Structural Basis of Disulfide Bond Formation in the Bacterial Periplasm and Mammalian ER


Cellular quality control systems rely on the maintenance of protein and redox homoeostasis, in which the formation, reduction and isomerisation of protein disulfide bonds play important roles. The biological kingdoms have evolved catalytic systems that greatly promote oxidative protein folding. Remarkable progress in structural biology in this field has provided molecular views on how protein disulfide bonds are generated de novo and transferred to downstream folding substrates. Very recently, a novel concept has emerged, proposing that multiple oxidative pathways work redundantly, distinctly and in a complementary manner in the endoplasmic reticulum of mammalian cells to efficiently produce large quantities, and a wide variety, of secretory proteins. Thus, the oxidative protein folding network is even more complicated in higher eukaryotes than previously thought.

Key Concepts:

  • Almost all organisms, from bacteria to humans, possess catalytic systems that promote protein‐disulfide bond formation.

  • Disulfide bond formation networks have diversified dramatically, as evidenced by the appearance of a large number of disulfide oxidoreductases in higher eukaryotes.

  • High‐resolution structures have been solved for an increasing number of disulfide oxidoreductases, thereby revealing the structural and mechanistic basis of cellular disulfide bond formation systems.

  • Disulfide bonds play an important role in folding, assembly and stabilisation of various proteins.

  • Disulfide bond formation networks are closely related to the protein and redox homeostasis in the cell.

Keywords: disulfide bond; crystal structure; DsbA ; DsbB ; PDI ; Ero1; Prx4; GPx; VKOR ; QSOX

Figure 1.

Crystal structures and mechanisms of action of E. coli DsbA and DsbB. (a) Ribbon diagram of E. coli DsbA (PDB ID: 1A2M). Cyan and magenta indicate the α‐helix and β‐strands, respectively. The Cys30–Cys33 redox‐active site is represented by sticks. (b) Ribbon diagram of E. coli DsbB (PDB ID: 2ZUQ). The Cys41, Cys44 and Cys104–Cys130 redox‐active sites and bound ubiquinone are represented by sticks. TM, transmembrane helix. (c) Chemical scheme of cooperative de novo disulfide bond generation by DsbB and quinone in E. coli. (d) Molecular mechanisms that prevent backward electron transfer from DsbB to DsbA. TM, transmembrane helix.

Figure 2.

Domain organisation and crystal structures of human PDI and other representative PDI‐family proteins. (a) Domain organisation of PDI, ERp57, ERp72, ERp44, ERp46, P5 and ERdj5 based on their amino acid sequences. Redox‐active and redox‐inactive thioredoxin domains are indicated by white and grey boxes, respectively. Active‐site sequences are indicated in the redox‐active domains. (b) Ribbon diagrams of oxidised (blue; PDB ID: 4EL1) and reduced (magenta; PDB ID: 4EKZ) forms of human PDI. The two structures are superimposed in the panel on the right, such that the root mean square distance of Cα atoms in their abb′ segments is minimised. The region involved in the redox‐dependent rearrangement of a′ is shown in inset. (c) Ribbon diagrams of ERp57 (PDB ID: 3F8U) (gold), ERp72 a0–a (PDB ID: 3IDV) and b–b′ (PDB ID: 3EC3) segments (green), ERp44 (PDB ID: 2R2J) (grey) and ERdj5 (PDB ID: 3APO) (teal). The side chains of Lys214, Lys274 and Arg282 in ERp57 are represented by sticks.

Figure 3.

Crystal structures of human Ero1α and Prx4. (a) Ribbon diagram of the active form of human Ero1α (PDB ID: 3AHQ). The regulatory loop, which could not be modelled due to the lack of information about electron density, is represented by a dotted line. Four regulatory cysteines (Cys94, Cys99, Cys104 and Cys131) converge in this loop. The FAD molecule is represented by sticks. The four helices that constitute the FAD‐embracing bundle are shown in green. This cofactor‐embracing architecture is conserved in bacterial DsbB and VKOR (see Figure b and Figure a). (b) Superimposition of oxidised (yellow, PDB ID: 3TJB) and reduced (cyan, PDB ID: 3TJF) human Prx4 (C51A) decamer structures. (c) Comparison of oxidised (left) and reduced (right) Prx4 dimer structures. Catalytic cysteines are indicated by yellow spheres. The C‐terminal region of oxidised Prx4, which could not be modelled due to a lack of information about electron density, is indicated by dotted line. Exposure of the active‐site disulfide in the oxidised form allows access (red circle) of the thioredoxin domains (Trxs) of PDIs; access is blocked (red ×) in reduced form.

Figure 4.

Overall structures of human GPx8, bacterial VKOR and trypanosomal QSOX. (a) Ribbon diagram of bacterial homologue of VKOR. The crystal structure of Synechococcus sp. VKOR (PDB ID: 3KPQ) is shown. The quinone molecule is represented by sticks. The quinone‐embracing four‐helix bundle is shown in green. Whereas bacterial VKOR is naturally fused to a thioredoxin‐like domain, human VKOR lacks this domain. (b) Crystal structure of the luminal domain of GPx8 (PDB ID: 3KIJ). The structure shows an extended thioredoxin fold. The active‐site cysteine (Cys79) is indicated by yellow spheres. (c) Crystal structure of Trypanosoma bruceiQSOX. The FAD molecule is represented by sticks. (Left) Crystal structure of wild‐type QSOX (PDB ID: 3QCP). Trypanosomal QSOX consists of Trx1 (green), a long helix (dark grey), Erv‐like domain (grey) and Erv domain (salmon). The Trx1 active site is 42 Å from the Erv active site (indicated by dashed line). (Right) Crystal structure of QSOX with the C72S/C353S mutation (PDB ID: 3QD9). In this mutant, the active sites in Trx1 and Erv were mutated to CXXC. The structure mimics an intermediate of the intramolecular disulfide transfer step. Compared with wild‐type QSOX, Trx1 is rotated, allowing two redox‐active sites to form an intramolecular disulfide bond. (Mammalian QSOX has a redox‐inactive Trx2 instead of a long helix.) Trx, thioredoxin domain.



Alon A , Grossman I , Gat Y et al. (2012) The dynamic disulphide relay of quiescin sulphydryl oxidase. Nature 488(7411): 414–418.

Anelli T , Alessio M , Bachi A et al. (2003) Thiol‐mediated protein retention in the endoplasmic reticulum: the role of ERp44. EMBO Journal 22(19): 5015–5022.

Anfinsen CB , Haber E , Sela M and White FH Jr (1961) The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proceedings of the National Academy of Sciences of the USA 47: 1309–1314.

Appenzeller‐Herzog C , Riemer J , Christensen B , Sorensen ES and Ellgaard L (2008) A novel disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells. EMBO Journal 27(22): 2977–2987.

Bader M , Muse W , Ballou DP , Gassner C and Bardwell JC (1999) Oxidative protein folding is driven by the electron transport system. Cell 98(2): 217–227.

Baker KM , Chakravarthi S , Langton KP et al. (2008) Low reduction potential of Ero1alpha regulatory disulphides ensures tight control of substrate oxidation. EMBO Journal 27(22): 2988–2997.

Cao Z , Tavender TJ , Roszak AW , Cogdell RJ and Bulleid NJ (2011) Crystal structure of reduced and of oxidized peroxiredoxin IV enzyme reveals a stable oxidized decamer and a non‐disulfide‐bonded intermediate in the catalytic cycle. Journal of Biological Chemistry 286(49): 42257–42266.

Chakravarthi S , Jessop CE , Willer M , Stirling CJ and Bulleid NJ (2007) Intracellular catalysis of disulfide bond formation by the human sulfhydryl oxidase, QSOX1. Biochemical Journal 404(3): 403–411.

Dias‐Gunasekara S , Gubbens J , van Lith M et al. (2005) Tissue‐specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1beta. Journal of Biological Chemistry 280(38): 33066–33075.

Dong G , Wearsch PA , Peaper DR , Cresswell P and Reinisch KM (2009) Insights into MHC class I peptide loading from the structure of the tapasin‐ERp57 thiol oxidoreductase heterodimer. Immunity 30(1): 21–32.

Frickel EM , Riek R , Jelesarov I et al. (2002) TROSY‐NMR reveals interaction between ERp57 and the tip of the calreticulin P‐domain. Proceedings of the National Academy of Sciences of the USA 99(4): 1954–1959.

Gross E , Kastner DB , Kaiser CA and Fass D (2004) Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell. Cell 117(5): 601–610.

Gross E , Sevier CS , Heldman N et al. (2006) Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p. Proceedings of the National Academy of Sciences of the USA 103(2): 299–304.

Hagiwara M , Maegawa K , Suzuki M et al. (2011) Structural basis of an ERAD pathway mediated by the ER‐resident protein disulfide reductase ERdj5. Molecular Cell 41(4): 432–444.

Inaba K , Masui S , Iida H et al. (2010) Crystal structures of human Ero1alpha reveal the mechanisms of regulated and targeted oxidation of PDI. EMBO Journal 29(19): 3330–3343.

Inaba K , Murakami S , Nakagawa A et al. (2009) Dynamic nature of disulphide bond formation catalysts revealed by crystal structures of DsbB. EMBO Journal 28(6): 779–791.

Inaba K , Murakami S , Suzuki M et al. (2006a) Crystal structure of the DsbB–DsbA complex reveals a mechanism of disulfide bond generation. Cell 127(4): 789–801.

Inaba K , Takahashi YH , Fujieda N et al. (2004) DsbB elicits a red‐shift of bound ubiquinone during the catalysis of DsbA oxidation. Journal of Biological Chemistry 279(8): 6761–6768.

Inaba K , Takahashi YH and Ito K (2005) Reactivities of quinone‐free DsbB from Escherichia coli . Journal of Biological Chemistry 280(38): 33035–33044.

Inaba K , Takahashi YH , Ito K and Hayashi S (2006b) Critical role of a thiolate‐quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB. Proceedings of the National Academy of Sciences of the USA 103(2): 287–292.

Jin DY , Tie JK and Stafford DW (2007) The conversion of vitamin K epoxide to vitamin K quinone and vitamin K quinone to vitamin K hydroquinone uses the same active site cysteines. Biochemistry 46(24): 7279–7283.

Kadokura H and Beckwith J (2002) Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA. EMBO Journal 21(10): 2354–2363.

Klappa P , Ruddock LW , Darby NJ and Freedman RB (1998) The b′ domain provides the principal peptide‐binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. EMBO Journal 17(4): 927–935.

Kobayashi T , Kishigami S , Sone M et al. (1997) Respiratory chain is required to maintain oxidized states of the DsbA–DsbB disulfide bond formation system in aerobically growing Escherichia coli cells. Proceedings of the National Academy of Sciences of the USA 94(22): 11857–11862.

Kozlov G , Azeroual S , Rosenauer A et al. (2010) Structure of the catalytic a(0)a fragment of the protein disulfide isomerase ERp72. Journal of Molecular Biology 401(4): 618–625.

Kozlov G , Maattanen P , Schrag JD et al. (2006) Crystal structure of the bb′ domains of the protein disulfide isomerase ERp57. Structure 14(8): 1331–1339.

Kozlov G , Maattanen P , Schrag JD et al. (2009) Structure of the noncatalytic domains and global fold of the protein disulfide isomerase ERp72. Structure 17(5): 651–659.

Li W , Schulman S , Dutton RJ et al. (2010) Structure of a bacterial homologue of vitamin K epoxide reductase. Nature 463(7280): 507–512.

Martin JL , Bardwell JC and Kuriyan J (1993) Crystal structure of the DsbA protein required for disulphide bond formation in vivo . Nature 365(6445): 464–468.

Masui S , Vavassori S , Fagioli C , Sitia R and Inaba K (2011) Molecular bases of cyclic and specific disulfide interchange between human ERO1alpha protein and protein‐disulfide isomerase (PDI). Journal of Biological Chemistry 286(18): 16261–16271.

Meunier L , Usherwood YK , Chung KT and Hendershot LM (2002) A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins. Molecular Biology of the Cell 13(12): 4456–4469.

Nelson JW and Creighton TE (1994) Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo . Biochemistry 33(19): 5974–5983.

Nguyen VD , Saaranen MJ , Karala AR et al. (2011) Two endoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation. Journal of Molecular Biology 406(3): 503–515.

Oliver JD , Roderick HL , Llewllyn DH and High S (1999) ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin. Molecular Biology of the Cell 10(8): 2573–2582.

Pagani M , Fabbri M , Benedetti C et al. (2000) Endoplasmic reticulum oxidoreductin 1‐lbeta (ERO1‐Lbeta), a human gene induced in the course of the unfolded protein response. Journal of Biological Chemistry 275(31): 23685–23692.

Rutkevich LA and Williams DB (2012) Vitamin K epoxide reductase contributes to protein disulfide formation and redox homeostasis within the endoplasmic reticulum. Molecular Biology of the Cell 23(11): 2017–2027.

Schulman S , Wang B , Li W and Rapoport TA (2010) Vitamin K epoxide reductase prefers ER membrane‐anchored thioredoxin‐like redox partners. Proceedings of the National Academy of Sciences of the USA 107(34): 15027–15032.

Solda T , Garbi N , Hammerling GJ and Molinari M (2006) Consequences of ERp57 deletion on oxidative folding of obligate and facultative clients of the calnexin cycle. Journal of Biological Chemistry 281(10): 6219–6226.

Tavender TJ and Bulleid NJ (2010) Peroxiredoxin IV protects cells from oxidative stress by removing H2O2 produced during disulphide formation. Journal of Cell Science 123(Pt 15): 2672–2679.

Tavender TJ , Springate JJ and Bulleid NJ (2010) Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum. EMBO Journal 29(24): 4185–4197.

Tian G , Kober FX , Lewandrowski U et al. (2008) The catalytic activity of protein‐disulfide isomerase requires a conformationally flexible molecule. Journal of Biological Chemistry 283(48): 33630–33640.

Tian G , Xiang S , Noiva R , Lennarz WJ and Schindelin H (2006) The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. (erratum appears in (2006) Cell. 124(5): 1085–1088). Cell 124(1): 61–73.

Ushioda R , Hoseki J , Araki K et al. (2008) ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER. Science 321(5888): 569–572.

Wang C , Li W , Ren J et al. (in press) Structural insights into the redox‐regulated dynamic conformations of human protein disulfide isomerase. Antioxidants and Redox Signaling.

Wang L , Vavassori S , Li S et al. (2008) Crystal structure of human ERp44 shows a dynamic functional modulation by its carboxy‐terminal tail. EMBO Reports 9(7): 642–647.

Wang X , Wang L , Sun F and Wang CC (2012) Structural insights into the peroxidase activity and inactivation of human peroxiredoxin 4. Biochemical Journal 441(1): 113–118.

Wunderlich M and Glockshuber R (1993) Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli . Protein Science 2(5): 717–726.

Zhou Y , Cierpicki T , Jimenez RH et al. (2008) NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB‐catalyzed disulfide bond formation. Molecular Cell 31(6): 896–908.

Zito E , Hansen HG , Yeo GS , Fujii J and Ron D (2012) Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice. Molecular Cell 48(1): 39–51.

Zito E , Melo EP , Yang Y et al. (2010) Oxidative protein folding by an endoplasmic reticulum‐localized peroxiredoxin. Molecular Cell 40(5): 787–797.

Further Reading

Anelli T and Sitia R (2008) Protein quality control in the early secretory pathway. EMBO Journal 27: 315–327.

Araki K and Inaba K (2012) Structure, mechanism and evolution of Ero1 family enzymes. Antioxidants and Redox Signaling 16(8): 790–799.

Bulleid NJ and Ellgaard L (2011) Multiple ways to make disulfides. Trends in Biochemical Sciences 36(9): 485–492.

Fass D (2012) Disulfide bonding in protein biophysics. Annual Review of Biophysics 41: 63–79.

Gruber CW , Cemazar M , Heras B , Martin J and Craik DJ (2006) Protein disulfide isomerase: the structure of oxidative folding. Trends in Biochemical Sciences 31: 455–464.

Hatahet F , Ruddock LW , Ahn K et al. (2009) Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation. Antioxidants and Redox Signaling 11(11): 2807–2850.

Hoseki J , Ushioda R and Nagata K (2009) Mechanism and components of endoplasmic reticulum‐associated degradation. Journal of Biochemistry 147(1): 19–25.

Inaba K (2009) Disulfide bond formation system in Escherichia coli . Journal of Biochemistry 146(5): 591–597.

Sato Y and Inaba K (2012) Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals. FEBS Journal 279: 2262–2271.

Sevier CS and Kaiser CA (2002) Formation and transfer of disulphide bonds in living cells. Nature Reviews Molecular Cell Biology 3(11): 836–847.

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Kojima, Rieko, Okumura, Masaki, and Inaba, Kenji(Sep 2013) Structural Basis of Disulfide Bond Formation in the Bacterial Periplasm and Mammalian ER . In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0024169]