Calcium signaling and pathogenesis of dysferlin C2 domains

Yuning Wang, The University of Western Ontario

Abstract

Failure to repair injured sarcolemmal membranes leads to muscular dystrophy, a degenerative disorder that results in increasing weakness and gradual wasting of skeletal muscles. Mutations in the gene encoding dysferlin are causative for limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) forms of the disease. Dysferlin is a Ca²⁺-sensitive membrane repair protein involved in trafficking of proteins and vesicles around injured membranes in skeletal muscle cells. It is a cytosolic-facing, membrane bound protein composed of seven intermittently spaced C2 domains (C2A-C2G). Dysferlin activity is mediated by the Ca²⁺-dependent actions of the C2 domains. The main goals of this thesis were to characterize the structure, dynamics and Ca²⁺-binding mechanisms of the C2 domains, and assess the impact of pathogenic substitutions on the C2 domains.

First, the dynamics of the C2A domain in both Ca²⁺-free and Ca²⁺-bound state was comprehensively probed using NMR spectroscopy, which revealed a remarkable flexibility change in the loop region upon Ca²⁺ binding. The Ca²⁺-binding properties of the C2A domain was studied on the basis of the crystal structure of the Ca²⁺-bound C2A, which determined the stoichiometry, binding sites and affinities. Further, mutagenesis study revealed the important role of the electrostatic potential contributed by non-Ca²⁺-coordinating residues, which provides novel insights into the mechanism of Ca²⁺ binding to the dysferlin C2A domain as a link for membrane repair.

To understand the consequences of pathogenic mutations, three substituted C2A proteins were generated and analyzed. It was demonstrated that there is dramatic decrease in stability resulted from the substitutions. The unfolding or improper folding of the substituted C2A domain is predicted to be responsible for impaired dysferlin function in the membrane repair process, and consequently the wasting of skeletal muscles in muscular dystrophy patients.

Finally, proteins encompassing the C2B and C2C domains of dysferlin were designed and generated using a combination of computational and experimental methods. The precise domain boundaries of the C2B and C2C domains were determined, which will provide useful information for the further characterization of dysferlin structure.