Characterization of the C‐terminal domain essential for toxic activity of adenylate cyclase toxin

Bejerano, Michal; Nisan, Israel; Ludwig, Albrecht; Goebel, Werner; Hanski, Emanuel

doi:10.1046/j.1365-2958.1999.01183.x

Cited by 39 publications

(47 citation statements)

References 31 publications

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“…Like other intrinsically disordered polypeptides it contains a high content of random coils and is highly hydrated (30)(31)(32). Upon binding calcium, the CyaA RD folds into a stable β-roll structure that facilitates interaction with a cell surface receptor and the translocation of an N-terminal adenylate cyclase domain across the plasma membrane (32)(33)(34)(35)(36). We found that the chimeric passenger domain was secreted efficiently even in the absence of calcium and thereby showed that translocation can proceed efficiently without passenger domain folding.…”

Section: Significancementioning

confidence: 80%

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Kang'ethe

Bernstein

2013

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

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Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular ("passenger") domain that folds into a β-helical structure and a C-terminal β-barrel ("β") domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the β-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction.bacterial pathogenesis | membrane protein assembly | type Va secretion

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Section: Significancementioning

confidence: 80%

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Kang'ethe

Bernstein

2013

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

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“…A number of previous studies suggest that a region between the repeat domain and the C terminus may be involved in binding this specific receptor. For example, Bejerano et al (14) have described two amino acid "blocks," located after the nonapeptide repeats, in the C terminus of the adenylate cyclase toxin (another member of the RTX family). Block A (15 amino acids) is essential for the toxic activity since it is required for the toxin binding and insertion into the membrane.…”

Section: ␣-Hemolysinmentioning

confidence: 99%

“…HlyA possesses homologous A and B blocks ( Fig. 1), and it has been shown (14) that deletion of a region that includes the last two residues of block A, the connection between the blocks, and the first nine amino acids of block B abolishes the hemolytic activity. Previously, Chervaux and Holland (15) had shown that five point mutations in that region (residues 918, 920, 921, 928, and 932) inhibited hemolysis without affecting protein export.…”

Section: ␣-Hemolysinmentioning

confidence: 99%

A Receptor-binding Region in Escherichia coli α-Haemolysin

Cortajarena¹,

Goñi

Ostolaza

2003

Journal of Biological Chemistry

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Escherichia coli ␣-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells. Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A. L., Goñ i, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513-12519). The present study was aimed at identifying the glycophorin-binding region in the toxin. Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain. Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914 -936 of HlyA. We therefore prepared a deletion mutant lacking these residues (HlyA⌬914 -936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type. This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column. 1 is a 107-kDa protein toxin secreted by pathogenic strains of Escherichia coli. It is a member of the so-called "RTX family," a group of proteins characterized by the presence of a Gly-and Asp-rich nonapeptide sequence repeated in tandem near the protein C terminus (for reviews, see Refs. 1-5). These repeats constitute a Ca 2ϩ -binding domain whose structure has been solved at high resolution for a non-toxin member of the RTX family, the alkaline protease from Pseudomonas aeruginosa (6). HlyA first binds a receptor on the cell surface, a ␤ 2 -integrin in leukocytes (7) or glycophorin in red blood cells (8), and then becomes inserted in the cell membrane. Recent data indicate that insertion may take place in the absence of Ca 2ϩ (9, 10), but Ca 2ϩ binding to the nonapeptide repeat domain is essential for membrane lysis (9,11,12). Note that the Ca 2ϩ -binding domain is located near the protein C terminus, whereas the membrane insertion domain is located near the N terminus (9, 10, 13). The present study is devoted to exploring the early stages of HlyA interaction with the target cell, namely its binding to the surface receptor. In particular, our investigation is aimed at the region(s) of the protein that bind(s) the receptor glycophorin on mammalian erythrocytes (8). A number of previous studies suggest that a region between the repeat domain and the C terminus may be involved in binding this specific receptor. For example, Bejerano et al. (14) have described two amino acid "blocks," located after the nonapeptide repeats, in the C terminus of the adenylate cyclase toxin (another member of the RTX family). Block A (15 amino acids) is essential for the toxic activity since it is required for the toxin binding and insertion into the membrane. Deletion of block B, however, does not affect the toxin activity. HlyA possesses homologous A and B blocks (Fig. 1), and it has been shown (14) that deletion of a region that includes the last two residues of block A, the connection between the blocks, and the first nine amino acids of block B...

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“…[86] Accordingly, Ca 2þ ions are required for folding and hence, cytotoxicity of the T1SS secreted proteins. [87][88][89][90] Recent studies provide evidence for an intramolecular translocation ratchet powered by folding of the RTX-domain: Within the "channel-tunnel" system of the T1SS that spans the entire cell envelope, the secreted proteins are transported in an unfolded state, as expected from the channel dimensions [91] ( Figure 4B). The unfolded protein is transported in a C to N direction, leading to initial exposure of the C-terminus and thus the first RTX repeat segment.…”

Section: Potentially Length-dependent Intramolecular Translocation Ramentioning

confidence: 93%

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

Hepp

Maier

2017

BioEssays

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Secretion systems enable bacteria to import and secrete large macromolecules including DNA and proteins. While most components of these systems have been identified, the molecular mechanisms of macromolecular transport remain poorly understood. Recent findings suggest that various bacterial secretion systems make use of the translocation ratchet mechanism for transporting polymers across the cell envelope. Translocation ratchets are powered by chemical potential differences generated by concentration gradients of ions or molecules that are specific to the respective secretion systems. Bacteria employ these potential differences for biasing Brownian motion of the macromolecules within the conduits of the secretion systems. Candidates for this mechanism include DNA import by the type II secretion/ type IV pilus system, DNA export by the type IV secretion system, and protein export by the type I secretion system. Here, we propose that these three secretion systems employ different molecular implementations of the translocation ratchet mechanism.

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Characterization of the C‐terminal domain essential for toxic activity of adenylate cyclase toxin

Cited by 39 publications

References 31 publications

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

A Receptor-binding Region in Escherichia coli α-Haemolysin

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

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