Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I:: Does Ca2+ induce a conformational change?

C-2 domains are widespread Ca2+-binding modules that are particularly abundant in proteins involved in membrane traffic and signal transduction. The C(2)A domain of synaptotagmin I is believed to play a key role in neurotransmitter release through its Ca2+-dependent interactions with syntaxin and phospholipids. Elucidating the structural consequences of Ca2+ binding to the C(2)A domain is critical for understanding its mechanism of action and for models of the functions of other C-2 domains. We have determined the solution structure of the Ca2+-free and Ca2+-bound forms of the C(2)A domain of synaptotagmin I by NMR spectroscopy. Our data represent the first structure determination of a C-2 domain in its Ca2+-free and Ca2+-bound forms. Three Ca2+ ions were included in the Ca2+-bound structure, yielding a Ca2+-binding motif that involves five aspartate side chains and one serine side chain. Ca2+ immobilizes the structure of the C(2)A domain but does not produce a significant conformational change from a well-defined conformation to another. Thus, the mechanism of action of the C(2)A domain of synaptotagmin I is different from that used by Ca2+-binding proteins of the EF-hand family. The main effect of Ca2+ binding on the C(2)A domain is to change its electrostatic potential rather than its structure. These results support a model whereby the C(2)A domain functions as an electrostatic switch in neurotransmitter release. The similarity between the structures of the synaptotagmin I C(2)A domain and the PLC-delta 1 C-2 domain suggests that the latter binds four Ca2+ ions and acts by a similar mechanism. This mechanism may also be valid for other C-2 domains that share the unusual ability to bind multiple Ca2+ ions in a tight cluster at the tip of the domain.