The Crystal Structure of the C2A Domain of Otoferlin Reveals an Unconventional Top Loop Region

Otoferlin (Otof), whose genetic mutations cause profound deafness in humans, is a protein composed of at least six C2 domains, which are known as Ca2+-binding and phospholipid-binding regions. Mammalian ferlin proteins are proposed to act in membrane fusion events, with Otof being specifically required for exocytosis in auditory hair cells. Ferlin C2 domains exhibit a rather low level of sequence similarity to those of synaptotagmins, protein kinase C isoforms, or phospholipases. Here, we report the crystal structure of the N-terminal C2 domain of Otof (C2A) at 1.95-Å resolution. In contrast to previous predictions, we found that this C2 domain is complete with eight β-strands. Comparing the structure of Otof C2A to those of other C2 domains revealed one top loop in Otof to be significantly shorter. This results in a depression of the surface, which is positively charged for the Otof C2A domain, and contrasts with the head-like protrusion surrounded by a negatively charged “neck” typically found in other C2 domains. Isothermal titration calorimetry and circular dichroism spectroscopy studies confirmed that Otof C2A is unable to bind Ca2+, while the synaptotagmin-1 C2A domain exhibited Ca2+ binding under the same conditions. Furthermore, floatation assays revealed a failure of Otof C2A to bind to phospholipid membranes. Accordingly, no positively charged β-groove-like surface structure, which is known to bind phosphatidylinositol-4,5-bisphosphate in other C2 domains, was found at the respective position in Otof C2A. Taken together, these data demonstrate that the Otof C2A domain differs structurally and functionally from other C2 domains.