C 2 -domains are widespread protein modules with diverse Ca 2⍣ -regulatory functions. Although multiple Ca 2⍣ ions are known to bind at the tip of several C 2 -domains, the exact number of Ca 2⍣ -binding sites and their functional relevance are unknown. The first C 2 -domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca 2⍣ -dependent interactions with syntaxin and phospholipids. We have studied the Ca 2⍣ -binding mode of this C 2 -domain as a prototypical C 2 -domain using NMR spectroscopy and site-directed mutagenesis. The C 2 -domain is an elliptical module composed of a β-sandwich with a long axis of 50 Å. Our results reveal that the C 2 -domain binds three Ca 2⍣ ions in a tight cluster spanning only 6 Å at the tip of the module. The Ca 2⍣ -binding region is formed by two loops whose conformation is stabilized by Ca 2ϩ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C 2 -domains. All three Ca 2⍣ ions are required for the interactions of the C 2 -domain with syntaxin and phospholipids. These results support an electrostatic switch model for C 2 -domain function whereby the β-sheets of the domain provide a fixed scaffold for the Ca 2⍣ -binding loops, and whereby interactions with target molecules are triggered by a Ca 2⍣ -induced switch in electrostatic potential.

C2 domains are found in many proteins involved in membrane traffic or signal transduction. Although C2 domains are thought to bind calcium ions, the structural basis for calcium binding is unclear. Analysis of calcium binding to C2 domains of synaptotagmin I and protein kinase C-beta by nuclear magnetic resonance spectroscopy revealed a bipartite calcium-binding motif that involves the coordination of two calcium ions by five aspartate residues located on two separate loops. Sequence comparisons indicated that this may be a widely used calcium-binding motif, designated here as the C2 motif.

of loop 2. These residues lie along a straight line on a surface ridge of the C 2 A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His 254 ; however, because His 254 is located on the other side of the molecule from the phospholipid docking site defined by the other residues , its shifts may result from nonspecific interactions. The results show that the "docking ridge" responsible for Ca 2-dependent membrane association is localized on the opposite side of the C 2 A domain from the transmembrane and C 2 B domains of synaptotagmin.