The molecular basis of ceramide-1-phosphate recognition by C2 domains

Abstract

Group IVA cytosolic phospholipase A₂ (cPLA₂α), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affinity for a cationic patch in the C2 domain β-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA₂α-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specificity is mediated by Arg⁵⁹, Arg⁶¹, and His⁶² (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confirm the origin of C1P specificity, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the first study demonstrating the molecular origin of C1P recognition.

Fig. 1.

Lipid binding properties of cPLA₂α-C2 and mutations. Quantitative binding analysis was performed for the cPLA₂α-C2 and respective mutations to POPC or POPC-C1P (97:3) vesicles at 10 μM Ca²⁺. A: SPR sensorgrams are shown for 10 nM WT cPLA₂α-C2 binding to POPC (light gray) or POPC:C1P (97:3) (black) vesicles. B: The equilibrium response (R_eq) from WT cPLA₂α-C2 binding at each respective protein concentration was plotted versus [cPLA₂α-C2] to fit with a nonlinear least-squares analysis of the binding isotherm (R_eq = R_max/(1 + K_d/C) to determine the K_d (see E). C: SPR sensorgrams are shown for 100 nM WT cPLA₂α-C2 (black) or 100 nM R59A binding to POPC-C1P (97:3) (gray) vesicles. D: Fold increase in K_d was normalized for each respective protein to the K_d value for WT cPLA₂α-C2 for POPC-C1P (97:3) -containing vesicles. The filled bars depict binding to POPC vesicles, and the gray bars display binding to POPC-C1P (97:3) vesicles. E: K_d values for WT and respective mutations binding to POPC or POPC-C1P (97:3) vesicles. The binding experiments were completed from independent experiments in triplicate and are listed with their respective standard deviation.

Fig. 2.

Determination of the C1P binding stoichiometry of cPLA₂α-C2. Titration of POPC-POPE-C1P (50:40:10) vesicles into 1 μM cPLA₂α-C2 as measured by tryptophan relative fluorescence (RFU) quenched. Through fitting the rise and saturation portions of the data with linear regression, a stoichiometric ratio of 1:1.16 ± 0.13 (cPLA₂α-C2-C1P) was determined in duplicate.

Fig. 3.

Mutation of C1P binding residues abrogates cPLA₂α’s ability to translocate in A549 cells. Cells were seeded, transfected at 24 h, and imaged at 48 h with confocal microscopy. A: Cells expressing EGFP-WT-cPLA₂α or respective mutations were treated with 10 μM A₂₃₁₈₇ in DMSO to induce cPLA₂α WT and mutant translocation. B: The percent translocation and standard deviation were calculated from three independent experiments, where 26–66 cells were counted in each experiment. The data are shown as an average ± the standard deviation. Statistical analysis was completed by employing a Student's t-test to determine the P value for each mutation with respect to WT. * = P < 0.0001, ** = P < 0.05. C: A display of the single point mutations mapped to the gray cPLA₂α-C2 crystal structure (PDB 1CJY), where the cationic β-groove is indicated by dark blue residues, control mutations in light blue, and the Ca²⁺ ions in yellow.

Fig. 4.

MD simulations of cPLA₂α-C2 docking PC- and C1P-containing bilayers. A: The MD simulations were performed on six different systems: POPC with both Ca²⁺ ions present (PC 2Ca²⁺), POPC and one Ca²⁺ ion present (PC 1Ca²⁺), POPC without Ca²⁺ present (PC), POPC-C1P with both Ca²⁺ ions present (C1P 2Ca²⁺), POPC-C1P with one Ca²⁺ ion present (C1P 1Ca²⁺), and POPC-C1P without Ca²⁺ present (C1P). B: The MD output of cPLA₂α-C2 docked to a POPC bilayer; or C: a POPC-C1P bilayer under variable conditions as defined in A.

Fig. 5.

MD simulations demonstrate the selectivity of basic residues in H-bonding with the C1P headgroup. A: Predicted number of H-bonds contributed by each residue in the basic patch during the MD simulation. B: SPR binding results for POPC (black bars), POPC-C1P (97:3) (white bars), or POPC-PI(4,5)P₂ (97:3) (gray bars) plotted as relative K_d value, with the K_d value for binding POPC-C1P vesicles at pH 7.4 set to 1. Each K_d value was determined in triplicate at each pH value to calculate and average and a standard deviation. C: The predicted H-bonds from the MD simulation depict a C1P binding site that is supported by in vitro C1P binding and cellular translocation data.

Fig. 6.

cPLA₂α coordinates the sphingosine backbone hydroxyl group to bind specifically to C1P. A: Chemical structures of PA, C1P, and deoxy-C1P. B: SPR responses for POPC-C1P (97:3) (black) and POPC-deoxy-C1P (97:3) (gray) at 50 nM cPLA₂α-C2 in buffer containing 20 mM HEPES, 160 mM KCl, pH 7.4. C: A549 cells were seeded, transfected with EGFP-cPLA₂α for 24 h, and treated with vehicle (98% dodecane:2% ethanol), 500 nM C1P or 500 nM deoxy-C1P for 2 h, then subsequently imaged with confocal microscopy. D: Statistical analysis of cells imaged in C. E: A549 cells were treated as stated in C, but were additionally treated with 10 μM A₂₃₁₈₇ in DMSO for 15 min. Vehicle corresponds to dodecane-ethanol (98:2) plus DMSO. F: Statistical analysis of cells imaged in E. Data were collected in triplicate and quantified using a Student's t-test. Error bars represent standard deviation. ** = P < 0.0002, * = P < 0.01.

Fig. 7.

The C2 domain rotates upon membrane docking to bind C1P embedded in the membrane. A: The structural output from the cPLA₂α-C2 MD simulation in a POPC membrane docked to a C1P molecule. B: Arg⁵⁹ and Arg⁶¹ shown in blue coordinating the C1P headgroup during the simulation. C: cPLA₂α-C2 is shown with hydrophobic membrane-penetrating residues (red), basic C1P binding β-groove residues (blue), and Ca²⁺ ions (yellow); the phosphorus atoms in the headgroups of the membrane lipids are represented by the gray dotted line. The shift in cPLA₂α-C2 required to bind to the phosphomonoester C1P is depicted, which results in a shift about the z-axis by ∼10° to reach the phosphate headgroup.

Fig. 8.

C2 domains bind different anionic lipids through their β-groove cationic residues. A: A model of cPLA₂α-C2 with a C1P molecule docked into the C1P binding site. The cPLA₂α-C2 crystal structure is depicted in gray, Ca²⁺ in yellow, cationic C1P binding site in blue, and C1P in green; the model displays the cPLA₂α-C2 binding C1P. B: Higher magnification shows more clearly the specific interactions between Arg⁵⁹, Arg⁶¹, and His⁶² and C1P in the C2 domain of cPLA₂α. C: The PKCα C2 domain is shown (3GPE) coordinated to Ins(1,4,5)P₃ with highlighted PI(4,5)P₂ binding residues. Basic residues are shown in dark blue, and hydrophobic residues are shown in teal. D: The rabphilin 3A C2 domain (2K3H) is shown with purported PI(4,5)P₂ binding residues in dark blue. E: The RxRH motif is conserved in cPLA₂ C2 domains from diverse organisms. Asterisks indicate identical residues, and the β-groove residues are bolded in blue. F: The RxRH motif is found in the β-groove of other C2 domains, including UVRAG, which binds C1P.