Use of EPR power saturation to analyze the membrane-docking geometries of peripheral proteins: applications to C2 domains

Abstract

Despite the central importance of peripheral membrane proteins to cellular signaling and metabolic pathways, the structures of protein-membrane interfaces remain largely inaccessible to high-resolution structural methods. In recent years a number of laboratories have contributed to the development of an electron paramagnetic resonance (EPR) power saturation approach that utilizes site-directed spin labeling to determine the key geometric parameters of membrane-docked proteins, including their penetration depths and angular orientations relative to the membrane surface. Representative applications to Ca(2+)-activated, membrane-docking C2 domains are described.

Figure 1

Use of site-directed spin labeling and power saturation to measure depth parameters (, –15, 19, 23, 24). (a) Structure of the MTSSL nitroxide probe used for site-directed spin labeling, illustrating its coupling to a cysteine via a disulfide linkage. (b) The two gradients of paramagnetic probes used to define the depth parameter. Molecular oxygen is found at highest concentration in the bilayer hydrocarbon core because of its a polarity: Its concentration gradient points toward the membrane center. The zwitterionic nickel(II) ethylenediaminediacetic acid (NiEDDA) complex is found at highest concentration in the aqueous phase because of its high polarity: Its concentration gradient points away from the membrane. (c) Continuous-wave power saturation curves for a membrane-docked cysteine variant of cPLA₂ C2 domain (Cys38-MTSSL) (24). The EPR signal amplitude is plotted as a function of the square root of microwave power. Saturation curves are shown for measurements of samples purged with nitrogen gas (closed circles), samples equilibrated with atmospheric oxygen (open circles), and samples containing 10 mM NiEDDA and purged with nitrogen gas (closed squares).

Figure 2

Plot of measured depth parameters versus modeled membrane depth for a membrane-docking C2 domain (24). The depth parameters of spin labels incorporated into the cPLA₂ C2 domain are plotted as a function of the modeled distance from the headgroup phosphate plane. Filled circles indicate spin label positions in the membrane interior where the depth parameter varies linearly: These depth parameters were used in the linear fitting procedure that generated the membrane-docking model. Open circles indicate spin label positions outside the linear region that were excluded from the fitting procedure. The side chain conformations of six spin labels were adjusted to optimize the linear fit. Open squares indicate the measured depth parameters for phosphatidylcholine lipids possessing spin labels at different carbons in the A2 fatty acid, plotted as a function of the known distance of the fatty acid carbon from the plane of membrane phosphates (squares). The solid curve represents the best-fit hyperbolic function (Equation 4), illustrating the ability of this function to accurately reproduce the distance-dependence of the depth parameter.

Figure 3

Orientation and depth of the C2 domain of cPLA₂ with respect to a membrane surface (24). The crystal structure of the C2 domain of cPLA₂ (31) is represented as the cyan ribbon, with two Ca²⁺ ions shown as yellow spheres. The horizontal lines represent the planar boundaries of the headgroup and hydrocarbon regions of the bilayer. Protein spin labels oriented in their final optimized conformations are colored according to their measured depth parameters, with positive depth parameters indicated by increasing red, and negative depth parameters indicated by increasing blue. Figure generated by Insight (Accelrys).

Figure 4

Comparison of the membrane-docking geometries of the cPLA₂, PKCα, and SytIA C2 domains (14, 15, 19, 23, 24). The cPLA₂ C2 domain penetrates significantly farther into the hydrocarbon core, explaining its hydrophobic docking mechanism, and the PKCα and SytIA C2 domains interact primarily with the charged headgroup region, explaining their electrostatic docking mechanism. The cPLA₂ C2 domain is oriented with its β-strands closer to the membrane normal than the other two domains. For all three domains the Ca²⁺ ions lie near the headgroup phosphate plane. Figure was generated by MacPymol (DeLano Scientific).

Figure 5

Molecular model of cPLA₂ C2 domain docked to a lipid bilayer showing two views of the domain in the preferred docking geometry. The three Ca²⁺-binding loops are shown in CPK format with loop 1 in red, loop 2 in orange, and loop 3 in yellow. Also highlighted in CPK is a representative phospholipid (S. Jaud, S. White, J. Falke & D. Tobias, unpublished data). The model was generated by equilibrating the C2 domain in the membrane at the depth and angular orientation specified by the EPR membrane depth analysis (24), followed by unconstrained molecular dynamics that revealed that the system was stable for the full length of the molecular dynamics simulation (10 ns; see text).