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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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pH stability and pKa determination for BdbD. A, plots of tryptophan fluorescence emission maxima and emission intensity at 346 nm as a function of pH for solutions of sBdbD (0.15 μm in PCTC buffer), as a function of pH. B, time-dependent increases in fluorescence at 510 nm upon reaction with wild-type sBdbD (1 μm) with badan (12 μm) in a mixed buffer system at pH values from 4.76 to 9.78, as indicated, at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant ko. C, plots of ko as a function of pH. The solid line show a fit to supplemental Equation S2.
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Figure 8: pH stability and pKa determination for BdbD. A, plots of tryptophan fluorescence emission maxima and emission intensity at 346 nm as a function of pH for solutions of sBdbD (0.15 μm in PCTC buffer), as a function of pH. B, time-dependent increases in fluorescence at 510 nm upon reaction with wild-type sBdbD (1 μm) with badan (12 μm) in a mixed buffer system at pH values from 4.76 to 9.78, as indicated, at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant ko. C, plots of ko as a function of pH. The solid line show a fit to supplemental Equation S2.

Mentions: To define the range over which the pKa values of the sBdbD active site cysteines can be measured, the pH stability of folded sBdbD was investigated using the intrinsic fluorescence of tryptophan. A plot of emission wavelength maximum versus pH (Fig. 8A) showed that pH-induced unfolding of sBdbD led to a sizeable shift in emission wavelength from around 353 nm at pH 7 to much longer wavelengths at extremes of pH. The apparent stability range of BdbD lies between pH 4.5 and pH 10 and is similar to both ResA and StoA from B. subtilis (23, 36). Within the stable range itself, there was little evidence for titration of other residues affecting the fluorescence of tryptophan residues. The data indicate that pKa values lower than 4.5 cannot be measured. This is particularly important for sBdbD because, as a DsbA-like protein, it is likely to have a highly nucleophilic N-terminal cysteine in the CXXC motif (11, 44).


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)

pH stability and pKa determination for BdbD. A, plots of tryptophan fluorescence emission maxima and emission intensity at 346 nm as a function of pH for solutions of sBdbD (0.15 μm in PCTC buffer), as a function of pH. B, time-dependent increases in fluorescence at 510 nm upon reaction with wild-type sBdbD (1 μm) with badan (12 μm) in a mixed buffer system at pH values from 4.76 to 9.78, as indicated, at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant ko. C, plots of ko as a function of pH. The solid line show a fit to supplemental Equation S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: pH stability and pKa determination for BdbD. A, plots of tryptophan fluorescence emission maxima and emission intensity at 346 nm as a function of pH for solutions of sBdbD (0.15 μm in PCTC buffer), as a function of pH. B, time-dependent increases in fluorescence at 510 nm upon reaction with wild-type sBdbD (1 μm) with badan (12 μm) in a mixed buffer system at pH values from 4.76 to 9.78, as indicated, at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant ko. C, plots of ko as a function of pH. The solid line show a fit to supplemental Equation S2.
Mentions: To define the range over which the pKa values of the sBdbD active site cysteines can be measured, the pH stability of folded sBdbD was investigated using the intrinsic fluorescence of tryptophan. A plot of emission wavelength maximum versus pH (Fig. 8A) showed that pH-induced unfolding of sBdbD led to a sizeable shift in emission wavelength from around 353 nm at pH 7 to much longer wavelengths at extremes of pH. The apparent stability range of BdbD lies between pH 4.5 and pH 10 and is similar to both ResA and StoA from B. subtilis (23, 36). Within the stable range itself, there was little evidence for titration of other residues affecting the fluorescence of tryptophan residues. The data indicate that pKa values lower than 4.5 cannot be measured. This is particularly important for sBdbD because, as a DsbA-like protein, it is likely to have a highly nucleophilic N-terminal cysteine in the CXXC motif (11, 44).

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