Investigating the Conformational Dynamics and Membrane Interaction Near the Catalytic Serine of Exou Upon Interaction with Diubiquitin and Membranes by EPR Spectroscopy

Pseudomonas aeruginosa (P. aeruginosa) is a Gram negative, opportunistic pathogen that is a rising health concern as an ESKAPE pathogen resistant to multiple antimicrobial agents. P. aeruginosa manipulates the host response by translocating effector proteins into the host cell via a type III secretion system (T3SS). P. aeruginosa's most cytotoxic T3SS effector, ExoU, is a potent patatin-like phospholipase with a catalytic serine-aspartate dyad that destroys host cell membranes once activated by non-covalent interaction with ubiquitin. Two crystal structures of ExoU in complex with its chaperone SpcU have been reported, however the activated conformational state of ExoU in the presence of ubiquitin remains unknown. Consequently, ExoU's mechanism of action remains poorly understood. This study focuses on elucidating the conformational dynamics, membrane interaction, and structure of sites near the catalytic serine upon interaction with a ubiquitin cofactor and membrane substrate. Site-directed spin labeling (SDSL) in conjunction with electron paramagnetic (EPR) spectroscopy was used to examine the motional dynamics of sites near the catalytic serine upon interaction with diubiquitin (diUb) and membranes. Membrane penetration of the same sites upon interaction with diUb and membranes was investigated using power saturation EPR spectroscopy in the presence of various paramagnetic relaxation agents. Changes in spin label motion and membrane penetration were observed for sites near the catalytic serine in the presence of diUb and membranes combined, but not in the presence of either diUb or membranes alone, suggesting that a synergistic interaction with both ubiquitin and membranes is necessary to form an active catalytic site. Supported by NIH grant GM114234.