Abstract
Group VIA calcium-independent phospholipase A(2) (iPLA(2)) is a member of the PLA(2) superfamily that exhibits calcium-independent activity in contrast to the other two major types, secreted phospholipase A(2) (sPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), which both require calcium for their enzymatic activity. Adenosine triphosphate (ATP) has been reported to allosterically activate iPLA(2), 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 by which 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 iPLA(2) 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 iPLA(2), 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 iPLA(2) consisting of residues 20 to 28. These two regulatory iPLA(2) sites open the road to the development of potential targets for therapeutic intervention.