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 beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A 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 beta-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.
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.
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Ubach
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Zhang
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Shao
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Südhof
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Rizo
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent; D008345:Manganese; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins; D055672:Static Electricity; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D008345:Manganese; D002118:Calcium