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.
Highlights
Few missense Duchenne muscular dystrophy (DMD)- or Becker muscular dystrophy (BMD)-linked point mutations have been reported in the dystrophin gene. 70 of such mutations were reviewed in the dystrophin gene encoding the rod domain [5]
We focused our study on the rod domain repeat 23 (R23) encoded by exon 59 [22] and corresponding to residues 2800 –2939 of the wild type full-length protein
Single mutation N2912D is mentioned in three cases from the Leiden data base with no mention of a pathology DMD, intermediary muscular dystrophy, or BMD pathology, but they are characterized as polymorphic variants [27]
Summary
The study of cloned wild type or mutated repeating single units from spectrin-like proteins represents a first significant step forward in the investigation of the rod domain (14 –20). We construct homology-based molecular model structures of the wild type and mutated repeat 23 of dystrophin.
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