Effect of PIP2 binding on the membrane docking geometry of PKC alpha C2 domain: an EPR site-directed spin-labeling and relaxation study.

Protein kinase C isoform alpha (PKCalpha) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca (2+) ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCalpha C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP 2, respectively. In both the absence and presence of PIP 2, the two bound Ca (2+) ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca (2+) and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP 2, the polybasic lipid binding site on the beta3-beta4 hairpin is occupied with PS, but in the presence of PIP 2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 +/- 10 degrees toward the bilayer normal. The ability of the beta3-beta4 hairpin site to bind PS as well as PIP 2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCalpha during a single membrane docking event. In principle, PIP 2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCalpha as it diffuses between membrane regions with different local PS and PIP 2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.

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

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.

Ca2+ activation of the cPLA2 C2 domain: ordered binding of two Ca2+ ions with positive cooperativity.

During Ca(2+) activation, the Ca(2+)-binding sites of C2 domains typically bind multiple Ca(2+) ions in close proximity. These binding events exhibit positive cooperativity, despite the strong charge repulsion between the adjacent divalent cations. Using both experimental and computational approaches, the present study probes the detailed mechanisms of Ca(2+) activation and positive cooperativity for the C2 domain of cytosolic phospholipase A(2), which binds two Ca(2+) ions in sites I and II, separated by only 4.1 A. First, each of the five coordinating side chains in the Ca(2+)-binding cleft is individually mutated and the effect on Ca(2+)-binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca(2+) affinity, while the two coordinating side chains that provide bridging coordination to both Ca(2+) ions, Asp 43 and Asp 40, are observed to make the largest contributions to positive cooperativity. Electrostatic calculations reveal that Asp 43 possesses the highest pseudo-pK(a) of the coordinating acidic residues, as well as the highest general cation affinity, due to its relatively buried location within 3.5 A of seven protein oxygens with full or partial negative charges. These calculations therefore explain the greater importance of Asp 43 in defining the Ca(2+) affinity. Overall, the experimental and computational results support an activation model in which the first Ca(2+) ion binds usually to site I, thereby preordering both bridging side chains Asp 40 and 43, and partially or fully deprotonating the three coordinating Asp residues. This initial binding event prepares the conformation and protonation state of the remaining site for Ca(2+) binding, enabling the second Ca(2+) ion to bind with higher affinity than the first as required for positive cooperativity.

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 Ca(2+) 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 Ca(2+)-binding loops and labeled with a methanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca(2+)-activated phospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence of Ca(2+) indicate that Ca(2+) binding triggers subtle changes in the dynamics of two localized regions within the Ca(2+)-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 Ca(2+) binding, nor upon Ca(2+)-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 Ca(2+) binding does not trigger large rearrangements of the membrane-docking loops favors the electrostatic switch model for Ca(2+) activation and disfavors, or places strong constraints on, the conformational switch model.

Membrane orientation and position of the C2 domain from cPLA2 by site-directed spin labeling.

The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.