Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P [Molecular Biophysics]

Dysferlin is a large transmembrane protein that plays a key role in cell membrane repair, and underlies a recessive form of inherited muscular dystrophy. Dysferlinopathy is characterized by absence or marked reduction of dysferlin protein, with 43% of reported pathogenic variants being missense variants that span the length of the dysferlin protein. The unique structure of dysferlin, with seven tandem C2-domains separated by linkers, suggest dysferlin may dynamically associate with phospholipid membranes in response to Ca2+ signaling. However, the overall conformation of the dysferlin protein is uncharacterized. To dissect the structural architecture of dysferlin, we have applied the method of limited proteolysis that allows non-specific digestion of unfolded peptides by trypsin. Using five antibodies spanning the dysferlin protein, we identified a highly reproducible jigsaw map of dysferlin fragments protected from digestion. Our data infers a modular architecture of four tertiary domains; 1) C2A which is readily removed as a solo domain, 2) mid-region C2B-C2C-Fer-DysF, commonly excised as an intact module, with sub-digestion to different fragments suggesting several dynamic folding options 3) C-terminal four C2 domain module and 4) calpain-cleaved mini-dysferlinC72, which is particularly resistant to proteolysis. Importantly, we reveal a patient missense variant L344P which largely escapes proteosomal surveillance, shows subtle but clear changes in tertiary conformation. Accompanying evidence from immunohisto-chemistry and flow cytometry, using antibodies with conformationally sensitive epitopes, supports proteolysis data. Collectively, we provide insight into the structural topology of dysferlin and show how a single missense mutation within dysferlin can exert local changes in tertiary conformation.

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