A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.
Synaptotagmin I is a Ca(2+)- and phospholipid-binding protein of synaptic vesicles with an essential function in neurotransmission. Ca2+/phospholipid binding by synaptotagmin I may be mediated by its C2 domains, sequence motifs that have been implicated in the Ca2+ regulation of a variety of proteins. However, it is currently unknown if C2 domains are sufficient for Ca2+/phospholipid binding or if they even directly participate in Ca2+/phospholipid binding. In order to address this question, we have studied the Ca2+/phospholipid-binding properties of the first C2 domain of synaptotagmin I. Our results show that this C2 domain by itself binds Ca2+ and phospholipids with high affinity (half-maximal binding at 4-6 microM free Ca2+) and exhibits strong positive cooperativity. The C2 domain is specific for negatively charged phospholipids and for those divalent cations that are known to stimulate synaptic vesicle exocytosis (Ca2+ > Sr2+, Ba2+ >>> Mg2+). These studies establish that C2 domains can serve as independently folding Ca2+/phospholipid-binding domains. Furthermore, the cation specificity and the cooperativity of Ca2+ binding by the C2 domain from synaptotagmin I support a role for this protein in mediating the Ca2+ signal in neurotransmitter release.
Davletov
B A
BA
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
Südhof
T C
TC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002244:Carbon / Q000378:metabolism; D017931:DNA Primers; D004789:Enzyme Activation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002244:Carbon; D011493:Protein Kinase C; D002118:Calcium