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Crystal structure and biophysical properties of Bacillus subtilis BdbD. An oxidizing thiol:disulfide oxidoreductase containing a novel metal site.

Crow A, Lewin A, Hecht O, Carlsson Möller M, Moore GR, Hederstedt L, Le Brun NE - J. Biol. Chem. (2009)

Bottom Line: The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR.However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein.Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

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Unfolding profiles of native and EDTA-treated sBdbD. 1 μm pre-reduced and pre-oxidized sBdbD protein was incubated in various concentrations of urea at 25 °C. Fraction unfolded was calculated from the fluorescence intensity. A and B show data from reduced (squares) and oxidized (circles) as-isolated protein, respectively, in 0.1 m Tris-HCl, pH 8.0. The folding (open symbols) and unfolding (filled symbols) data were analyzed using supplemental Equation S3, and the resulting fits are drawn as solid lines. C and D show folding and unfolding data from equivalent experiments performed with Ca2+-depleted sBdbD.
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Figure 7: Unfolding profiles of native and EDTA-treated sBdbD. 1 μm pre-reduced and pre-oxidized sBdbD protein was incubated in various concentrations of urea at 25 °C. Fraction unfolded was calculated from the fluorescence intensity. A and B show data from reduced (squares) and oxidized (circles) as-isolated protein, respectively, in 0.1 m Tris-HCl, pH 8.0. The folding (open symbols) and unfolding (filled symbols) data were analyzed using supplemental Equation S3, and the resulting fits are drawn as solid lines. C and D show folding and unfolding data from equivalent experiments performed with Ca2+-depleted sBdbD.

Mentions: The conformational stabilities of sBdbD in both reduced and oxidized states were investigated through equilibrium unfolding (and refolding) experiments using urea, in which the intrinsic tryptophan fluorescence of sBdbD was used to monitor the state of folding (Fig. 7, A and B, respectively). Urea-induced unfolding of sBdbD was found to be completely reversible and was analyzed in terms of a two-state model, yielding a free energy of stabilization of −30.4 ± 2.5 kJ mol−1 for the reduced protein and −11.8 ± 1.6 kJ mol−1 for the oxidized protein. The greater stability of the reduced state compared with the oxidized state is a characteristic of oxidizing TDORs. For sBdbD, ΔΔGox/red = 18.6 ± 4 kJ mol−1, a value similar to that measured for EcDsbA (14.8 ± 4 kJ mol−1) (65). The associated m values for the folding/unfolding transition were similar for both redox states (Table 2), indicating that the protein undergoes a similar degree of unfolding in each state. This is consistent with there being few structural changes between the reduced and oxidized states and that the disulfide bond is between two cysteines residues located close to one another and, therefore has little effect on the degree of unfolding.


Crystal structure and biophysical properties of Bacillus subtilis BdbD. An oxidizing thiol:disulfide oxidoreductase containing a novel metal site.

Crow A, Lewin A, Hecht O, Carlsson Möller M, Moore GR, Hederstedt L, Le Brun NE - J. Biol. Chem. (2009)

Unfolding profiles of native and EDTA-treated sBdbD. 1 μm pre-reduced and pre-oxidized sBdbD protein was incubated in various concentrations of urea at 25 °C. Fraction unfolded was calculated from the fluorescence intensity. A and B show data from reduced (squares) and oxidized (circles) as-isolated protein, respectively, in 0.1 m Tris-HCl, pH 8.0. The folding (open symbols) and unfolding (filled symbols) data were analyzed using supplemental Equation S3, and the resulting fits are drawn as solid lines. C and D show folding and unfolding data from equivalent experiments performed with Ca2+-depleted sBdbD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749146&req=5

Figure 7: Unfolding profiles of native and EDTA-treated sBdbD. 1 μm pre-reduced and pre-oxidized sBdbD protein was incubated in various concentrations of urea at 25 °C. Fraction unfolded was calculated from the fluorescence intensity. A and B show data from reduced (squares) and oxidized (circles) as-isolated protein, respectively, in 0.1 m Tris-HCl, pH 8.0. The folding (open symbols) and unfolding (filled symbols) data were analyzed using supplemental Equation S3, and the resulting fits are drawn as solid lines. C and D show folding and unfolding data from equivalent experiments performed with Ca2+-depleted sBdbD.
Mentions: The conformational stabilities of sBdbD in both reduced and oxidized states were investigated through equilibrium unfolding (and refolding) experiments using urea, in which the intrinsic tryptophan fluorescence of sBdbD was used to monitor the state of folding (Fig. 7, A and B, respectively). Urea-induced unfolding of sBdbD was found to be completely reversible and was analyzed in terms of a two-state model, yielding a free energy of stabilization of −30.4 ± 2.5 kJ mol−1 for the reduced protein and −11.8 ± 1.6 kJ mol−1 for the oxidized protein. The greater stability of the reduced state compared with the oxidized state is a characteristic of oxidizing TDORs. For sBdbD, ΔΔGox/red = 18.6 ± 4 kJ mol−1, a value similar to that measured for EcDsbA (14.8 ± 4 kJ mol−1) (65). The associated m values for the folding/unfolding transition were similar for both redox states (Table 2), indicating that the protein undergoes a similar degree of unfolding in each state. This is consistent with there being few structural changes between the reduced and oxidized states and that the disulfide bond is between two cysteines residues located close to one another and, therefore has little effect on the degree of unfolding.

Bottom Line: The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR.However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein.Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

Show MeSH
Related in: MedlinePlus