Publication Abstract from PubMed
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 (C(2)A) at 1.95-A 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 Ca(2+), while the synaptotagmin-1 C(2)A 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 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.
The crystal structure of the C(2)A domain of otoferlin reveals an unconventional top loop region.,Helfmann S, Neumann P, Tittmann K, Moser T, Ficner R, Reisinger E J Mol Biol. 2011 Feb 25;406(3):479-90. doi: 10.1016/j.jmb.2010.12.031. Epub 2011 , Jan 7. PMID:21216247[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
