Characterization of the ExoU activation mechanism using EPR and integrative modeling

Abstract

ExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa, serves as a prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used to identify an unstructured loop of approximately 20 residues in the ExoU amino acid sequence. Mutational analyses indicate the importance of specific loop amino acid residues in mediating catalytic activity. Engineered disulfide cross-links show that loop movement is required for activation. Site directed spin labeling EPR and DEER (double electron–electron resonance) studies of apo and holo states demonstrate local conformational changes at specific sites within the loop and a conformational shift of the loop during activation. These data are consistent with the formation of a substrate-binding pocket providing access to the catalytic site. DEER distance distributions were used as constraints in RosettaDEER to construct ensemble models of the loop in both apo and holo states, significantly extending the range for modeling a conformationally dynamic loop.

Integrated AlphaFold2 and DEER investigation of the conformational dynamics of a pH-dependent APC antiporter

“…Experimental distance distributions are then compared to predictions made from both the crystal structure and representative AF2 models. Unlike integrative modeling pipelines that use the data as modeling restraints ( 87 – 90 ), our approach was reinforced by the correspondence between DEER distributions predicted by the AF2 models and the experimentally resolved distance components.…”

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles

“…The different states characterized to date represent different relative orientations of the catalytic domain to the 4-α-helical bundle. According to a recent model, the association of the C-terminus with the membrane is accompanied by a complete unfolding of the 4-α-helical bundle [40,48].…”

Perspectives on the Pseudomonas aeruginosa Type III Secretion System Effector ExoU and Its Subversion of the Host Innate Immune Response to Infection

“…The membrane interacting loop 3 of the C-terminal 4-helix bundle is highlighted in yellow. Loops missing from the crystal structures were modeled as previously described [54].…”

“…Understanding the structural transitions between inactive and active states of these proteins may provide a target for rationally designed ligands that can prevent this conversion to a cell and tissue destructive virulence factor. The availability of crystal structures and recent use of biophysical and computational modeling techniques [54] open new horizons for understanding allosteric changes of dynamic enzymes in response to both cofactors and substrates.…”