Calcium Binding Preferentially Stabilizes Apical End of Dysferlin C2A

Abstract

Dysferlin is a crucial component of the eukaryotic membrane repair machinery, helping to seal microtears in the sarcolemmal membrane that arise from constant mechanical insult. Loss of this protein results in several muscle‐wasting phenotypes in both human patients and mouse models. Dysferlin contains seven C2 domains, which are well‐documented Ca2+‐dependent lipid sensors, which suggests a putative role in the calcium‐sensitive steps of membrane repair for dysferlin. A deeper understanding of the mechanisms that govern how dysferlin coordinates calcium signals with repair machinery mobilization is essential to develop therapeutic strategies. Out of the many C2 domains, the N‐terminal C2A domain is of particular interest because it has proved essential to the membrane repair process. Initial thermodynamic studies of this domain showed a curiously low energy of unfolding, suggesting that the domain is partially unfolded under resting calcium conditions. Here we use nuclear magnetic resonance spectroscopy and supporting biophysical techniques to examine the stabilizing effect of millimolar levels of calcium at the residue level and conclude that calcium binding primarily affects the apical end of the C2A domain of dysferlin. Calcium binding to C2 domains has been previously shown to induce subtle conformational shifts towards a more membrane‐competent fold, however, our studies provide the most drastic example of this phenomenon to date.