Disordered Ferlin C2A-C2B Linkers Bind Membranes and Encode Small Linear Motifs

Ferlins are vesicle trafficking proteins composed of folded C2 domains conjugated by linkers which are largely disordered. Although a role for the C2 domains as calcium sensors has been established it remains unclear whether the linkers function beyond acting as passive spacers. We examined the C2A-C2B linker sequences of vertebrate ferlins and found both putative short linear motifs (SLiMs) as well as membrane binding sequences for members of the protein family. Specifically, for otoferlin we identified an arginine-rich region proximal to an AP2 binding dileucine motif which interacts with negatively charged lipid membranes. Further, the linker region dominated the liposome binding properties of a larger recombinant C2A-C2B, two-C2 domain segment of otoferlin, suggesting a dominant role in mediating the membrane binding property of the N-terminus. We also found that alternative splicing of the otoferlin C2A-C2B linker adds an additional membrane binding segment and alters the affinity of membrane binding. Like otoferlin, a recombinant dysferlin linker interacted with liposomes. However, dysferlin encodes for SLiMs not detected in the otoferlin linker and interacted with both SH3- and WW- domain proteins as determined using fluorescence spectroscopy. We conclude that the C2A-C2B linker of vertebrate ferlins serves as a signaling platform by recruiting SLiM-binding partners. Membrane binding "hotspots" encoded in a subset of linkers including otoferlin may serve to localize protein complexes proximal to the cell membrane for activity.