Membranes serve as allosteric activators of phospholipase A ₂ , enabling it to extract, bind, and hydrolyze phospholipid substrates

Abstract

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. Phospholipase A 2 (PLA 2 ) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein-lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA 2 s: the cytosolic Group IVA cPLA 2 and calcium-independent Group VIA iPLA 2 . Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme-substrate-membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme's interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.GIVA cPLA 2 | GVIA iPLA 2 | PAPC | MD simulations | DXMS

Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease

“…These combined results led us to a complete model of the catalytic cycle of two different human PLA 2 s, cPLA 2 and iPLA 2 (147). After some forty years of studying PLA 2 , a most fulfilling moment for me was when our lab developed detailed simulations (and representative movies) depicting the association of PLA 2 with a membrane during which the membrane acts as an allosteric ligand of the enzyme's interfacial surface by shifting its conformation from a closed (inactive) state in water to an open (active) state.…”

“…This technique was used to create structural complexes of each enzyme with a single phospholipid substrate molecule in its catalytic site to visualize substrate extraction. Simulations of the enzyme-substrate-membrane system revealed important information about the mechanisms by which enzymes associate with the membrane (146) and then extract and bind their phospholipid substrate (147) (Fig. 7).…”

“…After some forty years of studying PLA 2 , a most fulfilling moment for me was when our lab developed detailed simulations (and representative movies) depicting the association of PLA 2 with a membrane during which the membrane acts as an allosteric ligand of the enzyme's interfacial surface by shifting its conformation from a closed (inactive) state in water to an open (active) state. This is followed by the extraction of a substrate phospholipid from the membrane into the active site of the enzyme, and then achievement of the energetically optimal conformation for catalysis and finally diffusion of the products into the membrane surface (147). My quest to match the quality of information available for "normal" water-soluble enzyme systems, the initial steps of which were recognized 15 years earlier by my receipt of an award from the ASBMB in lipid enzymology (Fig.…”

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

“…Overall, structural studies are currently limited to identification of the putative calmodulin binding sites 56 , molecular modeling, and mapping of the membraneinteraction loop using hydrogen/deuterium exchange mass spectrometry [57][58][59] .…”

Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange–MS (HDX–MS)

“…MS has become a useful tool to probe the architecture, structure and dynamics of proteins at membrane interfaces, including both integral and peripheral membrane proteins. This has been accomplished by a number of different MS-based approaches including chemical cross-linking [12], intact analysis of membrane proteins in the gas phase [13][14][15][16][17] and hydrogen deuterium exchange-MS (HDX-MS) [9,10,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. These studies have greatly expanded our understanding of the dynamics and regulation of large membrane protein assemblies.…”