Membrane Orientation and Position of the C2 Domain from cPLA2 by Site-Directed Spin Labeling

The C2 domain is a ubiquitous Ca2+-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca2+ signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A2 in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Φ, 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 Φ 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 Φ, 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 Ca2+-binding loops are inserted approximately 10 Å into the bilayer, with residues inserted as deep as 15 Å. The backbone of the second Ca2+-binding loop is positioned near the lipid phosphate, and the two β-sheets of the C2 domain are oriented so that the individual strands make angles of 30−45° with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca2+-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.