Solution Structures of the Ca2+-free and Ca2+-bound C2A Domain of Synaptotagmin I: Does Ca2+ Induce a Conformational Change?

Abstract

C₂ domains are widespread Ca²⁺-binding modules that are particularly abundant in proteins involved in membrane traffic and signal transduction. The C₂A 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 C₂A domain is critical for understanding its mechanism of action and for models of the functions of other C₂ domains. We have determined the solution structure of the Ca²⁺-free and Ca²⁺-bound forms of the C₂A domain of synaptotagmin I by NMR spectroscopy. Our data represent the first structure determination of a C₂ 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 C₂A domain but does not produce a significant conformational change from a well-defined conformation to another. Thus, the mechanism of action of the C₂A 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 C₂A domain is to change its electrostatic potential rather than its structure. These results support a model whereby the C₂A domain functions as an electrostatic switch in neurotransmitter release. The similarity between the structures of the synaptotagmin I C₂A domain and the PLC-δ1 C₂ domain suggests that the latter binds four Ca²⁺ ions and acts by a similar mechanism. This mechanism may also be valid for other C₂ domains that share the unusual ability to bind multiple Ca²⁺ ions in a tight cluster at the tip of the domain.