The mechanism of allosteric regulation of calcium-independent phospholipase A2 by ATP and calmodulin binding to the ankyrin domain

• 成果类型: Article
• 署名作者: Mouchlis, Varnavas D.; Hsu, Yuan-Hao; Hayashi, Daiki; Cao, Jian; Li, Sheng; Mccammon, J. Andrew; Dennis, Edward A.
• 署名单位: University of California System; University of California San Diego; University of California System; University of California San Diego; Kobe University; University of California System; University of California San Diego
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12988
• DOI: 10.1073/pnas.2411539121
• 发表日期: 2024-11-26
• 关键词: hydrogen/deuterium exchange; crystal-structure; a(2) enzymes; ipla(2)beta; inhibition; identification; cells

摘要

Group VIA calcium- independent phospholipase A2 (iPLA2) is a member of the PLA2 superfamily that exhibits calcium- independent activity in contrast to the other two major types, secreted phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2), which both require calcium for their enzymatic activity. Adenosine triphosphate (ATP) has been reported to allosterically activate iPLA2, and this has now been verified with a lipidomics-based mixed- micelle assay, but its mechanism of action has been unknown. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to identify ATP interaction peptide regions located within the ankyrin repeat domain at which ATP interacts. Molecular dynamics simulations revealed the mechanism bywhich ATP binds to its site and the main residues that interact. Site- directed mutagenesis was used to verify the importance of these residues in the role of ATP in regulating iPLA2 activity. Importantly, calcium was found to abolish the enhancing regulatory function of ATP and to promote the inhibitory activity by calmodulin. Given previous evidence that calcium does not bind directly to iPLA2, its effect appears to be indirect via association with ATP and/or calmodulin. Using HDX-MS, we found that calmodulin interacts with the N terminus peptide region of iPLA2 consisting of residues 20 to 28. These two regulatory iPLA2 sites open the road to the development of potential targets for therapeutic intervention.