Membrane orientation and depth of penetration of C2 domains using EPR spectroscopy

C2 domains are ubiquitous Ca+2-binding motifs that trigger the membrane docking of many key proteins involved in cell signaling. Site-directed spin labeling was carried out on C2 domains from synaptotagminI (synIC2A) and cytosolic phospholipase A₂ (cPLA₂) to investigate their membrane-bound positions. EPR spectroscopic results were used to model the membrane-bound structure for each C2 domain. Membrane depth parameters, Φ, were obtained by power saturating the EPR spectra. The values of Φ were combined with several constraints, including the solution NMR structure and Φ values for spin-labeled lipids and bacteriorhodopsin, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Φ, which defined the distance dependence of Φ from the center of the lipid bilayer to the bulk aqueous phase.; The polypeptide backbones of both the first and third Ca+2-binding loops of the cPLA₂ C2 domain are inserted approximately 10Å into the bilayer, with side chains inserted as deep as 15Å. The backbone of the second Ca+2-binding loop is positioned near the lipid phosphate. The modeled orientation for cPLA₂ C2 indicates that there are large desolvation effects upon membrane binding and that hydrophobic as well as electrostatic interactions contribute to the binding of the cPLA ₂ C2 domain.; The modeled orientation determined for synIC2A domain places the polypeptide backbones of both the first and third Ca+2-binding loops in contact with the membrane interface, with several side chains lying within the bilayer interior. All three Ca2+-binding sites lie near a plane defined by the lipid phosphates. This model indicates that there is some desolvation of the domain upon binding and that hydrophobic as well as electrostatic interactions contribute to the membrane binding of synIC2A. When compared to the C2 domain from cPLA₂, a similar orientation for the β-sandwich region is found for both synIC2A and cPLA₂ C2; however, the cPLA₂ C2 domain is translocated 5–7Å deeper into the membrane. This difference in depth is consistent with previous biophysical data and with the difference that long-range electrostatic interactions and desolvation are expected to make to the binding of these two C2 domains.