Synaptic vesicle exocytosis is mediated by the vesicular Ca2+-sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and with acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using both PC12 cells from Rattus norvegicus and artificial supported bilayers we now show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ via PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine (PS) in the vesicle membrane upon Ca2+-triggering. We determined the crystal structure of the C2B-domain of synaptotagmin-1 bound to phosphoserine, allowing for developing a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+-influx bringing the vesicle membrane close enough for membrane fusion.