Membrane Association Allosterically Regulates Phospholipase A(2) Enzymes and Their Specificity

[Image: see text] Water-soluble proteins as well as membrane-bound proteins associate with membrane surfaces and bind specific lipid molecules in specific sites on the protein. Membrane surfaces include the traditional bilayer membranes of cells and subcellular organelles formed by phospholipids. Monolayer membranes include the outer monolayer phospholipid surface of intracellular lipid droplets of triglycerides and various lipoproteins including HDL, LDL, VLDL, and chylomicrons. These lipoproteins circulate in our blood and lymph systems and contain triglycerides, cholesterol, cholesterol esters, and proteins in their interior, and these are sometimes interspersed on their surfaces. Similar lipid–water interfaces also occur in mixed micelles of phospholipids and bile acids in our digestive system, which may also include internalized triglycerides and cholesterol esters. Diacyl phospholipids constitute the defining molecules of biological membranes. Phospholipase A(1) (PLA(1)) hydrolyzes phospholipid acyl chains at the sn-1 position of membrane phospholipids, phospholipase A(2) (PLA(2)) hydrolyzes acyl chains at the sn-2 position, phospholipase C (PLC) hydrolyzes the glycerol–phosphodiester bond, and phospholipase D (PLD) hydrolyzes the polar group–phosphodiester bond. Of the phospholipases, the PLA(2)s have been the most well studied at the mechanistic level. The PLA(2) superfamily consists of 16 groups and numerous subgroups, and each is generally described as one of 6 types. The most well studied of the PLA(2)s include extensive genetic and mutational studies, complete lipidomics specificity characterization, and crystallographic structures. This Account will focus principally on results from deuterium exchange mass spectrometric (DXMS) studies of PLA(2) interactions with membranes and extensive molecular dynamics (MD) simulations of their interactions with membranes and specific phospholipids bound in their catalytic and allosteric sites. These enzymes either are membrane-bound or are water-soluble and associate with membranes before extracting their phospholipid substrate molecule into their active site to carry out their enzymatic hydrolytic reaction. We present evidence that when a PLA(2) associates with a membrane, the membrane association can result in a conformational change in the enzyme whereby the membrane association with an allosteric site on the enzyme stabilizes the enzyme in an active conformation on the membrane. We sometimes refer to this transition from a “closed” conformation in aqueous solution to an “open” conformation when associated with a membrane. The enzyme can then extract a single phospholipid substrate into its active site, and catalysis occurs. We have also employed DXMS and MD simulations to characterize how PLA(2)s interact with specific inhibitors that could lead to potential therapeutics. The PLA(2)s constitute a paradigm for how membranes interact allosterically with proteins, causing conformational changes and activation of the proteins to enable them to extract and bind a specific phospholipid from a membrane for catalysis, which is probably generalizable to intracellular and extracellular transport and phospholipid exchange processes as well as other specific biological functions. We will focus on the four main types of PLA(2), namely, the secreted (sPLA(2)), cytosolic (cPLA(2)), calcium-independent (iPLA(2)), and lipoprotein-associated PLA(2) (Lp-PLA(2)) also known as platelet-activating factor acetyl hydrolase (PAF-AH). Studies on a well-studied specific example of each of the four major types of the PLA(2) superfamily demonstrate clearly that protein subsites can show precise specificity for one of the phospholipid hydrophobic acyl chains, often the one at the sn-2 position, including exquisite sensitivity to the number and position of double bonds.