Lipid binding ridge on loops 2 and 3 of the C2A domain of synaptotagmin I as revealed by NMR spectroscopy.
The C2A domain of synaptotagmin I, which binds Ca2+ and anionic phospholipids, serves as a Ca2+ sensor during excitation-secretion coupling. We have used multidimensional NMR to locate the region of C2A from rat synaptotagmin I that interacts, in the presence of Ca2+, with phosphatidylserine. Untagged, recombinant C2A was double-labeled with 13C and 15N, and triple-resonance NMR data were collected from C2A samples containing either Ca2+ alone or Ca2+ plus 6:0 phosphatidylserine. Phospholipid binding led to changes in chemical shifts of backbone atoms in residues Arg233 and Phe234 of loop 3 (a loop that also binds Ca2+) and His198, Val205, and Phe206 of loop 2. These residues lie along a straight line on a surface ridge of the C2A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His254; however, because His254 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 Ca2+-dependent membrane association is localized on the opposite side of the C2A domain from the transmembrane and C2B domains of synaptotagmin.
Chae
Y K
YK
National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Abildgaard
F
F
Chapman
E R
ER
Markley
J L
JL
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D008055:Lipids / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D054803:Phospholipase C delta; D010741:Phospholipases A / Q000737:chemistry; D010743:Phospholipids / Q000494:pharmacology; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D050863:Synaptotagmin I; D050857:Synaptotagmins; D010738:Type C Phospholipases / Q000737:chemistry
D002135:Calcium-Binding Proteins; D007527:Isoenzymes; D008055:Lipids; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; C494310:SYT1 protein, human; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D010741:Phospholipases A; D010738:Type C Phospholipases; D054803:Phospholipase C delta