Inversion of Ligand Binding Preferences in Re-Engineered Dysferlin C2Av1

Abstract

Dysferlin is a 237 kDa skeletal muscle protein that plays a key role in Ca2+-mediated membrane repair of muscle cells. The full length protein possesses seven tandem C2 domains and a single C-terminal transmembrane helix. Mutations in the dysferlin protein have been linked to Limb-Girdle Muscular Dystrophy in humans. The canonical C2A domain of dysferlin binds Ca2+ and phospholipid similar to other Ca2+-dependent phospholipid binding C2 domains; however, another co-expressed isoform of dysferlin C2A, C2A variant 1 (C2Av1), binds negatively changed phospholipid, but not Ca2+. To test whether we could modulate the binding behavior of the alternate domain with a simple mutation, we mutated two Arg residues (R20 and R21) in Loop 1 of C2Av1 to Asp, and tested the resulting protein for Ca2+ and phospholipid binding. Interestingly, the resulting domain (C2Av1 R20D,R21D) bound a single calcium ion but not phospholipid, a complete inversion of binding preference. Our result also supports the hypothesis that the C2A domains of dysferlin interact with negatively charged phospholipid surfaces using only electrostatic attraction. The 3D structure of the domain including ligand was obtained using X-Ray Crystallography.