Structure and functional properties of Bacillus subtilis endospore biogenesis factor StoA.
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.
Crow
Allister
A
Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Liu
Yiming
Y
Möller
Mirja Carlsson
MC
Le Brun
Nick E
NE
Hederstedt
Lars
L
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001703:Biophysics / Q000379:methods; D020134:Catalytic Domain; D045304:Cytochromes c / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013171:Spores, Bacterial / Q000737:chemistry*; D013379:Substrate Specificity
D008565:Membrane Proteins; D018698:Glutamic Acid; D045304:Cytochromes c