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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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Active site region of BdbD. A, detailed view of the N terminus of helix α1 of sBdbD showing the Cys-Pro-Ser-Cys active site of sBdbD and the closely lying cis-proline (Pro193), which is invariant in all thioredoxin-like proteins. B and C, electron density (contoured at 1.0σ) of the active site region of sBdbD in oxidized and reduced states, respectively.
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Figure 3: Active site region of BdbD. A, detailed view of the N terminus of helix α1 of sBdbD showing the Cys-Pro-Ser-Cys active site of sBdbD and the closely lying cis-proline (Pro193), which is invariant in all thioredoxin-like proteins. B and C, electron density (contoured at 1.0σ) of the active site region of sBdbD in oxidized and reduced states, respectively.

Mentions: Structure of the soluble domain of B. subtilis BdbD. A, three-dimensional structure of sBdbD showing that the protein exhibits a thioredoxin-like fold with an inserted helical domain. Secondary structure elements are labeled from the N terminus with the predicted transmembrane helix of the full-length protein (covered by residues 1–36 that were not present in the protein used for crystallization and therefore not indicated on the figure) being 0. The active site and Ca2+-binding sites are indicated. The thioredoxin-like part of BdbD is colored green, and the all-helical domain is colored teal. Note that the same color scheme is used in Figs. 3 and 4. B, surface representation of sBdbD. Regions colored red indicate areas of high negative electrostatic potential, and blue areas indicate areas of high positive potential. Neutral regions are in white. Electrostatic potentials of surfaces were calculated using PyMOL (73). All structural figures were prepared with PyMOL and annotated with GIMP. C, amino acid residue sequence comparison of B. subtilis BdbD (BsBdbD) with other DsbA-like proteins from Bacillus anthracis (BaBdbD), Bacillus cereus (BcBdbD), S. aureus (SaDsbA), and E. coli (EcDsbA). Invariant residues are marked in black and highly conserved residues in gray. Diamonds indicate the active site residues Cys69 and Cys72, and asterisks indicate the residues acting as ligands to the Ca2+ ion. A black circle indicates the near-active site glutamate residue (Glu63), and an inverted triangle indicates the beginning of the soluble domain of B. subtilis BdbD studied here. Secondary structural elements, derived from the structure of BdbD, are indicated above the sequence. Elements belonging to the thioredoxin-fold are indicated in dark gray; those belonging to the helical domain are indicated in light gray, and those not belonging to either are indicated in white. Residue numbers are indicated on the right; these refer to the non-processed predicted translated sequence. The alignment was obtained using ClustalW (74) and annotated in Genedoc (75).


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)

Active site region of BdbD. A, detailed view of the N terminus of helix α1 of sBdbD showing the Cys-Pro-Ser-Cys active site of sBdbD and the closely lying cis-proline (Pro193), which is invariant in all thioredoxin-like proteins. B and C, electron density (contoured at 1.0σ) of the active site region of sBdbD in oxidized and reduced states, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Active site region of BdbD. A, detailed view of the N terminus of helix α1 of sBdbD showing the Cys-Pro-Ser-Cys active site of sBdbD and the closely lying cis-proline (Pro193), which is invariant in all thioredoxin-like proteins. B and C, electron density (contoured at 1.0σ) of the active site region of sBdbD in oxidized and reduced states, respectively.
Mentions: Structure of the soluble domain of B. subtilis BdbD. A, three-dimensional structure of sBdbD showing that the protein exhibits a thioredoxin-like fold with an inserted helical domain. Secondary structure elements are labeled from the N terminus with the predicted transmembrane helix of the full-length protein (covered by residues 1–36 that were not present in the protein used for crystallization and therefore not indicated on the figure) being 0. The active site and Ca2+-binding sites are indicated. The thioredoxin-like part of BdbD is colored green, and the all-helical domain is colored teal. Note that the same color scheme is used in Figs. 3 and 4. B, surface representation of sBdbD. Regions colored red indicate areas of high negative electrostatic potential, and blue areas indicate areas of high positive potential. Neutral regions are in white. Electrostatic potentials of surfaces were calculated using PyMOL (73). All structural figures were prepared with PyMOL and annotated with GIMP. C, amino acid residue sequence comparison of B. subtilis BdbD (BsBdbD) with other DsbA-like proteins from Bacillus anthracis (BaBdbD), Bacillus cereus (BcBdbD), S. aureus (SaDsbA), and E. coli (EcDsbA). Invariant residues are marked in black and highly conserved residues in gray. Diamonds indicate the active site residues Cys69 and Cys72, and asterisks indicate the residues acting as ligands to the Ca2+ ion. A black circle indicates the near-active site glutamate residue (Glu63), and an inverted triangle indicates the beginning of the soluble domain of B. subtilis BdbD studied here. Secondary structural elements, derived from the structure of BdbD, are indicated above the sequence. Elements belonging to the thioredoxin-fold are indicated in dark gray; those belonging to the helical domain are indicated in light gray, and those not belonging to either are indicated in white. Residue numbers are indicated on the right; these refer to the non-processed predicted translated sequence. The alignment was obtained using ClustalW (74) and annotated in Genedoc (75).

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