Mechanism of phospholipid binding by the C₂A-domain of synaptotagmin I

Synaptotagmin I is a synaptic vesicle membrane protein that probably functions as a Ca²⁺ sensor in neurotransmitter release and contains two C₂-domains which bind Ca²⁺. The first C₂-domain of synaptotagmin I (the C₂A-domain) binds phospholipids in a Ca²⁺-dependent manner similar to that of the C₂-domains of protein kinase C, cytoplasmic phospholipase A₂, and phospholipase Cδ1. Although the tertiary structure of these C₂-domains is known, the molecular basis for their Ca²⁺-dependent interactions with phospholipids is unclear. We have now investigated the mechanisms involved in Ca²⁺-dependent phospholipid binding by the C₂A-domain of synaptotagmin I. Our data show that the C₂A-domain binds negatively charged liposomes in an electrostatic interaction that is determined by the charge density of the liposome surface but not by the phospholipid headgroup. At the tip of the C₂A-domain, three tightly clustered Ca²⁺-binding sites are formed by five aspartates and one serine. Mutations in these aspartate and serine residues demonstrated that all three Ca²⁺-binding sites are required for phospholipid binding. The Ca²⁺ binding sites at the top of the C₂A-domain are surrounded by positively charged amino acids that were shown by mutagenesis to be also involved in phospholipid binding. Our results yield a molecular picture of the interactions between a C₂-domain and phospholipids. Binding is highly electrostatic and occurs between the surfaces of the phospholipid bilayer and of the tip of the C₂A-domain. The data suggest that the negatively charged phospholipid headgroups interact with the basic side chains surrounding the Ca²⁺-binding sites and with bound Ca²⁺ ions, thereby filling empty coordination sites and increasing the apparent affinity for Ca²⁺. In addition, insertion of hydrophobic side chains may contribute to phospholipid binding. This model is likely to be general for other C₂- domains, with the relative contributions of electrostatic and hydrophobic interactions dictated by the exposed side chains surrounding the Ca²⁺- binding region.