Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning.1 Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that comprise the membrane bilayer, provide the ideal system for studying protein–lipid interactions.2 Our current study focuses on understanding the catalytic cycle of two different recombinant human intracellular PLA2s: the GIVA cPLA2 and GVIA iPLA2, which are responsible for arachidonic acid release for eicosanoid signaling and for membrane phospholipid remodeling, respectively. Molecular dynamics (MD) simulations, guided by hydrogen/deuterium exchange mass spectrometric (DXMS)3 experimental data, were used to show that the channel to the active sites of these PLA2s are opened upon allosteric interaction of the enzyme surface with the membrane to facilitate entry of the substrate phospholipid. This constitutes the first detailed study describing the binding and the interaction mechanism of intracellular PLA2s with the membrane bilayer as well as how they bind a single phospholipid molecule in the catalytic site. These enzymes are implicated in many diseases, and understanding their detailed mechanism of action will aid in the discovery of new therapeutics. References 1. Mouchlis, V. D.; Bucher, D.; McCammon, J. A.; Dennis, E. A., Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates. PNAS 2015, 112, E516-E525. 2. Dennis, E.; Cao, J.; Hsu, Y.-H.; Magrioti, V.; Kokotos, G., Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem. Rev. 2011, 111, 6130-6185. 3. Cao, J.; Burke, J.; Dennis, E., Using hydrogen/deuterium exchange mass spectrometry to define the specific interactions of the phospholipase A2 superfamily with lipid substrates, inhibitors, and membranes. J. Biol. Chem. 2013, 288, 1806-1813.

Evolution of phospholipases A2 in catalysis and allosteric regulation by membranes

251st National Meeting (2016)

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