C2 domain membrane penetration by group IVA cytosolic phospholipase A₂ induces membrane curvature changes

Abstract

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA(2)α binds zwitterionic lipids such as phosphatidylcholine in a Ca(2+)-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA(2)α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA(2)α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA(2)α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA(2)α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA(2)α.

Fig. 1.

Structural depiction of the C2 domain of cPLA₂α. A: The C2 domain (PDB ID 1CJY) is shown in gray in surface transparency mode to depict hydrophobic amino acids in calcium binding loops 1 and 3 (red). The two Ca²⁺ ions bound to the C2 domain are shown in yellow. B: A close-up view of the calcium-binding and membrane penetration regions of the C2 domain of cPLA₂α. Amino acids mutated in this study to assess membrane penetration and membrane curvature are shown in red (Phe³⁵, Met³⁸, and Leu³⁹ in CBL1 and Tyr⁹⁶ and Met⁹⁸ in CBL3). Ca²⁺ ions crystalized with the protein are shown in yellow. C: The C2 domain has been shown to deeply penetrate zwitterionic membranes. Here the C2 domain is shown with the depth of penetration and orientation previously resolved by EPR (16) deeply penetrating hydrophobic and aromatic residues are shown in red (Phe³⁵, Met³⁸, and Leu³⁹ in CBL1 and Tyr⁹⁶ and Met⁹⁸ in CBL3) and 2 Ca²⁺ ions in yellow. The domain was docked to the membrane according to previous biophysical studies, which provided molecular insight into the depth and orientation of the C2 domain binding to membranes (16). The protein shown is docked to a POPC membrane, which displays the importance of Phe³⁵, Met³⁸, and Leu³⁹ in CBL1 and Tyr⁹⁶ and Met⁹⁸ in CBL3 in penetrating the lipid bilayer.

Fig. 2.

The C2 domain induces membrane tubulation of POPC LUVs. Transmission electron microscopy was used to assess the ability of the C2 domain and mutants to induce changes to liposome morphology. All measurements were done with 10 μM protein in 20 mM HEPES (pH 7.4) containing 160 mM KCl and either 100 μM CaCl₂ or 100 μM EGTA. WT C2 induced extensive tubulation of POPC liposomes in a Ca²⁺-dependent manner. However, mutations F35A/L39A and Y96A greatly reduced changes in liposome morphology, whereas M38A and M98A induced formation of tubules from liposomes but to a lesser extent than WT. Incubation of the C2 domain in 100 μM EGTA in place of CaCl₂ with POPC liposomes did not induce appreciable changes in liposome morphology. Scale bars = 500 nm.

Fig. 3.

The GUV assay demonstrates the C2 domain's ability to induce membrane budding. A: Experiments with POPC:POPE:POPS (60:20:20) GUVs were performed in triplicate with a minimum of 60 GUVs assessed per measurement to provide a quantitative representation of membrane curvature changes shown in B. B: Quantitative representation of GUV vesiculation induced by C2 domain and respective mutants. WT induced significant vesiculation of GUVs in the presence of 10 μM CaCl₂ compared with control experiments performed in 100 μM EGTA. M38A and M98A displayed some induction of GUV vesiculation and membrane reorganization but to a significantly lesser extent than WT. F35A/L39A and Y96A did not appreciably induce GUV vesiculation when compared with the control. C: The WT C2 domain and full-length cPLA₂α were assessed at 200 nM protein concentration for their ability to induce membrane curvature changes in the presence of 500 nM CaCl₂ to GUVs containing POPC:POPE:POPS (60:20:20). The WT C2 domain induced substantial vesiculation, whereas full-length cPLA₂α induced vesiculation and long tubule formation from GUVs. D: Quantification of vesiculation in control versus WT C2 experiments shown in C. The P value for each protein was determined in comparison to the control in C and D (ns, not significant; *P < 0.001; **P < 0.0001) using an unpaired Student t-test. Scale bars = 5 μm.

Fig. 4.

The C2 domain induces lipid fragmentation of membrane sheets. POPC membrane sheets were used to test the ability of the C2 domain to induce membrane fragmentation to relatively flat membrane surfaces. Membranes were hydrated and then incubated with 2 μM WT or mutant C2 domain for 15 min. A: The WT C2 domain induced extensive membrane fragmentation from membrane sheets, which was not observed in control experiments with buffer alone. B: Hydrophobic and aromatic mutations reduced C2 domain membrane fragmentation. Only M98A displayed detectable membrane fragmentation compared with WT, F35A/L39A, M38A, and Y96A. All membrane sheets were imaged before and after protein incubation. Scale bars = 25 μm.

Fig. 5.

Mutations that reduce or abrogate membrane deformation reduce or abolish membrane penetration and membrane affinity. A: Insertion of the wild-type C2 domain in the presence of Ca²⁺ (filled circles) or EGTA (open circles) into a POPC monolayer monitored as a function of π₀. Insertion of F35A/L39A (filled squares), M38A (filled triangles), or D43N (filled diamonds) was also monitored in the presence of Ca²⁺. B: Insertion of the wild-type C2 domain (filled circles), Y96A (filled squares), or M98A (filled triangles). All measurements were performed in the presence of Ca²⁺. C: The normalized saturation response (R_eq) from WT cPLA₂α-C2 (filled circles), M38A (filled circles), or Y96A (filled squares) binding at each respective protein concentration was plotted versus 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. D: K_d values for WT and respective mutations binding to POPC vesicles. The binding experiments were completed from independent experiments in triplicate and are listed with their respective K_d ± SD.

Fig. 6.

Membrane penetration by the C2 domain of cPLA₂α is sufficient to induce membrane curvature changes. The hydrophobic residues essential in penetrating the membrane are also key for membrane curvature generation. The deep ∼15 Å penetration of these hydrophobic and aliphatic residues as well as a significant area of insertion (∼2110 Ų) are sufficient to reduce the energetic barrier to bend the membrane as deletion of one of these key residues abolishes this effect, as shown for the F35A/L39A and Y96A mutants. Although the overall mechanism is unknown, our data suggest that membrane penetration of the C2 domain is vital for membrane bending, tubulation, vesiculation, and fragmentation, depending on the initial curvature of the membrane.