Structure and Function of POTRA Domains of Omp85/TPS Superfamily

Simmerman, Richard; Dave, Ashita; Bruce, Barry D.

doi:10.1016/b978-0-12-800097-7.00001-4

Cited by 31 publications

(27 citation statements)

References 132 publications

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“…Indeed, only a P. aeruginosa strain harboring both exlB - exlA genes, expressed from an arabinose-inducible promoter, was cytolytic on epithelial and endothelial cells (6). As with other TPS systems, the TpsB homologue ExlB, in addition to the β-barrel-forming domain, possesses two predicted conserved domains predicted to be oriented toward the bacterial periplasm and is responsible for TpsA (ExlA) export (18, 19). These domains are called the POTRA domains (for po lypeptide-associated tra nsport) and are located in ExlB between amino acids 85 to 160 and 162 to 230 for POTRA1 and POTRA2, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

Basso

Ragno

Elsen

et al. 2017

mBio

113

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Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication.

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Section: Resultsmentioning

confidence: 99%

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

Basso

Ragno

Elsen

et al. 2017

mBio

113

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“…Although Toc75 proteins from green and red algae also are predicted to contain three POTRA domains based on secondary structure predictions, the available algal sequences are highly divergent from those in land plants, as noted previously (17), and the lack of structural information for these proteins makes it difficult to determine whether the conserved charged and hydrophobic regions in land plants are present. Nonetheless, the P2-helix insertion is clearly absent from available algae sequences, and it was previously speculated that the P2-helix is an evolutionary adaptation of Toc75 in land plants (17,25).…”

Section: Discussionmentioning

confidence: 99%

“…The structure reveals that the POTRA domains form an L-shaped structure very similar to BamA, with a unique extended helix within POTRA2 that is not observed in the POTRA domains of BamA and other Omp85 family members (25,28,(35)(36)(37)(38). Based on these unique structural properties, we investigated the contribution of individual POTRA repeats in binding chloroplast precursors.…”

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The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

O'Neil

Richardson

Paila

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a β-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded β-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transportassociated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperonelike activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.protein import | chloroplast | outer membrane | Toc75 | POTRA domain

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“…Although the b-barrel domain follows the evolutionary path in its divergence, phylogenetic analysis of the POTRA domains revealed a functional classification (5). POTRA domains of Omp85 proteins have been implicated in complex assembly and recognition of substrate proteins (6).…”

Section: Introductionmentioning

confidence: 99%

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

Dastvan

Brouwer

Schuetz

et al. 2016

Biophysical Journal

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Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a b-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal b-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRAdomain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spinlabeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

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Structure and Function of POTRA Domains of Omp85/TPS Superfamily

Cited by 31 publications

References 132 publications

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

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