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

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 the high severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in the 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, 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 exon 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 in the goal of a successful gene therapy for DMD.

Fine mapping of hydrophobic contacts reassesses the organization of the first three dystrophin coiled‐coil repeats

“…Finally, while the first structure prediction computed for dystrophin and based on spectrin patterns revealed only a majority of straight linker regions, 19 it was later observed experimentally that the dystrophin central domain was presenting many kinks at linker regions. 27 Our study of hydrophobic contacts through the first coiled-coil repeats of dystrophin, confirms its singularity towards both mathematical model of triple-helix coiled-coil 7 and spectrin regular pattern, confirming previous observations. 11,12 Indeed, the definition of the dystrophin coiled-coil repeat seems related to a dense, asymmetric and highly adaptable network of inter-helices hydrophobic contacts, a network also strengthened by local bending of Helices B.…”

“…Moreover, unlike many coiled‐coil proteins forming multimeric structures among them spectrin, dystrophin is known to be monomeric and this specificity appears to be partly related to this loosely conserved heptad pattern. Finally, while the first structure prediction computed for dystrophin and based on spectrin patterns revealed only a majority of straight linker regions, it was later observed experimentally that the dystrophin central domain was presenting many kinks at linker regions . Our study of hydrophobic contacts through the first coiled‐coil repeats of dystrophin, confirms its singularity towards both mathematical model of triple‐helix coiled‐coil and spectrin regular pattern, confirming previous observations .…”

“…From an exhaustive biochemical characterization of the dystrophin central domain, Mirza et al did already show that the R2 and R3 repeats of dystrophin were defining an integrated site . This was structurally confirmed in our study of dystrophin fragments by SAXS, by the observation of various large kinks between R1 and R2 but not between R2 and R3 . Analysis of the hydrophobic networks involving the R1–R2 and R2–R3 linkers brings some clues about the involvement of specific residues in these differentiated organizations.…”

“…Indeed, the differences inherent to the dimeric and monomeric nature of spectrin and dystrophin, respectively, raised the question about the relevance of a comparison between their dynamical properties. A recent study of multi‐repeat fragments of dystrophin in solution by small angle X‐ray scattering revealed the existence of unexpected high degrees of bending at some linker regions, in contrast to the findings in spectrin. Modulations in the bending amplitude and directionality of linker have been notably observed between dystrophin repeats R1 and R2 that is 40° in the R1–R2 fragment and 75° in the R1–R3 fragment.…”

“…SAXS‐derived models of the dystrophin fragments R1–R2 and R1–R3 were optimized following the previously described procedure based on interactive flexible fitting simulations driven by low‐resolution molecular shapes . The theoretical SAXS curves computed from these atomic molecular models were all fitting to the experimental SAXS data at a high level of confidence.…”