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.
Highlights
Loop regions are often recognized as key regulatory elements of protein function, with their dynamic nature impacting characteristics such as enzyme a ctivity[1,2], substrate s pecificity[3,4] and m ore[5]
Using new molecular modeling techniques coupled with Double electron–electron resonance (DEER) restraints, we develop ensemble models of the flexible loop in both the apo and holo states
Given the homology of the ExoU ubiquitin binding domain (UBD) to the VipD Rab binding domain (RBD), we postulated that ExoU may share a similar activation mechanism
Summary
Loop regions are often recognized as key regulatory elements of protein function, with their dynamic nature impacting characteristics such as enzyme a ctivity[1,2], substrate s pecificity[3,4] and m ore[5] Due to their intrinsic flexibility, the structural properties of protein loops are often difficult to study. One of the most promising techniques couples DEER distances with the Rosetta molecular modeling suite[13,14] This approach was used to develop high-resolution models of T4 lysozyme and αA-crystallin[15]. Another example is the use of RosettaDock and DEER distance restraints to identify the ubiquitin binding interface associated with the bacterial A2 phospholipase, E xoU16. Using new molecular modeling techniques coupled with DEER restraints, we develop ensemble models of the flexible loop in both the apo and holo states
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