Dataset Information

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

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.

SUBMITTER: Feix JB

PROVIDER: S-EPMC6347562 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Publications

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

Feix Jimmy B JB Kohn Samantha S Tessmer Maxx H MH Anderson David M DM Frank Dara W DW

Cell biochemistry and biophysics 20180725 1

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 ...[more]

PMID: 30047043

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Project description:Protein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein-protein and protein-ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined. Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-directed spin labeling (SDSL) is an EPR based approach that is particularly well-suited to high molecular-weight systems such as membrane proteins. Because of the relatively fast time scale for EPR spectroscopy, it is an excellent method to examine exchange. Conformations that are in exchange are captured as distinct populations in the EPR spectrum, and this feature when combined with the use of methods that can shift the free energy of conformational substates allows one to identify regions of proteins that are in dynamic exchange. In addition, modern pulse EPR methods have the ability to examine conformational heterogeneity, resolve discrete protein states, and identify the substates in exchange. Protein crystallography has provided high-resolution models for a number of membrane proteins; but because of conformational exchange, these models do not always reflect the structures that are present when the protein is in a native bilayer environment. In the case of the Escherichia coli vitamin B12 transporter, BtuB, the energy coupling segment of this protein undergoes a substrate-dependent unfolding, which acts to couple this outer-membrane protein to the inner-membrane protein TonB. EPR spectroscopy demonstrates that the energy coupling segment is in equilibrium between ordered and disordered states, and that substrate binding shifts this equilibrium to favor an unfolded state. However, in crystal structures of BtuB, this segment is resolved and folded within the protein, and neither the presence of this equilibrium nor the substrate-induced change is revealed. This is a result of the solute environment and the crystal lattice, both of which act to stabilize one conformational substate of the transporter. Using SDSL, it can be shown that conformational exchange is present in other regions of BtuB and in other members of this transporter family. Conformational exchange has also been examined in systems such as the plasma membrane SNARE protein, syntaxin 1A, where dynamics are controlled by regulatory proteins such as munc18. Regulating the microsecond to millisecond time scale dynamics in the neuronal SNAREs is likely to be a key feature that regulates assembly of the SNAREs and neurotransmitter release.

Dataset Information

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

Publications

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

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