Dystrophin's central domain forms a complex filament that becomes disorganized by in-frame deletions

Olivier Delalande,Marc Baaden,Marine Guilbaud,Christophe Tascon,Aurélie Nicolas,Arnaud Bondon,Émeline Pollet,Nicolas Férey,Mirjam Czjzek,Jean-François Hubert,Pierre Roblin,Angélique Chéron,Emmanuel Giudice,Anne-Elisabeth Molza,France Piétri‐Rouxel ,Javier Pérez ,Céline Raguénès-Nicol ,Raphaël Dos Santos Morais ,F Leturcq ,Élisabeth Le Rumeur

doi:10.1074/jbc.m117.809798

Abstract

Dystrophin, encoded by the DMD gene, is critical for maintaining plasma membrane integrity during muscle contraction events. Mutations in the DMD gene disrupting the reading frame prevent dystrophin production and result in severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in less severe Becker muscular dystrophy (BMD). Many known BMD deletions occur in dystrophin's central domain, generally considered to be a monotonous rod-shaped domain based on the knowledge of spectrin family proteins. However, the effects caused by these deletions, ranging from asymptomatic to severe BMD, argue against the central domain serving only as a featureless scaffold. We undertook structural studies combining small-angle X-ray scattering and molecular modeling in an effort to uncover the structure of the central domain, as dystrophin has been refractory to characterization. We show that this domain appears to be a tortuous and complex filament that is profoundly disorganized by the most severe BMD deletion (loss of exons 45-47). Despite the preservation of large parts of the binding site for neuronal nitric oxide synthase (nNOS) in this deletion, computational approaches failed to recreate the association of dystrophin with nNOS. This observation is in agreement with a strong decrease of nNOS immunolocalization in muscle biopsies, a parameter related to the severity of BMD phenotypes. The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs toward the goal of a successful gene therapy for DMD.

Highlights

The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs toward the goal of a successful gene therapy for Duchenne muscular dystrophy (DMD)
Dystrophin consists of an N-terminal actin-binding domain (ABD1), a central domain with 24 spectrin-like repeats interspaced by four hinge regions [2], and a cysteine-rich domain interacting with ␤-dystroglycan constituting the basis of the dystrophin– glycoprotein complex [3, 4] and a C-terminal domain
According to the Monaco rule [5], DMD is mostly a result of out-of-frame mutations in the DMD gene that result in a complete loss of the protein and a severe phenotype, whereas in-frame mutations of the DMD gene are mainly associated with Becker muscular dystrophy (BMD), where modified dystrophin is produced, resulting in less severe phenotypes

Summary

To whom correspondence should be addressed

Structural effects of in-frame deletion of the DMD gene of the severity of certain in-frame mutations to anticipate future therapies This could be achieved by structural and functional studies of the dystrophin central domain and of its protein products related to in-frame mutations. This BMD-induced structural disorganization of dystrophin is associated with an alteration of nNOS binding and with a strong decrease of nNOS labeling in patient muscle biopsies These results show that the in-frame character of the deletion does not warrant a functional protein or a stable protein structure and that our structural characterization of the dystrophin central domain should lead to a better understanding of dystrophin-associated molecular assembly and help for the design of future DMD gene therapies

Results

Discussion

Experimental procedures