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：2022
卷号：119
期号：2
DOI：10.1073/pnas.2102953118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Lp-PLA
2 is a physiologically important human enzyme and an inflammatory biomarker for assessing risk factors associated with cardiovascular diseases. It is associated with low- and high-density lipoproteins in human plasma and acts on the outside of the phospholipid monolayer that coats these particles, in stark contrast to traditional PLA
2 enzymes that act on bilayer membranes. This study addresses the allosteric activation of Lp-PLA
2 by phospholipid monolayers and membranes, its precise selectivity and specificity for particular oxidized and short acyl-chain phospholipid substrates not previously possible. Of particular importance, this work identifies and confirms by site-directed mutagenesis a phospholipid head-group binding pocket distinct from known drug inhibitor binding pockets that informs us about Lp-PLA
2’s mechanism of action and creates opportunities for additional therapeutic approaches.
Lipoprotein-associated phospholipase A
2 (Lp-PLA
2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein’s phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA
2 binding to a phospholipid surface. This allosteric regulation of an enzyme’s activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA
2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the
sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA
2‘s specificity toward oxidized phospholipids.
