Membrane orientation and position of the C2 domain from cPLA2 by site-directed spin labeling.
The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.
Frazier
April A
AA
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
Wisner
Mark A
MA
Malmberg
Nathan J
NJ
Victor
Kenneth G
KG
Fanucci
Gail E
GE
Nalefski
Eric A
EA
Falke
Joseph J
JJ
Cafiso
David S
DS
D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D003600:Cytosol / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008051:Lipid Bilayers / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010713:Phosphatidylcholines / Q000737:chemistry / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000235:genetics; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013113:Spin Labels; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013113:Spin Labels; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D010741:Phospholipases A; C028694:1-palmitoyl-2-oleoylphosphatidylcholine