Mode of disulfide bond formation of a heat‐stable enterotoxin (ST<sub>h</sub>) produced by a human strain of enterotoxigenic <i>Escherichia coli</i>

Shimonishi, Yasutsugu; Hidaka, Yuji; Koizumi, Michiyuki; Hane, Motomu; Aimoto, Saburo; Takeda, Tae; Miwatani, Toshio; Takeda, Yoshifumi

doi:10.1016/0014-5793(87)80134-5

Cited by 73 publications

(102 citation statements)

References 17 publications

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“…Subsequent analysis of a purified sample of the minor component (data not shown) confirmed this interpretation. The disulfide assignments made in this work are in complete agreement with those of Shimonishi et al (1987).…”

Section: C1c2elc5c6npac1'agsupporting

confidence: 84%

Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide Chain be split between close or adjacent Cys residues. The water-soluble tris-(2-~arboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra-and interchain disulfides; -Cys.Cys-pair), endothelin and apamin (two disulfides; -Cys.x.Cys-pair), conotoxin GI and isomers (two disulfides; -Cys.Cys-pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys-pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

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Section: C1c2elc5c6npac1'agsupporting

confidence: 84%

Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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“…In STp, three disulfide bonds are formed between the cysteines at positions 5 and 10, 6 and 14, and 9 and 17 (11). The same disulfide arrangement is found in STh (26). These intramolecular disulfide bonds are important for the correct conformation of the biologically active structure (8,18,21,35).…”

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confidence: 81%

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

et al. 1994

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The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions. Mature STp is released into the culture supernatant and is composed of 18-amino-acid residues which contain three intramolecular disulfide bonds. The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study. A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid. Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants. These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp. DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of the pro and mature regions. To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo. The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp. We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp.

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“…STas are 18-19-amino acid extracellular peptides that result from two independent proteolytic cleavages on a 72-amino acid precursor (pre-pro-STa) (Rasheed et al, 1990). The mature STa contains three disulfide bridges (Gariepy et ai., 1987;Shimonishi et al, 1987). The solution structure for this toxin has been determined by NMR methods and by X-ray diffraction studies (Gariepy et al, 1986;Ozaki et al, 1991).…”

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confidence: 99%

The structure ofEscherichia coliheat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism

Sarojadevi

Rizo²,

Wall

et al. 1995

Protein Sci.

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The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues I O and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two-and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMRderived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.

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Mode of disulfide bond formation of a heat‐stable enterotoxin (ST_h) produced by a human strain of enterotoxigenic Escherichia coli

Cited by 73 publications

References 17 publications

Disulfide structures of highly bridged peptides: A new strategy for analysis

Disulfide structures of highly bridged peptides: A new strategy for analysis

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

The structure ofEscherichia coliheat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism

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Mode of disulfide bond formation of a heat‐stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

Cited by 73 publications

References 17 publications

Disulfide structures of highly bridged peptides: A new strategy for analysis

Disulfide structures of highly bridged peptides: A new strategy for analysis

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

The structure ofEscherichia coliheat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism

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Mode of disulfide bond formation of a heat‐stable enterotoxin (ST_h) produced by a human strain of enterotoxigenic Escherichia coli