5.A.1 The Disufide Bond Oxidoreductase D (DsbD) Family
The best characterized member of the DsbD family is DsbD of E. coli (Katzen and Beckwith, 2000; Krupp et al., 2001). The DsbD protein is membrane-embedded with a putative N-terminal TMS plus 8 additional TMSs. The smallest homologues (190 aas with 6 putative TMSs) are found in archaea while the largest are found in both Gram-negative bacteria (758 aas with 9 putative TMSs) and Gram-positive bacteria (695 aas with 6 putative TMSs). In the E. coli DsbD system, electrons are transferred from NADPH in the cytoplasm to periplasmic dithiol/disulfide-containing proteins via an electron transfer chain that sequentially involves NADPH, thioredoxin reductase (TrxB), present in the cytoplasm, thioredoxin (TrxA), also in the cytoplasm, DsbD, the integral membrane constituent of the system, and the periplasmic electron acceptors, DsbC, DsbE (CcmG) and DsbG. All of these last three proteins can donate electrons to oxidized disulfide-containing proteins in the periplasm of a Gram-negative bacterium or presumably in the external milieu of a Gram-positive bacterium or an archaeon. Thus, the pathway is:
NADPH → TrxB → TrxA → DsbD → DsbC, DsbE, or DsbG → proteins.
Homologues include: (1) several thiol-disufide exchange proteins, (2) the cytochrome c-type biogenesis proteins, CcdA of Paracoccus pantotrophus and Bacillus subtilis (Bardischewsky and Friedrich, 2001; Le Brun et al., 2000), (3) the methylamine utilization proteins, MauF of Paracoccus denitrificans and P. versutus (Chistoserdov et al., 1992; Van Spanning et al., 1994), (4) the mercury resistance proteins (possibly Hg2+ transporters) of Mycobacterium tuberculosis and Streptomyces lividans (Brunker et al., 1996; Sedlmeier and Altenbuchner, 1992), (5) suppressors of copper sensitivity (copper tolerance proteins) of Salmonella typhimurium and Vibrio cholerae (Choudhury and Kumar, 1996; Gupta et al., 1997), (6) coomponents of peroxide reduction pathways, and (7) components of sulfenic acid reductases.
DsbD contains three domains, each containing two reactive cysteines. One membrane-embedded domain, DsbDγ, transfers electrons from thioredoxin to the carboxy-terminal thioredoxin-like periplasmic domain DsbDγ. Alanines were substituted for each of 19 conserved amino acid residues. 11 mutants caused defects in DsbC reduction. To analyze the redox state of each DsbD domain, a thrombin-cleavable DsbD (DsbDTH) was constructed from which all three domains as separate polypeptide chains were generated. Mutants with strong defects included one mutant class that could not receive electrons from cytoplasmic thioredoxin, resulting in a DsbD that has all six of its cysteines disulfide bonded. One mutant class could not transfer electrons from DsbDβ to DsbDγ (Cho and Beckwith, 2006).
DsbD contains three cysteine pairs that undergo reversible disulfide rearrangements (Krupps et al., 2001). TrxA donates electrons to the transmembrane cysteines C163 (C3) and C285 (C5) in putative TMSs 1 and 4 in the DsbD model proposed by Katzen and Beckwith (2000). This dithiol then donates electrons to the periplasmic C-terminal thioredoxin motif (CXXC) of DsbD, thereby reducing C461 and C464 (C6 and C7, respectively). This dithiol pair attacks the periplasmic N-terminal disulfide bridge at C103 and C109 (C1 and C2, respectively) which transfers electrons to DsbC and other protein electron acceptors as noted above.
DsbD catalyses an essentially irreversible reaction due to the fact that electrons flow down their electrochemical gradient from inside the cell (negative inside) to outside the cell (positive outside). In order to reverse the reaction, electrons are transferred from dithiol proteins in the periplasm to an electron acceptor in the cytoplasm as follows:
reduced proteinperiplasm → DsbAperiplasm → DsbBmembrane → quinonesmembrane → reductasemembrane
→ terminal electron acceptorcytoplasm (e.g., O2, NO3- or fumarate).
DsbB contains 4 essential cysteine residues, reversibly forming two disulfide bonds. Although DsbA displays no proofreading activity for repair of wrongly paired disulfides, this activity is displayed by DsbC, DsbE and DsbG (Krupp et al., 2001). Therefore, the two transmembrane pathways involving DsbD and DsbB together catalyze extracellular disulfide reduction (DsbD) and oxidation (DsbB) in a superficially reversible process that allows dithiol/disulfide exchange.
A class of DsbD proteins, named ScsB, are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, Cho et al. 2012 searched for the substrates of ScsB. ScsB provides electrons to a peroxide reduction pathway in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Additional proteins that depend on ScsB for reduction include a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC. Thus, the array of proteins involved in reductive pathways in the oxidative cell envelope is broad.
The best-known TM reductase is E. coli DsbD, which has a TM domain (DsbDβ) and two periplasmic domains (DsbDα and DsbDγ), each containing a redox-active cysteine pair. Electrons are transferred from cytoplasmic thioredoxin A (TrxA) to DsbDβ, relayed across the membrane and through its periplasmic domains, and transferred to various periplasmic targets to maintain their reductive activity. Redox activities (and their associated proteins) include protein disulfide isomerization (DsbC), cytochrome c maturation (CcmG) and defense against oxidative stress (DsbG) (Williamson et al. 2015).
CcdA is a minimal homolog of DsbDβ that has six TMSs instead of eight as in DsbDβ and can also reduce CcmG. Another DsbD homolog, ScsB, found in Salmonella typhimurium, also lacks the last two TMSs but maintains similar periplasmic domains and functionality, thus suggesting that the last two TMSs are dispensable. Separate DsbDα homologs exist in many bacteria with CcdA only. It has been proposed that CcdA has evolved into DsbD through fusion of the two periplasmic proteins to the TM domain, thus allowing greater electron-transfer efficiency (Williamson et al. 2015).
The transporter-like architecture of DsbDβ has been suggested from functional mutagenesis and biochemical studies probing solvent accessibility. The two functional cysteines of DsbDβ are on predicted TMSs 1 and 4. The sequence surrounding the cysteines is highly conserved, particularly the proline residues flanking each cysteine. Each proline in the PCX(2-3)P active sites is important for cysteine accessibility and redox activity. DsbDβ has an inverted pseudosymmetry, thus allowing access to the functional cysteines from both sides of the membrane, which allows the two cysteines to cycle through oxidized and reduced states and thus pass reducing power into the periplasm (Williamson et al. 2015).
The overall vectorial electron transfer reaction catalyzed by DsbD is:
2 e-cytoplasm → 2 e-periplasm
Disulfide bond oxidoreductase-D, DsbD. of 565 aas and 9 TMSs. DsbD provides reducing equivalents to a large array of periplasmic redox proteins. These proteins use the reducing power received from DsbD to correct non-native disulfides, mature c-type cytochromes, protect cysteines on secreted proteins from irreversible oxidation, reduce methionine sulfoxides, and scavenge reactive oxygen species such as hydrogen peroxide (Cho and Collet 2013). DsbD x-ray structures are known, revealing its unusual redox properties and extreme rigidity (Stirnimann et al. 2006).
Bacteria and Archaea
DsbD of E.coli
CcdA of 222 aas and 6 TMSs. The NMR structure in an oxidized and outward-facing state has been determined. CcdA consists of two inverted structural repeats of three transmembrane helices (2 x 3-TMSs). Zhou and Bushweller 2018 computationally modeled and experimentally validated an inward-facing state, which suggests that CcdA uses an elevator-type movement to shuttle the reactive cysteines across the membrane. Its structure may be relevant to other LysE superfamily transporters. Structure comparisons of CcdA, semiSWEET, Pnu, and major facilitator superfamily (MFS) transporters provide insights into membrane transporter architecture and mechanism (Zhou and Bushweller 2018).
CcdA of Thermus thermophilus
Cytochrome c biogenesis protein, CcdA
Cyt c biogenesis protein of Verrucosispora maris (F4FC13)
Cytochrome c biogenesis protein, CcdA
CcdA of Methanosarcina mazei (Q8PY72)
Cytochrome c biogenesis protein, CcdA
CcdA of Chlamydomonas reinhardtii (Q8S3X4)
Cytochrome c-type biogenesis CcdA-like chloroplastic protein (Cytochrome b6f biogenesis protein CCDA)
Cytochrome c biogenesis protein of 242 aas and 7 putative TMSs.
Cytochrome c biogenesis protein of Burkholderia sp. BT03
Transmembrane electron carrier, SoxV (CcdA-like protein), transferring electrons from the cytoplasm to SoxW, a perimplsmic thioredoxin; involved in thoisulfate oxidation (Appia-Ayme and Berks 2002).
SoxV of Rhodovulum sulfidophilum
Cytochrome c-type biogenesis protein (CcdA) of 190 aas and 6 TMSs. The NMR structure of a reduced-state mimic of CcdA that transfers electrons across the inner membrane has been determined (Williamson et al. 2015). The two cysteine positions in CcdA are separated by 20 Å. Whereas one is accessible to the cytoplasm, the other resides in the protein core, thus implying that conformational exchange is required for periplasmic accessibility, confirmed in vitro. The existence of multiple conformational states was demonstrated, suggesting a four-state model for relaying electrons from cytosolic to periplasmic redox substrates (Williamson et al. 2015).
CcdA of Archaeoglobus fulgidus
CcdA family member, the thiol:disulfide excange protein, DipZ of 695 aas and 7 TMSs. The 1.9Å resolution x-ray structure of the C-terminal ectodomain of Rv2874 revealed the predicted thioredoxin-like domain with its conserved Cys-X-X-Cys active-site motif, but this domain is combined with a second domain with a carbohydrate-binding module (CBM) fold (Goldstone et al. 2016). A cavity in the CBM adjacent to the thioredoxin active site suggested the presence of a carbohydrate-binding site. Possibly, this allows an expansion of the thioredoxin-domain functionality to carbohydrate modification.
DipZ of Mycobacterium tuberculosis
Suppressor of copper-sensitivity B, ScsB (Gupta et al. 1997). Part of a transmembrane peroxide reductase complex (Cho et al. 2012).
Bacteria and Archaea
ScsB of Salmonella typhimurium
Peroxide reductase complex component, ScsB (Cho et al. 2012).
ScsB opf Caulobacter crescentus
ScsB homologue of Parachlamydia acanthamoebae
Heavy metal transport detoxicification protein (499aas; 7TMSs). Has an N-terminal heavy metal binding domain ( 70 aas) resembling MerP (1.A.72.3) and the metal binding N-terminal domains of family 5 & 6-type P-ATPases (3.A.3.5 and 3.A.3.6), a central DsbD domain and a C-terminal COG4633 domain (Gupta et al., 1997). COG4633 may also contain a metal ion binding domain homologous to but more distant in sequence from those in copper-ATPases.
CycZ of Clostridium thermocellum (A3DGJ1)
Dsb2 of 228 aas
Dsb2 of Leptospira meyeri
Uncharacterized protein of 264 aas and 6 TMSs.
UP of Haloferax gibbonsii
Ferric reductase of 410 aas and 11 putative TMSs with a C-terminal YedZ domain (TC#9.B.43).
Ferric reductase of Anabaena cylindrica
Uncharacterized protein of 221 aas and 6 TMSs
UP of Bdellovibrio exovorus