Group VIA calcium-independent phospholipase A₂ (GVIA iPLA₂) is a distinctive member of the PLA₂ superfamily, characterized by its calcium-independent activity, in contrast to secreted (sPLA₂) and cytosolic (cPLA₂) phospholipases that require calcium for activation. Recent findings suggest that ATP can allosterically activate iPLA₂, confirmed through lipidomics-based mixed-micelle assays, but the precise mechanism remained unclear. In this study, we employed a multi-faceted approach emphasizing molecular dynamics simulations, alongside hydrogen/deuterium exchange mass spectrometry (HD-XMS), to elucidate the dynamics of ATP binding within the ankyrin repeat domain (ARD) of iPLA₂. Our molecular dynamics simulations revealed critical interacting residues and the structural dynamics of ATP binding, highlighting conformational changes that facilitate allosteric activation. These simulations were complemented by HD-XMS, which identified specific peptide regions where ATP interacts, thus pinpointing the structural basis for its regulatory role. Site-directed mutagenesis further validated the significance of these residues in mediating ATP's effects. Intriguingly, we found that calcium not only negates the ATP-mediated enhancement of iPLA₂ activity but also promotes inhibition via calmodulin, suggesting an indirect regulatory mechanism. Our HD-XMS data revealed that calmodulin binds to the N-terminal peptide region (residues 20-28) of iPLA₂, unveiling new regulatory sites. These findings offer a comprehensive understanding of iPLA₂ regulation through ATP and calcium interactions, paving the way for potential therapeutic interventions. This study underscores the powerful role of molecular dynamics in revealing the intricate dynamics and interactions of enzyme regulation.

Edward Dennis

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