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 C-2-domains which bind Ca2+. The first C-2-domain of synaptotagmin I (the C(2)A-domain) binds phospholipids in a Ca2+-dependent manner similar to that of the C-2-domains of protein kinase C, cytoplasmic phospholipase A(2), and phospholipase C delta 1. Although the tertiary structure of these C-2-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 C(2)A-domain of synaptotagmin I. Our data show that the C(2)A-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 C(2)A-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 C(2)A-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 C-2-domain and phospholipids. Binding is highly electrostatic and occurs between the surfaces of the phospholipid bilayer and of the tip of the C(2)A-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 C-2-domains, with the relative contributions of electrostatic and hydrophobic interactions dictated by the exposed side chains surrounding the Ca2+-binding region.