 2 III imagine the state of molecular biology II VIII XIII α helix 2 XII ends of the molecule. Although this model α helix 1 α helix 1 IX α helix 1 H4 and biotechnology today if Watson and Ι XI still holds true, the field has evolved in Crick’s publication of DNA structure had X nNOS binding site the last 15 y or so. We now know that been reported just a few decades earlier. RFHYDIKIFN despite their same general structure, in- So too is the case for translational research: Fig. 1. (A) Structure of dystrophin and utrophin. dividual STRs are not always functionally it is difficult to develop treatments for a See text for details. (B, Upper) This microdystrophin equivalent, and many possess distinct genetic disorder when we do not understand protects muscle membranes from damage during the affliction’s pathogenic mechanisms. functions beyond being a generic link in contraction and correctly localizes nNOS to the the rod-domain chain (5–10). The study Thus, the more basic knowledge we pos- . (B, Lower) Structural depiction of the sess about the function of a given disease micro-dystrophin 4 repeat rod domain. Colored published by Lai et al. in PNAS beautifully gene and its protein product, the more barrels indicate alpha helices from each respective underscores this point (2). ammunition we have to design rational STR. H1 and H4 represent hinge domains. The au- The current study builds on previous therapies. In PNAS, Lai et al. from the thors used this construct for domain swapping with work by this group, which showed that Duan laboratory at the University of comparable utrophin microdomains, the positions dystrophin STRs 16 and 17 were required of which are indicated by roman numerals. The for proper localization of neuronal nitric Missouri provide an exquisite example of starred IX marks the location of the 10-aa nNOS a basic study on protein structure and binding site. This site functions only in a proper oxide synthase (nNOS) to the muscle function with potentially important trans- structural environment. CR, cystine-rich domain; membrane. Why is nNOS important in lational implications for therapy of Du- CT, C-terminal domain; NT, N terminal domain. muscle and ? Muscles chenne muscular dystrophy (DMD) (2). comprise a significant portion of our DMD is a devastating form of muscular sequences. In truth, this work built on body mass and require a large amount of dystrophy caused by in the prior studies by this group and others in energy for proper function. Metabolites dystrophin gene (DMD). DMD holds many the DMD field over many years, so a brief and oxygen are brought to the muscles via distinctions, but its size is particularly re- summary of dystrophin structure/function the vasculature. nNOS primarily functions markable: the gene comprises 79 fl research will help put the current work in in regulating blood ow to ensure the spread across 2.4 million base pairs, mak- proper context. metabolic needs of contracting muscles ing it the largest human gene and roughly Dystrophin mutations were identified are met. Thus, DMD muscles that lack 90-times larger than average. The gene as the cause of DMD in 1986 (4). The functional dystrophin, and subsequently produces multiple protein isoforms, the fi have mis-localized nNOS, become sus- most predominant of which is a leviathan rst clues about its function came from examinations of the primary amino acid ceptible to both mechanical damage and consisting of 3,685 amino acids (aa) and fl sequence, which predicted a large, rod- exacerbating blood- ow restrictions (called measuring 427 kDa (3). Although the size functional ischemia). Although the mis- and complexity of this gene and protein shaped protein (3). Four major functional domains were identified: at the N terminus localization of nNOS in DMD was de- product may be impressive as a piece of scribed in 1995, the importance of this biological trivia to those not working in the is the eponymous N-terminal domain, phenomenon was not fully appreciated neuromuscular field, doing so here has with the COOH-end harboring the cyste- until more recently, and the Duan labora- a more immediate purpose: grasping the ine-rich and aptly described C-terminal tory has led the charge (2, 7, 11–14). This enormity of dystrophin is necessary to domain (Fig. 1) (3). These regions are appreciate the impressive work published separated by a long central rod, composed most recent report provides the most de- by Lai et al. in PNAS (2). In their work, of 24 structurally similar -type tailed characterization yet of the structural the authors perform an exhaustive set of repeats (STRs) and four flexible hinge elements in dystrophin required for proper structure/function studies to identify a regions (3) (Fig. 1). Binding sites for other nNOS localization and function. functionally important 10-aa microdomain proteins reside in the terminal regions of buried within the dystrophin protein dystrophin, including motifs that direct (representing a mere 0.2% of the entire critical interactions with cytoskeletal Author contributions: S.Q.H. wrote the paper. molecule), and further show that this motif and a group of transmembrane, cytoplas- The author declares no conflict of interest. functioned only when properly framed mic, and extracellular proteins called the See companion article on page 525. by a specific set of four α-helical peptide dystrophin glycoprotein complex. Thus, 1E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1220256110 PNAS | January 8, 2013 | vol. 110 | no. 2 | 387–388 Downloaded by guest on October 2, 2021 To accomplish this characterization, the pothesized that subtle structural differ- for a specific dystrophin function to date. authors cleverly use two major elements. ences in the flanking α-helices of R16-R17 Finding a functional motif that is 10-aa The first element is the minimal dystrophin could affect the way this R17α1 motif was long in a protein this size deserves some unit previously shown by this laboratory to positioned in 3D space, thereby affecting recognition. Third, the authors underscore bind nNOS: a microdystrophin they created the ability or inability of nNOS to locate the importance of mammalian in vivo called ΔR2-R15/ΔR18-R23/ΔC(Fig.1). and properly bind the site. To address assays for structure/function studies. The second critical element is utrophin, an this question, the authors again used a Historically, dystrophin structure studies autosomal paralog of dystrophin that con- domain-swapping strategy, but instead of were performed in vitro, typically using tains the same multidomain structure with using utrophin sequences, they inserted peptide fragments generated in bacteria, a structurally homologous 22-repeat rod individual α-helices from dystrophin R18, yeast, or insect cells. The differential domain, but lacks the ability to bind nNOS which is not required for nNOS binding. results reported by the Duan group using (Fig. 1A). The Duan group then proceeds to identical constructs in yeast versus mice methodically swap 14 different micro- should be “exhibit A” for anyone consid- domains from utrophin into homologous The structural elements ering the best strategy for performing regions of dystrophin, with the goal of structure/function studies of any protein identifying the critical regions for nNOS required for proper nNOS in the future. membrane localization. The authors use a localization should be Finally, this study has important thera- viral vector to deliver their domain-swapped peutic implications. Although DMD is microdystrophin/utrophin chimeras to dys- included in any DMD caused by the absence of functional dys- trophic mouse muscle (Fig. 1B). Of the trophin, not all dystrophin mutations 14 constructs they generate, only one therapy for which cause severe DMD phenotypes. A milder (IX; figures 2 and 3 of ref. 2) disrupted form of DMD, called Becker muscular the ability of microdystrophin to properly dystrophin restoration is dystrophy (BMD), arises from dystrophin localize nNOS. This small 10-aa sequence, mutations that typically cause in-frame, located in dystrophin repeat 17, thus repre- the goal. partial deletions of the rod domain. In sents the nNOS binding domain of dystro- these cases, smaller, subfunctional versions phin. With this sequence, the authors found of dystrophin protein are produced and the proverbial needle in the haystack. Thus, Lai et al. built multiple R16-R17 become properly localized at the sarco- Nevertheless, the authors dutifully constructs in which each individual helix lemma. One BMD patient was famously sought to confirm their finding, and used (of six possible) was replaced by the cor- ambulant well into his 60s, despite missing fi a reciprocal experimental strategy to ac- responding helix from R18 ( gures 4 and nearly two-thirds of the dystrophin rod complish it. Lai et al. (2) reason that if this 5 of ref. 2). Testing this strategy in an in domain (15). These truncated dystrophins 10-aa microdomain is the minimal unit vitro system failed to identify any effects in BMD patients formed the basis for required for nNOS binding, swapping it of these substitutions, so the authors went therapeutic development of DMD using into utrophin, which is otherwise lousy back to their tried-and-true method of in two strategies: dystrophin gene-re- for nNOS, should give the protein nNOS vivo expression using viral vectors. Using placement therapy and skipping. binding ability. Thus, the authors con- this strategy, they found that the 10-aa For the former, full-length dystrophin is structed a microutrophin analogous to the nNOS binding motif required the α2 and too large to fit into currently available ΔR2-R15/ΔR18-R23/ΔC microdystrophin, α3 helices from both R16 and R17 for viral vectors, but miniature versions of but with the nNOS binding motif from proper function. dystrophin (i.e., microdystrophins) can dystrophin R17. Following delivery to This study is noteworthy for several be packaged and effectively delivered to mouse muscle, they surprisingly found that reasons. First, it represents an incredibly muscle, with therapeutic benefit (6). For nNOS was absent from the membrane, large amount of painstaking work. Indeed, the latter, antisense oligonucleotides can despite that fact that microutrophin was the authors generated 48 different con- be designed to skip over mutated exons, abundantly localized there. This finding structs for delivery to mouse muscle. Sec- effectively converting a DMD transcript suggests that additional sequences within ond, Lai et al. encountered unexpected, to a BMD one (16). The data from the R16-R17 are required to support nNOS and at first glance, negative results that Duan laboratory, reported in this paper binding. The authors thus set out to could have signaled the end of the study and previous studies, demonstrates that identify these additional sequences. (figures 3 and 4 of ref. 2). Impressively, the the structural elements required for Each STR is composed of three α-helices. authors persisted, formed new hypotheses, proper nNOS localization should be in- The first α-helix of R17 (R17α1) contains and ultimately successfully identified the cluded in any DMD therapy for which the nNOS binding site. The authors hy- smallest known structural requirements dystrophin restoration is the goal.

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