期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：6
页码：E516-E525
DOI：10.1073/pnas.1424651112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe catalytic mechanisms of interfacial enzymes acting directly on the interfacial surface of the membrane are notoriously difficult to study experimentally with X-ray crystallography and other biophysical methods. This scientific study is, to our knowledge, the first to highlight similarities and differences in the extraction and binding of a phospholipid molecule into the substrate binding pocket of two human phospholipases A2 (PLA2s): the cytosolic Group IVA cPLA2 and the calcium-independent Group VIA iPLA2. Molecular dynamics simulations, guided by deuterium exchange experiments, are used to show that pathways to the active sites of these PLA2s are opened upon allosteric interaction with the membrane to facilitate entry of the substrate lipid. These enzymes are involved in various diseases, and understanding their mechanisms will aid in the discovery of therapeutics. 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 A2 (PLA2) 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 PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. 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.
