Schematic diagram of full-length otoferlin and the otoferlin fragments used in this study. (A) Otoferlin is composed of six tandem C2 domains followed by a C-terminal transmembrane domain (TMD). Recombinant proteins composed of either isolated C2 domains or three–C2 domain fragments were generated according to the amino acid designations listed on the right. (B) Conservation of putative calcium-binding ligands of the C2 domains of otoferlin (oto). Synaptotagmin I (syt) C2A and C2B are listed for comparison. The numbers correspond to the order of the five aspartate residues that function as ligands in the C2A (D172, 178, 230, 232, and 238) and C2B domains (D303, 309, 363, 365, 371) of synaptotagmin I (Sutton et al., 1995; Shao et al., 1998; Fernandez et al., 2001).