Dataset Information

C2 domain membrane penetration by group IVA cytosolic phospholipase A? induces membrane curvature changes.

ABSTRACT: Group IVA cytosolic phospholipase A(2) (cPLA(2)?) 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)? 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)? promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA(2)? 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)? 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)? 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)?.

SUBMITTER: Ward KE

PROVIDER: S-EPMC3494261 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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

Ward Katherine E KE Ropa James P JP Adu-Gyamfi Emmanuel E Stahelin Robert V RV

Journal of lipid research 20120918 12

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 ...[more]

PMID: 22991194

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Project description:The Group IVA (GIVA) phospholipase A(2) associates with natural membranes in response to an increase in intracellular Ca(2+) along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca(2+) binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A(2) irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

Dataset Information

C2 domain membrane penetration by group IVA cytosolic phospholipase A? induces membrane curvature changes.

Publications

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

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