Synaptotagmin-7 (Syt-7) is among the most Ca2+-sensitive synaptotagmins. It is one of two Ca2+-dependent synaptotagmins (along with Syt-9) that are abundant in pancreatic β-cells. Ca2+-dependent synaptotagmins bind Ca2+ and membranes through their C2A and C2B domains, located at their C-terminus. In 2018, our lab worked with collaborators to write a comprehensive review article about Syt-7.
Structure of the Syt-7 C2AB tandem domain. Ribbon diagram (blue) is a composite of published Syt-7 C2A (PDB 2D8K) and C2B (PDB 3N5A) structures. Calcium-binding loops are highlighted in magenta. From Vasquez et al, Biochemistry 2014, 53, 7904-7913.
In particular, the C2A domain of Syt-7 binds membranes with much stronger affinity than other C2 domains. This is part of how it achieves such high Ca2+ sensitivity. In 2012, we showed that the C2A domain of Syt-7 binds membranes much more strongly than the C2A domain of Syt-1, which is the best-studied neuronal synaptotagmin. The increased binding strength of Syt-7 C2A is due to a combination of electrostatic interactions and the hydrophobic effect. Later, in 2015, in collaboration with Hai Lin’s lab, we published two companion papers, one experimental and one computational, demonstrating that Syt-7 C2A inserts deeper into membranes than the Syt-1 C2A domain. We also collaborated with the lab of Scott Reed to show that the Syt-7 C2A domain can induce clustering and aggregation of liposomes and lipid-coated nanoparticles, something that the Syt-1 C2A domain is not known to do.
Syt-7 C2A domain inserts into membranes. Ribbon diagram (blue) of Syt-7 C2A domain structure (PDB 2D8K) inserted into a simulated lipid bilayer. The picture shows the depth and orientation determined using electron paramagnetic resonance experiments. Calcium ions are in orange. Phenylalanine residues on the tips of calcium-binding loops 1 and 3 (CBL1 and CBL3) are shown as green sticks. From Osterberg et al, Biochemistry 2015, 54, 5684-5695.
The C2B domain of Syt-7 is important too. We contributed to a 2018 paper from the lab of collaborator Arun Anantharam that showed how the Ca2+-binding loops of the C2 domain confer increased fusion pore lifetimes in chromaffin cells compared to Syt-1.
Molecular dynamics simulation of calcium binding by C2B domains. Images are a close-up of the calcium-binding regions of Syt-1 C2B (top) and a chimeric protein containing Syt-7 C2B loops on a Syt-1 C2B structure (bottom). Calcium ions are in orange numbered 1-3. Chloride ions are in green. The outermost calcium ion in the chimeric structure coordinates more chloride ions and fewer waters, consistent with its stronger attraction to anionic lipids. From Bendahmane et al, Mol. Biol. Cell 2018, 29, 834-845.
Of course, the C2A and C2B domains of synaptotagmins work together to bind membranes and trigger fusion in response to calcium. Our lab and others are still trying to figure out how they do that. Notably, Syt-7 appears to differ from Syt-1 in the way the two C2 domains work together. We showed in 2014 that the two C2 domains of Syt-7 bind membranes independently, like separate modules that are connected by a flexible linker. Our 2019 paper in Biophysical Journal followed up on this result by demonstrating that this is different from Syt-1, whose C2A and C2B domains bind membranes cooperatively, like a dimer of C2 domains that function as a single unit. The biological significance of this mechanistic difference between Syt-1 and Syt-7 is not yet clear.
Model of membrane dissociation mechanisms for individual and tandem C2 domains of Syt-1 and Syt-7. Arrow labels indicate off-rates of each species. From Tran et al, Biophys. J. 2019, 116, 1025-1036.
Despite the years of work by our lab and others, many open questions remain: Why does it matter that Syt-7 C2 domains work independently while Syt-1 C2 domains are cooperative? Are other Ca2+-dependent synaptotagmins cooperative or independent? How does Syt-7 induce membrane fusion? How does it produce long-lived fusion pores? Does it induce or stabilize saddle-curvature as we have hypothesized? What are the respective roles of Syt-7 and Syt-9 in the β-cell? We are approaching these questions and others in collaboration with other research groups through a combination of biophysical experiments, computations, and cell-based studies.
Hypothetical structural model of Syt-7 binding in a fusion pore neck to slow pore expansion. A: Composite structures of Syt-7 C2A and C2B domains with the polybasic regions shown as blue spheres (left) or as an electrostatic surface (right). B: With membrane binding surfaces potentially facing in three different directions simultaneously, the C2AB domain could bind and stabilize the high-curvature fusion pore as shown. From McDougall et al, J. Gen. Physiol. 2018, 150, 783-807.