Abstract
We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A2 specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A2. The differences between each enzyme’s specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme’s specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A2 for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A2, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A2 favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol.
Highlights
Phospholipase A2 (PLA2) constitutes a structurally and functionally diverse superfamily of enzymes, and distinct types can exhibit unique degradative, biosynthetic, and/or signaling functions and are implicated in a wide variety of diseases
This work constitutes the first detailed study elucidating the specificity of PLA2s toward membrane phospholipids and correlating it with the structural interactions of specific phospholipids bound to each enzyme’s active site, extending earlier studies on substrate and inhibitor binding.[4−11]
Journal of the American Chemical Society dynamics-based binding computations were employed to determine the structural features of each enzyme that contribute to its specificity
Summary
Phospholipase A2 (PLA2) constitutes a structurally and functionally diverse superfamily of enzymes, and distinct types can exhibit unique degradative, biosynthetic, and/or signaling functions and are implicated in a wide variety of diseases. Each PLA2 has a unique structure that contains two very important regions: the interfacial surface through which it associates and binds to the membrane and the active site where catalysis occurs.[4] Assaying the activity of PLA2 enzymes has posed significant challenges because their natural phospholipid substrates aggregate in aqueous solution to form micelles, vesicles, or liposomes.[12,13] To overcome these challenges, we have employed lipidomics to develop a novel mass spectrometricbased high-throughput assay toward both natural and synthetic membrane phospholipids in mixed micelles with a nonionic surfactant This assay was used to determine the substrate specificity of three human PLA2s toward the major phospholipid molecular species
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