Abstract
Ferlin proteins participate in such diverse biological events as vesicle fusion in C. elegans, fusion of myoblast membranes to form myotubes, Ca2+-sensing during exocytosis in the hair cells of the inner ear, and Ca2+-dependent membrane repair in skeletal muscle cells. Ferlins are Ca2+-dependent, phospholipid-binding, multi-C2 domain-containing proteins with a single transmembrane helix that spans a vesicle membrane. The overall domain composition of the ferlins resembles the proteins involved in exocytosis; therefore, it is thought that they participate in membrane fusion at some level. But if ferlins do fuse membranes, then they are distinct from other known fusion proteins. Here we show that the central FerA domain from dysferlin, myoferlin, and otoferlin is a novel four-helix bundle fold with its own Ca2+-dependent phospholipid-binding activity. Small-angle X-ray scattering (SAXS), spectroscopic, and thermodynamic analysis of the dysferlin, myoferlin, and otoferlin FerA domains, in addition to clinically-defined dysferlin FerA mutations, suggests that the FerA domain interacts with the membrane and that this interaction is enhanced by the presence of Ca2+.
Highlights
Vesicle via a single C-terminal transmembrane helix
We studied the thermal unfolding of dysferlin FerA and the two disease-linked mutations by differential scanning calorimetry (DSC) (Table 1) and circular dichroism (CD) (Table S2, Fig. S3)
The stability of the FerA domain is on the same order as that of dysferlin C2A
Summary
The recruitment and fusion of these patching vesicles by dysferlin to the site of injury occur rapidly, usually within one second[14]. A lack of dysferlin in muscle cells causes an accumulation of unfused vesicles near sites of injury[13]. Myoferlin is expressed in myoblasts and shares a considerable amount of sequence similarity with dysferlin, yet their physiological roles are different[19]. From a physiological perspective, ferlin proteins serve an essential role in membrane fusion. What is not yet understood is whether ferlins possess the fusion machinery to join disparate phospholipid membranes by themselves, or whether they require other partners as a part of a larger multi-protein fusion complex. Unlike other four-helix bundles, FerA domains are unique in that they can bind to, and perhaps insert into, phospholipid membranes in a Ca2+dependent manner. The ferlin fusion mechanism may share similar biophysical characteristics to both eukaryotic SNARE-based proteins and viral fusion proteins
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