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Conformational Changes and Membrane Interaction of the Bacterial Phospholipase, ExoU: Characterization by Site-Directed Spin Labeling

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Abstract

Numerous pathogenic bacteria produce proteins evolved to facilitate their survival and dissemination by modifying the host environment. These proteins, termed effectors, often play a significant role in determining the virulence of the infection. Consequently, bacterial effectors constitute an important class of targets for the development of novel antibiotics. ExoU is a potent phospholipase effector produced by the opportunistic pathogen Pseudomonas aeruginosa. Previous studies have established that the phospholipase activity of ExoU requires non-covalent interaction with ubiquitin, however the molecular details of the mechanism of activation and the manner in which ExoU associates with a target lipid bilayer are not understood. In this review we describe our recent studies using site-directed spin labeling (SDSL) and EPR spectroscopy to elucidate the conformational changes and membrane interactions that accompany activation of ExoU. We find that ubiquitin binding and membrane interaction act synergistically to produce structural transitions that occur upon ExoU activation, and that the C-terminal four-helix bundle of ExoU functions as a phospholipid-binding domain, facilitating the association of ExoU with the membrane surface.

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Acknowledgements

This work is supported by National Institutes of Health grants GM114234 (J.B.F.) and AI104922 (D.W.F.). DEER instrumentation was supported by NIH grants S10RR022422 and S10OD011937.

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Authors and Affiliations

  1. Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Jimmy B. Feix & Samantha Kohn

  2. Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Maxx H. Tessmer & Dara W. Frank

  3. Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA

    David M. Anderson

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Correspondence to Jimmy B. Feix.

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Feix, J.B., Kohn, S., Tessmer, M.H. et al. Conformational Changes and Membrane Interaction of the Bacterial Phospholipase, ExoU: Characterization by Site-Directed Spin Labeling. Cell Biochem Biophys 77, 79–87 (2019). https://doi.org/10.1007/s12013-018-0851-8

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