title: The crystal structure of the C₂A domain of otoferlin reveals an unconventional top loop region.
abstract: 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 Ca(2)(+)-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 (C₂A) at 1.95-Å resolution. In contrast to previous predictions, we found that this C(2) domain is complete with eight β-strands. Comparing the structure of Otof C₂A 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 C₂A 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 C₂A is unable to bind Ca(2+), while the synaptotagmin-1 C₂A domain exhibited Ca(2+) 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 β-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 C₂A. Taken together, these data demonstrate that the Otof C₂A domain differs structurally and functionally from other C(2) domains.
authors.affiliation: Molecular Biology of Cochlear Neurotransmission Junior Research Group, University Medical Center Göttingen, Robert-Koch-Str. 40, 37099 Göttingen, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Helfmann
authors.forename: Piotr
authors.initials: P
authors.lastname: Neumann
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
authors.forename: Tobias
authors.initials: T
authors.lastname: Moser
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
authors.forename: Ellen
authors.initials: E
authors.lastname: Reisinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005089:Exocytosis; D006198:Hair Cells, Auditory / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D008565:Membrane Proteins; C533993:Otof protein, rat; D010743:Phospholipids