Structural and biochemical characterisation of utrophin and dystrophin spectrin repeat domains : submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry, Massey University, Palmerston North, NZ

Abstract

Duchenne and Becker muscular dystrophies are muscle-wasting disorders caused by mutations in the X-linked dystrophin gene. Dystrophin is a large cytoskeletal protein belonging to the spectrin superfamily, that links intracellular F-actin to the extracellular matrix via a membrane-associated glyco-protein complex thus maintaining structural rigidity and flexibility. Utrophin is a widely expressed protein that has been shown to functionally compensate for dystrophin in cultured muscle cells as well as in the muscular dystrophy mice model. Both utrophin and dystrophin share a similar domain architecture with N-terminal actin-binding domains and C-terminal variable domains separated by 22 or 24 spectrin-like repeats respectively. Therapeutic strategies to replace individuals having defective dystrophin with utrophin require full characterisation of these proteins. In this thesis, high-resolution structures of the N-terminal first spectrin repeat domains from utrophin and dystrophin have been determined by x-ray crystallography to 1.95 and 2.3 Å. Despite multiple structures of spectrin repeats in the Protein Data Bank from a-actinin, spectrin and plectin these are the first structures determined for any of the spectrin repeats from utrophin and dystrophin. These structures are similar to one another and display a three-helix bundle spectrin repeat fold. The repeat domain structure reveals the relationship between the canonical spectrin repeat domain sequence motif and the structural domain for utrophin and dystrophin, showing the N-terminal first spectrin repeat to be extended at the C-terminal end. Earlier biochemical studies revealed that the extension at the C-terminus was required for the protein’s stability. Studies have also shown that spectrin repeats of utrophin are required for a higher affinity interaction of the actin-binding domain with F-actin. However, it was unclear whether the N-terminal repeat domain has an intrinsic affinity for F-actin. In the present study the actin-binding properties of these spectrin repeats are elucidated. Previous experiments using molecular dynamic simulations and atomic force microscopy of tandem spectrin repeat domains from erythroid spectrin and a-actinin suggested that flexibility of multiple repeats depends upon the linker region. However under higher extension, the triple helical domain further undergoes unfolding and refolding and thus functions as an elastic element within the cell. Studies using steered molecular dynamics suggested that the force required for unfolding the N-terminal first spectrin repeat domain from utrophin is higher in comparison to that of the N-terminal first spectrin repeat from dystrophin.