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 C-2 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 C-2 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 C-2 domain of Otof (C(2)A) at 1.95-angstrom resolution. In contrast to previous predictions, we found that this C-2 domain is complete with eight beta-strands. Comparing the structure of Otof C(2)A to those of other C-2 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 C(2)A domain, and contrasts with the head-like protrusion surrounded by a negatively charged "neck" typically found in other C-2 domains. Isothermal titration calorimetry and circular dichroism spectroscopy studies confirmed that Otof C(2)A is unable to bind Ca2+, while the synaptotagmin-1 C(2)A domain exhibited Ca2+ binding under the same conditions. Furthermore, floatation assays revealed a failure of Otof C(2)A to bind to phospholipid membranes. Accordingly, no positively charged beta-groove-like surface structure, which is known to bind phosphatidylinositol-4,5-bisphosphate in other C-2 domains, was found at the respective position in Otof C(2)A. Taken together, these data demonstrate that the Otof C(2)A domain differs structurally and functionally from other C-2 domains. (C) 2011 Elsevier Ltd. All rights reserved.