Membrane-docking loops of the cPLA2 C2 domain: Detailed structural analysis of the protein-membrane interface via site-directed spin-labeling

C2 domains are protein modules found in numerous eukaryotic signaling proteins, where their function is to target the protein to cell membranes in response to a Ca2+ signal. Currently, the structure of the interface formed between the protein and the phospholipid bilayer is inaccessible to high-resolution structure determination, but EPR site-directed spin-labeling can provide a detailed medium-resolution view of this interface. To apply this approach to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)), single cysteines were introduced at all 27 positions in the three Ca2+-binding loops and labeled with a methanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca2+-activated phospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence of Ca2+ indicate that Ca2+ binding triggers subtle changes in the dynamics of two localized regions within the Ca2+-binding loops: one face of the loop 1 helix and the junction between loops 1 and 2. However, no significant changes in loop structure were detected upon Ca2+ binding, nor upon Ca2+-triggered docking to membranes. EPR depth parameters measured in the membrane-docked state allow determination of the penetration depth of each residue with respect to the membrane surface. Analysis of these depth parameters, using an improved, generalizable geometric approach, provides the most accurate picture of penetration depth and angular orientation currently available for a membrane-docked peripheral protein. Finally, the observation that Ca2+ binding does not trigger large rearrangements of the membrane-docking loops favors the electrostatic switch model for Ca2+ activation and disfavors, or places strong constraints on, the conformational switch model.