Contacts between the endoplasmic reticulum and the plasma membrane involve extended synaptotagmins (E-Syts) in mammals or tricalbins in yeast, proteins with multiple C₂ domains. One of the tandem C₂ domains of E-Syt2 is predicted to bind Ca²⁺, but no Ca²⁺-dependent function has been attributed to this protein. We have determined the crystal structures of the tandem C₂ domains of E-Syt2 in the absence and presence of Ca²⁺ and analyzed their Ca²⁺-binding properties by nuclear magnetic resonance spectroscopy. Our data reveal an unexpected V-shaped structure with a rigid orientation between the two C₂ domains that is not substantially altered by Ca²⁺. The E-Syt2 C2A domain binds up to four Ca²⁺ ions, whereas the C₂B domain does not bind Ca²⁺. These results suggest that E-Syt2 performs an as yet unidentified Ca²⁺-dependent function through its C₂A domain and uncover fundamental differences between the properties of the tandem C₂ domains of E-Syts and synaptotagmins.
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