Membrane Allostery and Unique Hydrophobic Sites Promote Enzyme Substrate Specificity

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.

Small-molecule inhibitors as potential therapeutics and as tools to understand the role of phospholipases A2

“…In a continuation of that work, Mouchlis et al further explored the substrate specificity by lipidomics and MD simulations [136]. They discovered that a unique hydrophobic area in the binding site accommodates the cleaved fatty acid and it thus drives the substrate specificity.…”

“…Most recently, a novel mass spectrometric-based high-throughput assay (96 well-plate assay) toward both natural and synthetic membrane phospholipids in mixed micelles with a nonionic surfactant has been reported [136]. In this lipidomics-based HPLC/MS assay, a HILIC column and multiple reaction monitoring (MRM) were used for targeted quantification of the assay components including the surfactant (octaethylene glycol monododecyl ether, C12E8), a free fatty acid (AA), a phospholipid (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, PAPC), a lysophospholipid (1-palmitoyl-sn-glycero-3-phosphocholine, 16:0 LPC), and an internal standard (1-heptadecanoyl-snglycero-3-phosphocholine, 17:0 LPC).…”

2-Oxoester Phospholipase A2 Inhibitors with Enhanced Metabolic Stability

“…Group specific PLA 2 assays were employed to determine the inhibitory activity using a lipidomics-based mixed micelle assay as previously described [4,25]. The substrate for each enzyme consisted of 100 µM PAPC (except for GIVA cPLA 2 as noted), 400 µM of C12E8 surfactant, and 2.5 µM of 17:0 LPC internal standard.…”

“…Phospholipase A 2 (PLA 2 ) enzymes are involved in a variety of inflammatory diseases, which has stimulated the interest of the scientific community in exploring the pathophysiological role of each type of PLA 2 and developing strategies for the modulation of their activity [1][2][3]. Among the various PLA 2 s present in mammals, the cytosolic calcium-dependent PLA 2 s (cPLA 2 ) is characterized by a marked preference for the hydrolysis of arachidonic acid from the phospholipid substrates initiating the eicosanoid cascade [4]. The most well studied enzyme of this group is Group IVA (GIVA) cPLA 2 and it is responsible for the biosynthesis of many diverse lipid signaling molecules contributing to inflammation [5].…”

Phospholipid arachidonic acid remodeling in macrophages: role of plasmalogen species

“…Phospholipid AA remodeling is a key aspect in eicosanoid regulation because the nature and the amount of eicosanoids produced under activation conditions may depend on the composition and subcellular localization of the phospholipid pool where the AA-hydrolyzing phospholipase A2 acts. In vitro studies suggest that cPLA2α does not show preference for the sn-3 position headgroup, whereas recent studies proposed a minor preference for PC and PE species (256). As discussed in preceding paragraphs, during stimulation conditions AA is hydrolyzed from PE and the pools are replenished with AA from PC via CoA-IT-mediated transacylation reactions.…”