| Autor: |
Tessmer MH; Department of Chemistry, University of Washington, Seattle, WA, USA., DeCero SA; Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, USA., Del Alamo D; Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN, USA.; Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA., Riegert MO; Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, USA., Meiler J; Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN, USA.; Institute for Drug Discovery, Leipzig University Medical School, Leipzig SAC, Germany., Frank DW; Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, USA. frankd@mcw.edu., Feix JB; Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA. |
| Abstrakt: |
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. |
