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Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Forster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein–membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca2+ requirements: the threshold Ca2+ concentration for membrane binding is lowest, followed by apposit...

Synaptotagmin 1 (Syt1), a Ca2+ sensor involved in exocytosis in pre-synaptic neurons, contains a characteristic tandem C2 sequence (C2AB) which inserts into anionic membranes in the presence of Ca2+. Previous studies have shown that the C2AB fragment of Syt1 inserts into target membranes more deeply than either individual C2A or C2B domain, suggesting cooperative interaction between C2A and C2B in the membrane-docked state. This behavior stands in apparent contrast to that of another family member, Syt7, whose C2A and C2B domains have been shown to bind membranes independently. To compare the cooperative behaviors of the C2 domains in these two isoforms, the dissociation kinetics (off-rates) of protein-liposome complexes were measured upon addition of the Ca2+ chelator EDTA using stopped-flow fluorescence spectroscopy. Using liposomes composed of a 1:1 mixture of phosphatidylcholine and phosphatidylserine (PC/PS), the Syt1 C2AB tandem domain exhibits a much slower off-rate than either of the single domains. In contrast, dissociation kinetics for Syt7 C2AB were best fit to a two-step model in which each rate constant matches those from the individual domains. This preliminary result supports the presence of interdomain interactions in the membrane-docked state of Syt1 C2AB but not Syt7 C2AB. These experiments were performed with protein domains purified using affinity chromatography with high-salt washes and verified to be >95% free of nucleic acid contaminants; ongoing work aims to assess effects of polyanions on this apparent cooperative behavior, including ion-exchange steps in the protein purifications.

The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca(2+) to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.

Synaptotagmin (Syt) triggers Ca(2+)-dependent membrane fusion via its tandem C2 domains, C2A and C2B. The 17 known human isoforms are active in different secretory cell types, including neurons (Syt1 and others) and pancreatic β cells (Syt7 and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking mechanisms of Syt1 C2A and Syt7 C2A to vesicles comprised of physiological lipid mixtures. In agreement with previous studies, the Ca(2+) sensitivity of membrane binding is much higher for Syt7 C2A. We report here for the first time that this increased sensitivity is due to the slower target membrane dissociation of Syt7 C2A. Association and dissociation rate constants for Syt7 C2A are found to be ~2-fold and ~60-fold slower than Syt1 C2A, respectively. Furthermore, the membrane dissociation of Syt7 C2A but not Syt1 C2A is slowed by Na(2)SO(4) and trehalose, solutes that enhance the hydrophobic effect. Overall, the simplest model consistent with these findings proposes that Syt7 C2A first docks electrostatically to the target membrane surface and then inserts into the bilayer via a slow hydrophobic mechanism. In contrast, the membrane docking of Syt1 C2A is known to be predominantly electrostatic. Thus, these two highly homologous domains exhibit distinct mechanisms of membrane binding correlated with their known differences in function.

Synaptotagmin (Syt) triggers Ca2+-dependent membrane fusion during secretion via its tandem C2 domains, termed C2A and C2B. The seventeen known human isoforms are active in different secretory cell types, including neurons (SytI and others) and pancreatic β cells (SytVII and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking affinities, kinetics, and molecular driving forces for C2A and C2B domains from SytI and SytVII, using vesicles comprised of physiological target lipid mixtures. In agreement with previous studies, the Ca2+ sensitivity of membrane binding is greater for both domains from SytVII than for their counterparts in SytI. We demonstrate that for C2A, this increased sensitivity is due to a stronger SytVIIC2A membrane interaction, which involves substantial contribution from the hydrophobic effect. Association and dissociation rate constants for both SytVII domains are found to be significantly slower than their counterparts in SytI. For SytVIIC2A, the dissociation rate constant is ∼50-fold slower than SytIC2A and is reminiscent of the cPLA2C2 domain that is known to insert deeply into membranes. Addition of sodium sulfate decreases the dissociation rate of SytVIIC2A but not SytIC2A, further indicating that hydrophobic contacts play a major role in SytVIIC2A membrane docking. Thus, SytVIIC2A docks to membranes via both hydrophobic and electrostatic interactions, while the membrane docking interaction of SytIC2A is predominantly electrostatic. The inclusion of phosphatidylinositol-4,5-bisphosphate (PIP2) in membrane mixtures leads to increased affinity and slower dissociation for both C2B domains, but has minimal effects on C2A domains. Overall, highly homologous domains from these two proteins exhibit distinct mechanisms of membrane binding that may reflect their functions in different cell types.

Synaptotagmin (Syt) proteins serve as Ca2+ sensitive triggers in many exocytotic pathways, with the seventeen different human isoforms active in various cell types. Syt proteins contain two C2 domains, C2A and C2B, which bind membranes in response to Ca2+ to drive vesicle fusion. Much is known about the biophysical mechanism of function for Syt1, which triggers fusion for rapid neurotransmitter secretion, but less mechanistic information is available for other isoforms. Syt7 typically operates in slower pathways requiring smaller peak Ca2+ concentrations, and its C2 domains are known to bind membranes with a much higher Ca2+ sensitivity compared to Syt1. using kinetic and equilibrium fluorescence measurements of C2A domain docking to synthetic liposomes approximating the lipid composition of physiological membranes, we report that the differences between the two isoforms include kinetic and solute effects consistent with much greater hydrophobic membrane contact for Syt7 C2A. A strong hydrophobic contribution to the membrane docking mechanism of Syt7 C2A stands in contrast to the known electrostatic membrane interaction of Syt1 C2A, and is somewhat surprising given the 90% conserved amino acid polarity between the two domains. In order to test our proposed hydrophobic docking model for Syt7 C2A and probe its structural origins, we use a combination of site-directed mutagenesis, equilibrium and kinetic protein-membrane docking assays, and electron paramagnetic resonance-based depth measurements. In addition, single-molecule measurement of protein lateral diffusion on supported lipid bilayers is used to report on contributions of intra- and intermolecular protein-protein contact to the membrane-docked states of individual C2 domains and C2AB tandems. The results are interpreted to provide information on the structural origins of differences in function between these two isoforms.