Prevalent mechanism of membrane bridging by synaptotagmin-1

Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release through its two C-2 domains (the C(2)A and C2B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble N-ethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C(2)AB) was shown to bind simultaneously to two membranes via the Ca2+-binding loops at the top of both domains and basic residues at the bottom of the C2B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC(2)AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC(2)AB oligomers bound to each membrane via the Ca2+-binding loops, with no contact of the bottom of the C2B domain with the membranes. We now show that lnC(2)AB containing impurities indeed aggregates in solution, but properly purified lnC(2)AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC(2)AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C2B domain contacts the membrane in a sizeable population of molecules of both membrane-bound C(2)AB and membrane-bound lnC(2)AB. NMR data on nanodiscs show that a fraction of C(2)AB molecules bind to membranes with antiparallel orientations of the C-2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C(2)AB and lnC(2)AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.