Identification of a ubiquitin-binding interface using Rosetta and DEER

Tessmer, Maxx H.; Anderson, David M.; Pickrum, Adam M.; Riegert, Molly O.; Moretti, Rocco; Meiler, Jens; Feix, Jimmy B.; Frank, Dara W.

doi:10.1073/pnas.1716861115

Cited by 27 publications

(27 citation statements)

References 55 publications

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“…Such approaches have become popular for pharmacological targeting of disease-associated protein kinases [103]. Exploiting the ubiquitin-binding site of ExoU, which has been modelled in silico with the use of biophysical analysis and available crystallographic evidence [104], could serve as a point of allosteric inhibition that is selective and potent to ExoU activity inhibition. Such inhibitors could offer the advantage of being non-substrate competitive and, therefore, potentially, achieve superior potency.…”

Section: Allosteric Inhibitorsmentioning

confidence: 99%

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections

et al. 2019

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The opportunistic pathogen Pseudomonas aeruginosa employs the type III secretion system (T3SS) and four effector proteins, ExoS, ExoT, ExoU, and ExoY, to disrupt cellular physiology and subvert the host’s innate immune response. Of the effector proteins delivered by the T3SS, ExoU is the most toxic. In P. aeruginosa infections, where the ExoU gene is expressed, disease severity is increased with poorer prognoses. This is considered to be due to the rapid and irreversible damage exerted by the phospholipase activity of ExoU, which cannot be halted before conventional antibiotics can successfully eliminate the pathogen. This review will discuss what is currently known about ExoU and explore its potential as a therapeutic target, highlighting some of the small molecule ExoU inhibitors that have been discovered from screening approaches.

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Section: Allosteric Inhibitorsmentioning

confidence: 99%

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections

et al. 2019

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“…Loops 1 and 3 penetrate into the membrane and the helix bundle unfolds facilitating the association of the helices on the membrane surface and ExoU oligomerisation (6–8 monomers). The role of ExoU oligomerisation is not yet understood (Feix, Kohn, Tessmer, Anderson, & Frank, 2019; Springer, Reid, Gies, & Feix, 2019; Tessmer et al, 2018; Tessmer, Anderson, Buchaklian, Frank, & Feix, 2017; Tyson et al, 2015; Veesenmeyer et al, 2010; Zhang, Veesenmeyer, & Hauser, 2018). The model of the ExoU 4HBM insertion into membrane is possibly shared by toxins containing a 4HBM.…”

Section: Membrane Localisation Domain In Other Bacterial Toxins and Vmentioning

confidence: 99%

Bacterial intracellularly active toxins: Membrane localisation of the active domain

Varela-Chavez

Blondel

Popoff

2020

Cellular Microbiology

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Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras-GTPases, certain virulence factors of Gram negative bacteria, Rho-GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G-proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four-helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine. K E Y W O R D S Bacterial virulence factors, cell membrane, large clostridial glucosylating toxins, MARTX toxins, Pasteurella multocida toxin, phospholipids, Rho/Ras-GTPases

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“…The C-terminal domain of AxoU is far shorter than that of ExoU. In ExoU, this region consists of a bridging domain that has been shown to interact with ubiquitin (14) and a four-helix bundle that serves as a MLD (9-11). We utilized Rosetta to perform ab initio modeling of the C-terminal domain of AxoU (27,28).…”

Section: Figmentioning

confidence: 99%

Identification and Verification of Ubiquitin-Activated Bacterial Phospholipases

et al. 2019

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ExoU is a potent type III secretion system effector that is injected directly into mammalian cells by the opportunistic pathogen Pseudomonas aeruginosa. As a ubiquitin-activated phospholipase A2 (PLA2), ExoU exhibits cytotoxicity by cleaving membrane phospholipids, resulting in lysis of the host cells and inhibition of the innate immune response. Recently, ExoU has been established as a model protein for a group of ubiquitin-activated PLA2 enzymes encoded by a variety of bacteria. Bioinformatic analyses of homologous proteins is a powerful approach that can complement and enhance the overall understanding of protein structure and function. To conduct homology studies, it is important to have efficient and effective tools to screen and to validate the putative homologs of interest. Here we make use of an Escherichia coli-based dual expression system to screen putative ubiquitin-activated PLA2 enzymes from a variety of bacteria that are known to colonize humans and to cause human infections. The screen effectively identified multiple ubiquitin-activated phospholipases, which were validated using both biological and biochemical techniques. In this study, two new ExoU orthologs were identified and the ubiquitin activation of the rickettsial enzyme RP534 was verified. Conversely, ubiquitin was not found to regulate the activity of several other tested enzymes. Based on structural homology analyses, functional properties were predicted for AxoU, a unique member of the group expressed by Achromobacter xylosoxidans. IMPORTANCE Bacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers and inflammation during infections. Identifying enzymes with a common mechanism of activation is an initial step in understanding structural and functional properties. These properties serve as critical information for the design of specific inhibitors to reduce enzymatic activity and ameliorate host cell death. In this study, we identify and verify cytotoxic PLA2 enzymes from several bacterial pathogens. Similar to the founding member of the group, ExoU, these enzymes share the property of ubiquitin-mediated activation. The identification and validation of potential toxins from multiple bacterial species provide additional proteins from which to derive structural insights that could lead to paninhibitors useful for treating a variety of infections.

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Identification of a ubiquitin-binding interface using Rosetta and DEER

Cited by 27 publications

References 55 publications

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections

Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections

Bacterial intracellularly active toxins: Membrane localisation of the active domain

Identification and Verification of Ubiquitin-Activated Bacterial Phospholipases

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