Cryo-EM sheds light on membrane-associated NAPE-PLD

The membrane-associated enzyme, N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), generates bioactive signaling molecules and has a pivotal player in various physiological and cellular pathways crucial for organismal protection. We recently reported the crystal structure of NAPE-PLD revealing details of its dimeric structure, enzymatic catalysis, and interaction with bile acid molecules1- 5 . Despite its key role, a nanometer view of the association of NAPE-PLD to membranes remains elusive 6-7 . This makes it hard to understand the functional role of its unusual internal dimeric channel and the physiological link between bioactive lipid amide signaling and bile acid signaling. Within my doctoral thesis scope, the research focused on the intricate interaction between NAPE-PLD, bile acid molecules and membrane lipids, using nanodisc technology and sp cryo-EM 8-10 . We showed that NAPE-PLD, with its internal channel, can create membrane-pores as dynamic conductive pathways through which the charged essential cell cofactor pyridoxal phosphate, PLP, can diffuse through cell membranes, a key physiological process poorly understood in mammalian cells 11 . We investigated the process characterizing the affinity and binding kinetics of PLP and other ligands that bind the PLP-binding site. The crystal structure of the complex between NAPE-PLD and PLP contributes to define the membrane transport mechanism at atomic resolution.