Defining the details of the molecular recognition between water-soluble enzymes and membranes is fundamental to understanding protein-lipid interactions, molecular complementarity and membrane functioning. Phospholipases A2 (PLA2), which catalyze the hydrolysis of phospholipids that comprise the membrane bilayer, provide an ideal system for studying protein-lipid binding and interactions. Our current study focuses on understanding the catalytic cycle of three human PLA2 enzymes including the cytosolic (cPLA2), calcium-independent (iPLA2) and lipoprotein-associated (Lp-PLA2). cPLA2 is the main arachidonic acid provider for the eicosanoid pathway, while iPLA2 is involved in membrane phospholipid remodeling. Lp-PLA2 was found to associate with LDL and HDL in human plasma in order to hydrolyze phospholipids containing short-chain and oxidized fatty acids as well as F2-isoprostanes esterified at the sn-2 position. Hydrogen/deuterium exchange mass spectrometry was used to experimentally identify distinct PLA2 peptide regions that interact with phospholipid vesicles. Molecular dynamics simulations, guided and validated by the experimental data, showed that the active sites of these enzymes are allosterically regulated by membranes. Membrane phospholipids bind to allosteric sites located on the interfacial surface of PLA2s shifting their conformation from the “closed” to the “open” state. This process facilitates extraction and binding of a phospholipid molecule in the active site where the hydrolysis occur. This constitutes the first detailed study describing the binding and interaction mechanism of PLA2s with the membrane bilayer as well as how they bind a single phospholipid molecule in the catalytic site. These enzymes are implicated in chronic inflammatory diseases and understanding their association with membranes, mechanism of action and interactions at the molecular level will allow us to identify potent and selective inhibitors that can be further developed as anti-inflammatory agents.

Phospholipase A2: A unique paradigm of allosteric regulation by membranes

254th National Meeting (2017)

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