MECHANISM OF PHOSPHOLIPID-BINDING BY THE C(2)A-DOMAIN OF SYNAPTOTAGMIN-I

Synaptotagmin I is a synaptic vesicle membrane protein that probably f unctions 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 simi lar to that of the C-2-domains of protein kinase C, cytoplasmic phosph olipase A(2), and phospholipase C delta 1. Although the tertiary struc ture of these C-2-domains is known, the molecular basis for their Ca2-dependent interactions with phospholipids is unclear. We have now inv estigated the mechanisms involved in Ca2+-dependent phospholipid bindi ng 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 intera ction 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 aspart ates and one serine. Mutations in these aspartate and serine residues demonstrated that all three Ca2+-binding sites are required for phosph olipid 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 mu tagenesis to be also involved in phospholipid binding. Our results yie ld a molecular picture of the interactions between a C-2-domain and ph ospholipids. Binding is highly electrostatic and occurs between the su rfaces 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 an d 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.