Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance

Abstract

The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.

Figure 1

Regression analysis of the C2_cPLA2-membrane orientation. The solid line shows values of simulated Φ obtained by using the nonlinear Poisson–Boltzmann equation as a function of distance from the spin label nitrogen to the membrane (r). The circles are the values of Φ calculated from the experimental EPR data as a function of the modeled distance from the spin label to the membrane for each residue (the residue number is shown next to each circle). Residues 34, 39, 55, and 97 are all close to the membrane, and those that penetrate the interior of the bilayer were distinguished by the oxygen effect (see text).

Figure 2

Stereoview of EPR-based docked C2_cPLA2 on DOPM membranes by using the data in Fig. 1. The position of the protein with respect to the membrane (plane of electrostatic potential −77 mV, shown as an array of yellow balls 6 Å apart) is as shown in Fig. 2. The protein backbone is shown as a white ribbon, and native residues that were replaced with spin labels are shown as van der Waals surfaces in red except for residues 88 and 110, which are shown in pink. The two calcium ions are shown as green spheres. CBR1 contains residues 34 and 39 and also F35 and M38 (shown as yellow sticks) and D40 (cyan stick). CBR3 contains V97 and also Y96 and M98 (yellow sticks) and N95 and D99 (cyan sticks). Basic residues R57, K58, and R59 are shown as blue-gray sticks.

Figure 3

Comparison of the optimal C2_cPLA2-membrane orientation (given by the fit in Fig. 1, ribbon diagram), which excludes data from residues 88 and 110, to that given by the alternative regression fit described in the text (ball-and-stick diagram). The membrane planes of the two docked structures are superimposed and shown edge on as a row of crosses.