title: A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin.
abstract: Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three-dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.
authors.affiliation: UMR CNRS 6026 Interactions Cellulaires et Moléculaires,Equipe RMN et Interactions Lipides-Protéines and UMR CNRS 6061 Génétique et Développement, Université de Rennes 1, IFR 140, FacultédeMédecine, CS 34317, 35043 Rennes Cedex, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Legardinier
authors.forename: Baptiste
authors.initials: B
authors.lastname: Legrand
authors.forename: Céline
authors.initials: C
authors.lastname: Raguénès-Nicol
authors.forename: Arnaud
authors.initials: A
authors.lastname: Bondon
authors.forename: Serge
authors.initials: S
authors.lastname: Hardy
authors.forename: Christophe
authors.initials: C
authors.lastname: Tascon
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Le Rumeur
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Hubert
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016189:Dystrophin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D020388:Muscular Dystrophy, Duchenne; D020125:Mutation, Missense; D011489:Protein Denaturation / Q000235:genetics; D055550:Protein Stability; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D016189:Dystrophin