Published on Wed Jun 23 2021

Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling

Bleffert, F., Granzin, J., Caliskan, M., Schott-Verdugo, S., Siebers, M., Thiele, B., Rahme, L., Felgner, S., Doermann, P., Gohlke, H., Batra-Safferling, R., Jaeger, K.-E., Kovacic, F.

Pseudomonas aeruginosa has first cytoplasmic membrane-bound phospholipase A1 (PlaF) involved in the Lands cycle. PlaF is an important virulence factor, as the P. aerug inosa mutant showed strongly attenuated virulence in Galleria mellonella and macrophages.

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Abstract

Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa {Delta}plaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-[A]-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. A dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network which likely explains the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the GPL remodeling pathway for virulence and pave the way for the development of a novel therapeutic class of antibiotics targeting PlaF-mediated membrane GPL remodeling.