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Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue.

Ren G, Stephan D, Xu Z, Zheng Y, Tang D, Harrison RS, Kurz M, Jarrott R, Shouldice SR, Hiniker A, Martin JL, Heras B, Bardwell JC - J. Biol. Chem. (2009)

Bottom Line: The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties.It is isoleucine 75 in the case of thioredoxin.Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Departments of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

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Characterization of DsbA and mutant V150T-DsbB interaction in vivo and in vitro. A, E. coli disulfide bond formation pathway in the periplasm. B, DsbA  mutants are very sensitive to cadmium. In vivo DsbA oxidase activity was examined by measuring the cadmium resistance of DsbA  mutant strains expressing various mutants of DsbA on a plasmid. Spot titers were performed on LB plates with 40 μm cadmium. C33S, represents the DsbA active site mutant of CXXS. C, an acid trapping assay shows the slow oxidation of DsbA V150T by DsbB in vivo. Expression of DsbA and mutants was induced 10 min before AMS acid trapping. Reduced DsbA shows a 1-kDa upshift on SDS-PAGE. D, in vitro measurement of Km and kcat of DsbB catalyzing the oxidation of DsbA and variants by multi-turnover assay. The curves were fit from the average of three independent experiments. Original data are shown in supplemental Fig. S3A.
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fig4: Characterization of DsbA and mutant V150T-DsbB interaction in vivo and in vitro. A, E. coli disulfide bond formation pathway in the periplasm. B, DsbA mutants are very sensitive to cadmium. In vivo DsbA oxidase activity was examined by measuring the cadmium resistance of DsbA mutant strains expressing various mutants of DsbA on a plasmid. Spot titers were performed on LB plates with 40 μm cadmium. C33S, represents the DsbA active site mutant of CXXS. C, an acid trapping assay shows the slow oxidation of DsbA V150T by DsbB in vivo. Expression of DsbA and mutants was induced 10 min before AMS acid trapping. Reduced DsbA shows a 1-kDa upshift on SDS-PAGE. D, in vitro measurement of Km and kcat of DsbB catalyzing the oxidation of DsbA and variants by multi-turnover assay. The curves were fit from the average of three independent experiments. Original data are shown in supplemental Fig. S3A.

Mentions: The cisPro-minus1 Residue Is Involved in Interactions with Upstream Partner Proteins—We also investigated the role of the cisPro-minus1 residue in contributing to interactions with partner proteins. One way to do this is by studying the in vivo phenotype of DsbA V150T and its ability to be reoxidized by the membrane protein DsbB (Fig. 4) (43). Negatively influencing the interaction between DsbA and its downstream substrates (partially folded proteins) or its upstream partner (DsbB) should impair disulfide bond formation in vivo so that the in vivo effects would be similar to those seen in dsbA- strains. For example, dsbA- strains are sensitive to cadmium because of the high affinity of Cd2+ for protein thiol groups (11, 44). Similarly, E. coli-expressing DsbA V150T is cadmium-sensitive (Fig. 4B) showing that the cisPro-minus1 residue is important for in vivo DsbA function. Given that DsbA V150T is equivalent to WT DsbA (Fig. 4B) in oxidizing downstream substrates such as hirudin, at least in vitro, it is possible that the in vivo phenotype is a result of reduced affinity of DsbA V150T for its upstream partner DsbB. Supporting this explanation, the Km for interaction with DsbB is 5-fold higher for the V150T variant than for WT DsbA (98 versus 16 μm) (Fig. 4D). Similarly, the variant DsbA V150G is 40-fold slower than WT DsbA in the rate at which it is reoxidized by DsbB (10). Moreover, the DsbG T200L mutant is reduced only very slowly by its upstream partner, DsbD (supplemental Fig. S3C). Thus, mutation of the cisPro-minus1 residue in both DsbA and DsbG appears to result in a defect in the interaction with their respective upstream partners.


Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue.

Ren G, Stephan D, Xu Z, Zheng Y, Tang D, Harrison RS, Kurz M, Jarrott R, Shouldice SR, Hiniker A, Martin JL, Heras B, Bardwell JC - J. Biol. Chem. (2009)

Characterization of DsbA and mutant V150T-DsbB interaction in vivo and in vitro. A, E. coli disulfide bond formation pathway in the periplasm. B, DsbA  mutants are very sensitive to cadmium. In vivo DsbA oxidase activity was examined by measuring the cadmium resistance of DsbA  mutant strains expressing various mutants of DsbA on a plasmid. Spot titers were performed on LB plates with 40 μm cadmium. C33S, represents the DsbA active site mutant of CXXS. C, an acid trapping assay shows the slow oxidation of DsbA V150T by DsbB in vivo. Expression of DsbA and mutants was induced 10 min before AMS acid trapping. Reduced DsbA shows a 1-kDa upshift on SDS-PAGE. D, in vitro measurement of Km and kcat of DsbB catalyzing the oxidation of DsbA and variants by multi-turnover assay. The curves were fit from the average of three independent experiments. Original data are shown in supplemental Fig. S3A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665069&req=5

fig4: Characterization of DsbA and mutant V150T-DsbB interaction in vivo and in vitro. A, E. coli disulfide bond formation pathway in the periplasm. B, DsbA mutants are very sensitive to cadmium. In vivo DsbA oxidase activity was examined by measuring the cadmium resistance of DsbA mutant strains expressing various mutants of DsbA on a plasmid. Spot titers were performed on LB plates with 40 μm cadmium. C33S, represents the DsbA active site mutant of CXXS. C, an acid trapping assay shows the slow oxidation of DsbA V150T by DsbB in vivo. Expression of DsbA and mutants was induced 10 min before AMS acid trapping. Reduced DsbA shows a 1-kDa upshift on SDS-PAGE. D, in vitro measurement of Km and kcat of DsbB catalyzing the oxidation of DsbA and variants by multi-turnover assay. The curves were fit from the average of three independent experiments. Original data are shown in supplemental Fig. S3A.
Mentions: The cisPro-minus1 Residue Is Involved in Interactions with Upstream Partner Proteins—We also investigated the role of the cisPro-minus1 residue in contributing to interactions with partner proteins. One way to do this is by studying the in vivo phenotype of DsbA V150T and its ability to be reoxidized by the membrane protein DsbB (Fig. 4) (43). Negatively influencing the interaction between DsbA and its downstream substrates (partially folded proteins) or its upstream partner (DsbB) should impair disulfide bond formation in vivo so that the in vivo effects would be similar to those seen in dsbA- strains. For example, dsbA- strains are sensitive to cadmium because of the high affinity of Cd2+ for protein thiol groups (11, 44). Similarly, E. coli-expressing DsbA V150T is cadmium-sensitive (Fig. 4B) showing that the cisPro-minus1 residue is important for in vivo DsbA function. Given that DsbA V150T is equivalent to WT DsbA (Fig. 4B) in oxidizing downstream substrates such as hirudin, at least in vitro, it is possible that the in vivo phenotype is a result of reduced affinity of DsbA V150T for its upstream partner DsbB. Supporting this explanation, the Km for interaction with DsbB is 5-fold higher for the V150T variant than for WT DsbA (98 versus 16 μm) (Fig. 4D). Similarly, the variant DsbA V150G is 40-fold slower than WT DsbA in the rate at which it is reoxidized by DsbB (10). Moreover, the DsbG T200L mutant is reduced only very slowly by its upstream partner, DsbD (supplemental Fig. S3C). Thus, mutation of the cisPro-minus1 residue in both DsbA and DsbG appears to result in a defect in the interaction with their respective upstream partners.

Bottom Line: The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties.It is isoleucine 75 in the case of thioredoxin.Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Departments of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Show MeSH
Related in: MedlinePlus