Crystal Structure and Biophysical Properties of Bacillus subtilis BdbD: AN OXIDIZING THIOL:DISULFIDE OXIDOREDUCTASE CONTAINING A NOVEL METAL SITE*

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 Ca²⁺, at an inter-domain position some 14 Å 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 Ca²⁺-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca²⁺-depleted BdbD was significantly less stable than reduced Ca²⁺-containing protein, and the midpoint reduction potential was shifted by approximately −20 mV, suggesting that Ca²⁺ 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.Disulfide bonds, formed upon oxidation of two cysteine residue side chain thiols, are key for the stability and/or function of many secreted and membrane-bound peptides and proteins in bacteria, and the failure to insert these correctly has wide ranging effects (1–4). To regulate the redox state of cysteine residues on the outside of the cytoplasmic membrane, intricate disulfide bond regulatory systems have evolved. These involve enzymes of the thiol:disulfide oxidoreductase (TDOR)³ family, which contain cysteine residues often arranged in a Cys-Xaa-Xaa-Cys thioredoxin motif (5, 6). These enzymes function in pathways that lead to the formation of disulfide bonds, rearrangement of incorrectly positioned disulfide bonds, or the removal of unwanted disulfide bonds, and the redox properties of the enzymes appear to correlate closely with function.The paradigm system for disulfide bond formation is the DsbA-DsbB system of Escherichia coli, which has been characterized in great detail (7–11). DsbA is a soluble periplasmic TDOR, which has a thioredoxin-like fold with an additional helical domain (8). The protein oxidizes the di-thiol motifs of a range of substrates, generating in each a disulfide bond. Reduced DsbA is rapidly re-oxidized by DsbB, a membrane-bound TDOR (12) that channels the resulting electrons into the membrane quinol pool (13, 14). Dsb-like homologues appear to be extremely widespread in Gram-negative bacteria.Gram-positive bacteria, unlike Gram-negatives, do not have an outer membrane and so have no spatially defined periplasmic compartment. This places different functional and structural demands on extra-cytoplasmic proteins, and one consequence of this is that extra-cytoplasmic TDORs are generally membrane-anchored in Gram-positive bacteria. Systems for the introduction of disulfide bonds appear to be variable in these organisms (15, 16). As an example, Mycobacterium tuberculosis does not contain close homologues of DsbA/B but contains another thioredoxin-like TDOR, DsbE, which has been shown to have redox properties similar to those of DsbA (17). Some Gram-positive bacteria, including Staphylococcus aureus, contain a DsbA homologue but no homologue of DsbB (18). The recent structural and biochemical characterization of S. aureus DsbA revealed major similarities with the E. coli protein but was also consistent with a distinct mechanism of re-oxidation (16).Some Gram-positive bacteria, however, contain clear homologues of both DsbA and DsbB. In the model organism Bacillus subtilis, BdbD and BdbC/BdbB have been identified as homologues of DsbA and DsbB, respectively, and demonstrated to be involved in processes such as natural competence development, which requires the insertion of disulfide bonds (19). BdbD and BdbC are also involved in a number of other pathways that do not require the insertion of a disulfide bond; B. subtilis contains several extra-cytoplasmic TDORs, for example ResA and StoA (required for cytochrome c maturation and endospore biogenesis, respectively), which function to specifically reduce disulfide bonds introduced by BdbD (20, 21). To understand disulfide bond management systems in Gram-positive bacteria, detailed information on each of the different systems found is required.Here we present the crystal structure of the catalytic domain of B. subtilis BdbD in both reduced and oxidized states. The structure is broadly similar to that of E. coli DsbA, but it also reveals the presence of a novel Ca²⁺-binding site remote from the CXXC active site. Using two-dimensional NMR methods and fluorescence kinetic studies of thiolate alkylation, we report the reduction potential and pK_a properties of the soluble protein, which are entirely consistent with an oxidizing function for the protein in vivo. Conformational stability studies and NMR studies showed that the occupancy of the metal site by Ca²⁺ ion is not required for folding/stability but leads to a significant increase of the midpoint reduction potential. The possibility that the principal function of the metal site is to boost the oxidizing power of the protein is discussed.