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Structure and functional properties of Bacillus subtilis endospore biogenesis factor StoA.

Crow A, Liu Y, Möller MC, Le Brun NE, Hederstedt L - J. Biol. Chem. (2009)

Bottom Line: The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states.Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents.In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs.

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
Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.

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The active site of StoA in oxidized and reduced states. A, electron density (contoured at 1.2 σ) of the active site CPPC motif of StoA reveals a mixture of oxidized and reduced states. B and C, separated representations of the active site region in oxidized and reduced states, respectively. Intercysteine sulfur distances are indicated (in Å).
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fig4: The active site of StoA in oxidized and reduced states. A, electron density (contoured at 1.2 σ) of the active site CPPC motif of StoA reveals a mixture of oxidized and reduced states. B and C, separated representations of the active site region in oxidized and reduced states, respectively. Intercysteine sulfur distances are indicated (in Å).

Mentions: In the structure determined here, the active site cysteines of sStoA appear as a mixture of oxidized and reduced redox states (in each monomer). Crystallization of sStoA utilized solely the oxidized form of the protein, and thus it is likely that partial reduction of the disulfide bond was induced by photoreduction in the x-ray beam. The electron density associated with the partially broken disulfide is shown in Fig. 4A along with separated models of the oxidized and reduced conformations shown in Fig. 4, B and C, respectively.


Structure and functional properties of Bacillus subtilis endospore biogenesis factor StoA.

Crow A, Liu Y, Möller MC, Le Brun NE, Hederstedt L - J. Biol. Chem. (2009)

The active site of StoA in oxidized and reduced states. A, electron density (contoured at 1.2 σ) of the active site CPPC motif of StoA reveals a mixture of oxidized and reduced states. B and C, separated representations of the active site region in oxidized and reduced states, respectively. Intercysteine sulfur distances are indicated (in Å).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665060&req=5

fig4: The active site of StoA in oxidized and reduced states. A, electron density (contoured at 1.2 σ) of the active site CPPC motif of StoA reveals a mixture of oxidized and reduced states. B and C, separated representations of the active site region in oxidized and reduced states, respectively. Intercysteine sulfur distances are indicated (in Å).
Mentions: In the structure determined here, the active site cysteines of sStoA appear as a mixture of oxidized and reduced redox states (in each monomer). Crystallization of sStoA utilized solely the oxidized form of the protein, and thus it is likely that partial reduction of the disulfide bond was induced by photoreduction in the x-ray beam. The electron density associated with the partially broken disulfide is shown in Fig. 4A along with separated models of the oxidized and reduced conformations shown in Fig. 4, B and C, respectively.

Bottom Line: The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states.Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents.In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs.

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
Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.

Show MeSH