Ca²⁺ binding to synaptotagmin: How many Ca²⁺ ions bind to the tip of a C₂-domain?

C₂-domains are widespread protein modules with diverse Ca²⁺-regulatory functions. Although multiple Ca²⁺ ions are known to bind at the tip of several C₂-domains, the exact number of Ca²⁺-binding sites and their functional relevance are unknown. The first C₂-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca²⁺-dependent interactions with syntaxin and phospholipids. We have studied the Ca²⁺-binding mode of this C₂-domain as a prototypical C₂-domain using NMR spectroscopy and site-directed mutagenesis. The C₂-domain is an elliptical module composed of a β-sandwich with a long axis of 50 Å. Our results reveal that the C₂-domain binds three Ca²⁺ ions in a tight cluster spanning only 6 Å at the tip of the module. The Ca²⁺-binding region is formed by two loops whose conformation is stabilized by Ca²⁺ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C₂-domains. All three Ca²⁺ ions are required for the interactions of the C₂-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C₂-domain function whereby the β-sheets of the domain provide a fixed scaffold for the Ca²⁺ binding loops, and whereby interactions with target molecules are triggered by a Ca²⁺-induced switch in electrostatic potential.