title: Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling.
abstract: Site-directed spin labeling is used to determine the orientation and depth of insertion of the second C2 domain from synaptotagmin I (C2B) into membrane vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS). EPR line shapes of spin-labeled mutants located with the Ca(2+)-binding loops of C2B broaden in the presence of Ca(2+) and PC/PS vesicles, indicating that these loops undergo a Ca(2+)-dependent insertion into the membrane interface. Power saturation of the EPR spectra provides a position for each spin-labeled site along the bilayer normal, and these EPR-derived distance constraints, along with a high-resolution structure of the C2B domain, are used to generate a model for the domain orientation and position at the membrane interface. Our data show that the isolated C2B domain from synaptotagmin I penetrates PC/PS membranes, and that the backbone of Ca(2+)-binding loops 1 and 3 is inserted below the level of a plane defined by the lipid phosphates. The side chains of several loop residues are within the bilayer interior, and both Ca(2+)-binding sites are positioned near a plane defined by the lipid phosphates. A Tb(3+)-based fluorescence assay is used to compare the membrane affinity of the C2B domain to that of the first synaptotagmin C2 domain (C2A). Both C2A and C2B bind PC/PS (75:25) membrane vesicles with a micromolar lipid affinity in the presence of metal ion. These results indicate that C2A and C2B have a similar membrane affinity and position when bound to PC/PS (75:25) membrane vesicles. EPR spectroscopy indicates that the C2B domain has different interactions with PC/PS membranes containing 1 mol % phosphatidylinositol 4,5-bisphosphate.
authors.affiliation: Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Rufener
authors.forename: April A
authors.initials: AA
authors.lastname: Frazier
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Wieser
authors.forename: Anne
authors.initials: A
authors.lastname: Hinderliter
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D002135:Calcium-Binding Proteins; D008081:Liposomes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D013113:Spin Labels; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins