Lipid versatility of the C2C domain of Extended Synaptotagmin 3 safeguards plasma membrane binding
Membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) are critical hubs for lipid homeostasis and signal transduction. Extended Synaptotagmins (E-Syts) tether the ER to the PM and mediate lipid transport from the PM to the ER following Ca2+ signaling. Although E-Syts are essential for maintaining ER-PM contact sites during PI(4,5)P2 resynthesis, the molecular mechanisms governing their membrane association remain poorly understood. In particular, it is unclear how these contact sites are maintained following PI(4,5)P2 hydrolysis, when their principal membrane-binding ligand is transiently depleted. Here, we characterize the membrane-binding mechanism of the C-terminal C2 domain of extended synaptotagmin 3 (E-Syt3) by combining coarse-grained molecular dynamics simulations with live-cell experiments. We identify distinct lipid binding sites with specific affinities for phosphoinositides as well as for phosphatidylinositol (PI) and phosphatidylserine (PS). While phosphoinositide interactions dominate membrane binding under resting conditions, PI and PS binding may provide an alternative membrane-binding mechanism that helps preserve ER-PM contacts during PI(4,5)P2 depletion. Our simulations further reveal that phosphoinositide binding is reinforced by the insertion of a tryptophan residue into the membrane hydrophobic core, providing a mechanistic basis for the high membrane affinity of the C2C domain. Together, our findings uncover the molecular determinants of E-Syt3 C2C-mediated membrane binding and provide mechanistic insight into the stability of E-Syt-mediated ER-PM contact sites during dynamic changes in membrane phosphoinositide composition.