Investigating Structural Properties of Pseudomonas Aeruginosa Exou Toxin Upon Interaction with Liposome and Nanodisc Bilayers by EPR Spectroscopy

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen that commonly infects the lungs of cystic fibrosis (CF) patients and severe burn victims. One of the four type III secreted effector proteins of P. aeruginosa is ExoU, a bacterial phospholipase A2 (PLA2) enzyme evolved to utilize host ubiquitin (Ub) and ubiquitinated proteins for enzymatic activation, leading to the destruction of host membranes. ExoU has three major domains: a catalytic domain, a linker domain, and a four-helix bundle located in the C-terminus. Sequence and structural homology of the four-helix bundle are weak across various genus and species, suggesting unique structural and functional features that may play a role in the molecular mechanism of activation. Previous work identified regions within helix IV of the four-helix bundle that are necessary for both activation and localization of ExoU to the lipid bilayer, and suggested a synergistic mechanism of activation involving ubiquitin and membrane binding. We are interested in understanding conformational changes in ExoU upon membrane binding in the presence and absence of ubiquitin. We hypothesize that the C-terminal four-helix bundle maintains essential intramolecular interactions with the catalytic domain that are necessary for enzymatic activation. In this study, site-directed mutagenesis, continuous wave (CW) EPR spectroscopy, CW power saturation, and double electron-electron resonance (DEER) were used to investigate the structure and membrane localization of helix IV in the presence of LUVs and nanodiscs. Results show that ubiquitin binding facilitates membrane penetration by helix IV, and demonstrate the utility of nanodisc bilayers for the study of protein-membrane interactions. Supported by NIH grant GM114234.