Abstract
Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.
Highlights
The X-linked Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by mutations in dystrophin (Dys) associated with muscle plasma membrane fragility, increased intracellular Ca2+ levels and proteolytic activity [1,2] leading to muscle myofibrillar decomposition with subsequent replacement by fibrous and fat tissue
Dys and Utr potentially act as cellular biomechanical shock-absorbers limiting damage to the plasma membrane with the spectrin repeat domains acting as spring units by undergoing force-induced unfolding [10]
We present the first structures of Dys and Utr spectrin repeat domains that reveal the phasing of the helices with respect to the sequence motif and its underlying heptad repeat
Summary
The X-linked Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by mutations in dystrophin (Dys) associated with muscle plasma membrane fragility, increased intracellular Ca2+ levels and proteolytic activity [1,2] leading to muscle myofibrillar decomposition with subsequent replacement by fibrous and fat tissue. The importance of individual spectrin repeat domains for overall Dys function was initially assumed to be low owing to the observation that in-frame deletion of repeats 4–19, (nearly half of Dys) gave rise to a mild BMD phenotype [11]. This assumption is becoming increasingly challenged; e.g. an inframe two amino acid deletion that destabilizes repeat 23 is associated with a severe DMD phenotype [12]
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