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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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pKa plots for wild-type and single cysteine sStoA variants. A, time-dependent increases in fluorescence at 510 nm upon reaction of wild-type (wt) sStoA (1 μm) with 6-bromoacetyl-2-dimethylaminonaphthalene (15 μm) in PCTC buffer system at pH values from 5 to 9 as indicated at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant, ko. B, plot of ko as a function of pH for wild-type sStoA. The solid line shows a fit to supplemental Equation S4. C and D, plots of ko as a function of pH obtained from similar experiments with C65A and C68A sStoA, respectively. Solid lines represent fits of the data to supplemental Equation S3.
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fig8: pKa plots for wild-type and single cysteine sStoA variants. A, time-dependent increases in fluorescence at 510 nm upon reaction of wild-type (wt) sStoA (1 μm) with 6-bromoacetyl-2-dimethylaminonaphthalene (15 μm) in PCTC buffer system at pH values from 5 to 9 as indicated at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant, ko. B, plot of ko as a function of pH for wild-type sStoA. The solid line shows a fit to supplemental Equation S4. C and D, plots of ko as a function of pH obtained from similar experiments with C65A and C68A sStoA, respectively. Solid lines represent fits of the data to supplemental Equation S3.

Mentions: The acid-base properties of the active site cysteines of wild-type sStoA and the C65A and C68A variants were investigated by measuring rates of reaction with the fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene as described under “Experimental Procedures” (see Fig. 8A). The fluorescence is sensitive to the environment of the modified cysteine with emission occurring in the range of ∼440-550 nm, depending on the solvent exposure of the modified cysteine (34). Here the emission maxima for Cys-65 and Cys-68 were 510 nm, indicating that the fluorescent group of both modified residues is located in a relatively solvent-exposed position. For the single cysteine variants of sStoA, data fitted well to an equation describing a single protonation/deprotonation event, giving pKa values of 7.0 ± 0.1 and 7.1 ± 0.1 for Cys-65 and Cys-68, respectively (see Fig. 8, C and D). For the wild-type protein in which both cysteine residues are intact, the data fitted well to two independent protonation/deprotonation events, giving pKa values of 5.5 ± 0.4 and 7.8 ± 0.2 (Fig. 8B). We tentatively ascribe the first transition to Cys-65 and the latter to Cys-68. Both of these values are lower than the typical value of ∼8.5-9.0 observed for cysteine, and the data are consistent with the reactivity of both residues toward alkylating reagents (which react with the deprotonated form only). A low pKa value is normally observed for the N-terminal active site cysteine of thioredoxin-like proteins, which in some cases exhibit pKa values as low as 3.5 (35). However, the pKa of the second cysteine is normally estimated to be >9 (32, 36, 37). In this respect, StoA is similar to ResA in that the C-terminal cysteinyl has a pKa value low enough to be measurable in the stable pH range of the protein.


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)

pKa plots for wild-type and single cysteine sStoA variants. A, time-dependent increases in fluorescence at 510 nm upon reaction of wild-type (wt) sStoA (1 μm) with 6-bromoacetyl-2-dimethylaminonaphthalene (15 μm) in PCTC buffer system at pH values from 5 to 9 as indicated at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant, ko. B, plot of ko as a function of pH for wild-type sStoA. The solid line shows a fit to supplemental Equation S4. C and D, plots of ko as a function of pH obtained from similar experiments with C65A and C68A sStoA, respectively. Solid lines represent fits of the data to supplemental Equation S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig8: pKa plots for wild-type and single cysteine sStoA variants. A, time-dependent increases in fluorescence at 510 nm upon reaction of wild-type (wt) sStoA (1 μm) with 6-bromoacetyl-2-dimethylaminonaphthalene (15 μm) in PCTC buffer system at pH values from 5 to 9 as indicated at 25 °C. Plots were fitted (solid lines) to obtain an observed, pseudo-first order rate constant, ko. B, plot of ko as a function of pH for wild-type sStoA. The solid line shows a fit to supplemental Equation S4. C and D, plots of ko as a function of pH obtained from similar experiments with C65A and C68A sStoA, respectively. Solid lines represent fits of the data to supplemental Equation S3.
Mentions: The acid-base properties of the active site cysteines of wild-type sStoA and the C65A and C68A variants were investigated by measuring rates of reaction with the fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene as described under “Experimental Procedures” (see Fig. 8A). The fluorescence is sensitive to the environment of the modified cysteine with emission occurring in the range of ∼440-550 nm, depending on the solvent exposure of the modified cysteine (34). Here the emission maxima for Cys-65 and Cys-68 were 510 nm, indicating that the fluorescent group of both modified residues is located in a relatively solvent-exposed position. For the single cysteine variants of sStoA, data fitted well to an equation describing a single protonation/deprotonation event, giving pKa values of 7.0 ± 0.1 and 7.1 ± 0.1 for Cys-65 and Cys-68, respectively (see Fig. 8, C and D). For the wild-type protein in which both cysteine residues are intact, the data fitted well to two independent protonation/deprotonation events, giving pKa values of 5.5 ± 0.4 and 7.8 ± 0.2 (Fig. 8B). We tentatively ascribe the first transition to Cys-65 and the latter to Cys-68. Both of these values are lower than the typical value of ∼8.5-9.0 observed for cysteine, and the data are consistent with the reactivity of both residues toward alkylating reagents (which react with the deprotonated form only). A low pKa value is normally observed for the N-terminal active site cysteine of thioredoxin-like proteins, which in some cases exhibit pKa values as low as 3.5 (35). However, the pKa of the second cysteine is normally estimated to be >9 (32, 36, 37). In this respect, StoA is similar to ResA in that the C-terminal cysteinyl has a pKa value low enough to be measurable in the stable pH range of the protein.

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
Related in: MedlinePlus