Structural Studies of a Novel Extended Synaptotagmin with Only Two C2 Domains from Trypanosoma brucei

Extended synaptotagmins (E-Syts) are a group of evolutionarily conserved proteins localizing at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate inter-membrane lipid transfer and control plasma membrane lipid homeostasis. There are three E-Syts in mammals, which all contain an N-terminal transmembrane (TM) hairpin for the association with the ER membrane, and a central synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain as a carrier for phospholipids. Additionally, mammalian E-Syt1 and E-Syt2/E-Syt3 respectively have five and three C2 domains at their C-terminus, which mediate their interaction with the plasma membrane. Here we report a novel E-Syt from the protist parasite Trypanosoma brucei, TbE-Syt. Despite also having the TM hairpin and the SMP domain, TbE-Syt contains only two C2 domains, which makes it the shortest E-Syt currently identified. We determined a 1.5 A resolution crystal structure of the TbE-Syt C2B domain, which showed that it binds two Ca2+ ions. Our mutagenesis studies demonstrated that TbE-Syt-C2B binds lipids via both Ca2+- and PI(4,5)P2-dependent means. Our bioinformatics analyses showed that TbE-Syt-C2A lacks the Ca2+-binding site found in C2B but may still interact with lipids via a basic surface patch. Furthermore, in contrast to the rigid V-shaped arrangement of the C2A and C2B domains in human E-Syt2, our analysis suggests that the two C2 domains in TbE-Syt are connected by a long flexible linker. We propose a working model for how TbE-Syt might tether the ER membrane and the plasma membrane to transfer lipids between the two organelles.