Electrostatic Control of the Membrane Targeting of C2 Domains Diana Murray, Barry Honig  Molecular Cell  Volume 9, Issue 1, Pages 145-154 (January 2002) DOI: 10.1016/S1097-2765(01)00426-9

Figure 1 Electrostatic Equipotential Profiles of SytI-C2A and cPLA2-C2 Domains in the Absence and Presence of Calcium (A) Calcium-free SytI-C2A. (B) SytI-C2A bound to three calcium ions. (C) Calcium-free cPLA2-C2. (D) cPLA2-C2 bound to two calcium ions. The arrows denote the calcium binding loops, the regions responsible for the membrane association of these C2 domains. The red and blue meshes represent, respectively, the −25 and +25 mV electrostatic equipotential contours in 0.1 M KCl as calculated and visualized in the program GRASP (Nicholls et al., 1991). The Protein Data Bank identifiers for the structures are 1byn for SytI C2A and 1rlw for cPLA2 C2, both of which contain bound calcium ions which were removed for the electrostatic calculations of the calcium-free states exhibited in (A) and (B). The dotted line corresponds to the location of the surface of the membrane slab (see text). Molecular Cell 2002 9, 145-154DOI: (10.1016/S1097-2765(01)00426-9)

Figure 2 Model of the Membrane Interaction of SytI-C2A (A) SytI-C2A at the surface of a 2:1 PC/PS bilayer in 0.1 M KCl. This is an example of the atomic-level systems studied in the FDPB calculations. The C2 domain (cyan; bound calcium ions are magenta) is in the orientation of minimum free energy in which the electrostatic free energy of interaction is strongest (see Figure 3A). (B) Electrostatic properties of the system in (A). The red and blue meshes depict the −25 and +25 mV equipotential contours as calculated using the FDPB methodology (Ben-Tal et al., 1996a) and visualized in GRASP (Nicholls et al., 1991). Molecular Cell 2002 9, 145-154DOI: (10.1016/S1097-2765(01)00426-9)

Figure 3 Prediction of the Electrostatic Components of SytI-C2A Membrane Association (A) Electrostatic free energy of interaction (ΔWel) between SytI-C2A and a 2:1 PC/PS in 0.1 M KCl. ΔWel is plotted as a function of distance between the van der Waals surfaces of the C2 domain and the membrane; the orientation of the C2 domain with respect to the membrane is depicted in Figure 2. Circles and squares represent, respectively, the calculations in the absence and presence of calcium. The side chain of Phe-234, which has been shown to penetrate the membrane interface (Chapman and Davis, 1998), was removed for this calculation in order to calculate the full electrostatic contribution as done for other systems in previous work (Arbuzova et al., 2000). Nonpolar partitioning of the Phe-234 side chain is predicted to contribute about −1 kcal/mol to the membrane interaction (data not shown; Wimley and White, 1996). There are alternative orientations for the calcium-free state that have a more favorable ΔWel than the calcium-free orientation whose energy curve is illustrated here, but these still interact significantly more weakly with the bilayer than the calcium-bound state. (B) Dependence of the electrostatic free energy of interaction of SytI-C2A with PC/PS bilayers as a function of mole percent acidic lipid. The calculations were performed for 0.1 M KCl and assumed that there is no redistribution of the acidic lipids due to the presence of the protein. Molecular Cell 2002 9, 145-154DOI: (10.1016/S1097-2765(01)00426-9)

Figure 4 Electrostatic Properties of Different C2 Domains (A and B) The C2 domain of PKCβ (1a25) in the absence (A) and presence (B) of calcium. (C and D) The C2 domain of PLCδ1 (1djg) in the absence (C) and presence (D) of calcium. (E) The C2 domain of the PTEN tumor suppressor (1d5r). The red and blue meshes represent, respectively, the −25 and +25 mV electrostatic equipotential contours in 0.1 M KCl as calculated and visualized in the program GRASP (Nicholls et al., 1991). The domains are positioned in their minimum free energy orientations with the membrane surface below each domain. Molecular Cell 2002 9, 145-154DOI: (10.1016/S1097-2765(01)00426-9)

Figure 5 Properties of the Electrostatic Free Energy of Interaction between cPLA2-C2 and Different Membrane Models in 0.1 M KCl (A and B) ΔWel is plotted as a function of distance between the van der Waals surfaces of the C2 domain and the surface of an atomic model of a 1:0 PC/PS (A) or 2:1 PC/PS (B) bilayer. Squares and circles represent, respectively, the calculations in the absence and presence of calcium. The calculations are similar to those for basic peptides enriched in aromatic residues (Arbuzova et al., 2000), and, therefore, the side chains of the aromatic residues that penetrate into the membrane hydrocarbon were removed (Ben-Tal et al., 1997; Murray et al., 1998). (C) The difference in ΔWel for cPLA2-C2 in the presence and absence of calcium as the domain penetrates slab models of a an electrically neutral membrane. The quantities plotted are ΔWel (calcium-bound)-ΔWel (calcium-free) for slabs with dielectric constants of 2 (circles), 10 (squares), and 30 (triangles). The vertical line denotes the position of the slab's upper surface. The horizontal axis denotes the distance between the van der Waals surface of the tip of the calcium binding loops of cPLA2-C2 (Figure 1, arrows); positive distances correspond to the domain being located outside the slab and negative values correspond to penetration of the domain into the slab. Molecular Cell 2002 9, 145-154DOI: (10.1016/S1097-2765(01)00426-9)