Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding

Article metrics


Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)—the most versatile disulfide-introducing enzyme in the endoplasmic reticulum—during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Redox-dependent regulation of PDI dynamics.
Fig. 2: Significance of redox-dependent regulation of PDI dynamics in catalysis of oxidative protein folding.
Fig. 3: Unfolded substrate-induced dimerization of oxidized PDI.
Fig. 4: Structural diversity of substrate-induced PDI dimers.
Fig. 5: Physiological significance of PDI dimers in oxidative folding of BPTI.
Fig. 6: PDI dimerization induced by unfolded RNase A.

Data availability

None of the data in this paper have been deposited in public databases. All data in this study are available upon reasonable request.


  1. 1.

    Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

  2. 2.

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

  3. 3.

    Bulleid, N. J. & Ellgaard, L. Multiple ways to make disulfides. Trends Biochem. Sci. 36, 485–492 (2011).

  4. 4.

    Chen, Y. et al. SPD—a web-based secreted protein database. Nucleic Acids Res. 33, D169–D173 (2005).

  5. 5.

    Arolas, J. L., Aviles, F. X., Chang, J. Y. & Ventura, S. Folding of small disulfide-rich proteins: clarifying the puzzle. Trends Biochem. Sci. 31, 292–301 (2006).

  6. 6.

    Okumura, M., Shimamoto, S. & Hidaka, Y. Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation. Curr. Protoc. Protein Sci. 76, 7.1–7.13 (2014).

  7. 7.

    Weissman, J. S. & Kim, P. S. Reexamination of the folding of BPTI: predominance of native intermediates. Science 253, 1386–1393 (1991).

  8. 8.

    Uehara, T. et al. S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 441, 513–517 (2006).

  9. 9.

    Hoffstrom, B. G. et al. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nat. Chem. Biol. 6, 900–906 (2010).

  10. 10.

    Woehlbier, U. et al. ALS-linked protein disulfide isomerase variants cause motor dysfunction. EMBO J. 35, 845–865 (2016).

  11. 11.

    Okumura, M., Kadokura, H. & Inaba, K. Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum. Free Radic. Biol. Med. 83, 314–322 (2015).

  12. 12.

    Hatahet, F. & Ruddock, L. W. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation. Antioxid. Redox Signal. 11, 2807–2850 (2009).

  13. 13.

    Lyles, M. M. & Gilbert, H. F. Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry 30, 613–619 (1991).

  14. 14.

    van den Berg, B., Chung, E. W., Robinson, C. V., Mateo, P. L. & Dobson, C. M. The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase. EMBO J. 18, 4794–4803 (1999).

  15. 15.

    Weissman, J. S. & Kim, P. S. Efficient catalysis of disulphide bond rearrangements by protein disulphide isomerase. Nature 365, 185–188 (1993).

  16. 16.

    Kojima, R. et al. Radically different thioredoxin domain arrangement of ERp46, an efficient disulfide bond introducer of the mammalian PDI family. Structure 22, 431–443 (2014).

  17. 17.

    Sato, Y. et al. Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding. Sci. Rep. 3, 2456 (2013).

  18. 18.

    Inaba, K. et al. Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI. EMBO J. 29, 3330–3343 (2010).

  19. 19.

    Kanemura, S. et al. Human ER oxidoreductin-1α (Ero1α) undergoes dual regulation through complementary redox interactions with protein-disulfide isomerase. J. Biol. Chem. 291, 23952–23964 (2016).

  20. 20.

    Masui, S., Vavassori, S., Fagioli, C., Sitia, R. & Inaba, K. Molecular bases of cyclic and specific disulfide interchange between human ERO1α protein and protein-disulfide isomerase (PDI). J. Biol. Chem. 286, 16261–16271 (2011).

  21. 21.

    Wang, L. et al. Reconstitution of human Ero1-Lα/protein-disulfide isomerase oxidative folding pathway in vitro. Position-dependent differences in role between the a and aʹ domains of protein-disulfide isomerase. J. Biol. Chem. 284, 199–206 (2009).

  22. 22.

    Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A. & Rutter, W. J. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature 317, 267–270 (1985).

  23. 23.

    Klappa, P., Ruddock, L. W., Darby, N. J. & Freedman, R. B. The bʹ domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. EMBO J. 17, 927–935 (1998).

  24. 24.

    Tian, G., Xiang, S., Noiva, R., Lennarz, W. J. & Schindelin, H. The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. Cell 124, 61–73 (2006).

  25. 25.

    Tian, G. et al. The catalytic activity of protein-disulfide isomerase requires a conformationally flexible molecule. J. Biol. Chem. 283, 33630–33640 (2008).

  26. 26.

    Okumura, M. et al. Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A. J. Biol. Chem. 289, 27004–27018 (2014).

  27. 27.

    Wang, C. et al. Structural insights into the redox-regulated dynamic conformations of human protein disulfide isomerase. Antioxid. Redox Signal. 19, 36–45 (2013).

  28. 28.

    Serve, O. et al. Redox-dependent domain rearrangement of protein disulfide isomerase coupled with exposure of its substrate-binding hydrophobic surface. J. Mol. Biol. 396, 361–374 (2010).

  29. 29.

    Tsai, B., Rodighiero, C., Lencer, W. I. & Rapoport, T. A. Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin. Cell 104, 937–948 (2001).

  30. 30.

    Lumb, R. A. & Bulleid, N. J. Is protein disulfide isomerase a redox-dependent molecular chaperone? EMBO J. 21, 6763–6770 (2002).

  31. 31.

    Cho, K. et al. Redox-regulated peptide transfer from the transporter associated with antigen processing to major histocompatibility complex class I molecules by protein disulfide isomerase. Antioxid. Redox Signal. 15, 621–633 (2011).

  32. 32.

    Kosuri, P. et al. Protein folding drives disulfide formation. Cell 151, 794–806 (2012).

  33. 33.

    Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).

  34. 34.

    Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755–758 (2011).

  35. 35.

    Uchihashi, T., Kodera, N. & Ando, T. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat. Protoc. 7, 1193–1206 (2012).

  36. 36.

    Noi, K. et al. High-speed atomic force microscopic observation of ATP-dependent rotation of the AAA + chaperone p97. Structure 21, 1992–2002 (2013).

  37. 37.

    Irvine, A. G. et al. Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway. PLoS ONE 9, e82511 (2014).

  38. 38.

    Ido, H. et al. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins. J. Biol. Chem. 282, 11144–11154 (2007).

  39. 39.

    Bastos-Aristizabal, S., Kozlov, G. & Gehring, K. Structural insight into the dimerization of human protein disulfide isomerase. Protein Sci. 23, 618–626 (2014).

  40. 40.

    Wallis, A. K. et al. The ligand-binding bʹ domain of human protein disulphide-isomerase mediates homodimerization. Protein Sci. 18, 2569–2577 (2009).

  41. 41.

    Maegawa, K. I. et al. The highly dynamic nature of ERdj5 is key to efficient elimination of aberrant protein oligomers through ER-associated degradation. Structure 25, 846–857.e4 (2017).

  42. 42.

    Wendel, M., Lorenz, H. & Kotthaus, J. P. Sharpened electron beam deposited tips for high resolution atomic force microscope lithography and imaging. Appl. Phys. Lett. 67, 3732–3734 (1995).

  43. 43.

    Rodrı́guez, T. R. & Garcı́a, R. Theory of Q control in atomic force microscopy. Appl. Phys. Lett. 82, 4821–4823 (2003).

  44. 44.

    Akiyama, S. Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering. J. Appl. Crystallogr. 43, 237–243 (2010).

  45. 45.

    Svergun, D. Mathematical methods in small-angle scattering data analysis. J. Appl. Crystallogr. 24, 485–492 (1991).

  46. 46.

    Svergun, D., Barberato, C. & Koch, M. H. J. CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).

  47. 47.

    Kinoshita, M. et al. Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin. Biochim. Biophys. Acta 1847, 1200–1211 (2015).

  48. 48.

    Saio, T., Guan, X., Rossi, P., Economou, A. & Kalodimos, C. G. Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344, 1250494 (2014).

  49. 49.

    Akaike, H. A new look at the statistical model identification. IEEE Trans. Automat. Contr. 19, 716–723 (1974).

Download references


Synchrotron radiation experiments were performed on BL45XU in SPring-8 with the approval of RIKEN (proposal no. 2014A1345). We are grateful to M. Matsusaki, S. Kanbayashi and S. Ogawa for their experimental assistance. This work was supported by funding from CREST (to T.O. (JPMJCR13M1) and K.I. (JPMJCR13M6)), Grant-in-Aids for Scientific Research on Innovative Areas from MEXT (to K.I. (26116005) and M.O. (15641922)), the Takeda Science Foundation (to K.I. and M.O.), the Uehara Memorial Foundation (to K.I. and M.O.), the Naito Foundation (to M.O.), a Grant-in-Aid for JSPS Fellows (to M.O. and K.S.), the Building of Consortia for the Development of Human Resources in Science and Technology (to M.O.), the program of the Joint Usage/Research Center for Developmental Medicine (IMEG, Kumamoto University) (to M.O. and K.I.), and the Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT (to M.O., S.K., S.A. and K.I.).

Author information

M.O. designed and performed almost all experiments including the SAXS, HS-AFM and oxidative protein folding experiments. K.N. performed AFM measurements, and analyzed the HS-AFM data. S.K. performed SAXS experiments. M.K. performed ITC experiments and statistical analysis using AIC scores. T.S. performed SEC-MALS experiments. Y.I. analyzed the AFM images. T.H. assisted the SAXS experiment. S.A. analyzed the SAXS data. T.O. assisted with HS-AFM experiments and reviewed the manuscript. K.I. supervised the study. K.I. and M.O. wrote the manuscript. M.O. prepared figures. All authors discussed the results and approved the manuscript.

Correspondence to Masaki Okumura or Teru Ogura or Kenji Inaba.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Table 1, Supplementary Figs. 1–14 and Supplementary Video legends.

Reporting Summary

Supplementary Video 1

HS-AFM movies showing closed conformations of the reduced form of PDI.

Supplementary Video 2

HS-AFM movies showing conformational dynamics of oxidized PDI.

Supplementary Video 3

HS-AFM movies showing transient dimerization of PDI in the presence of reduced and denatured BPTI.

Supplementary Video 4

High-speed AFM movies showing long-lived and transformable PDI dimers in the presence of reduced and denatured RNase A

Supplementary Video 5

HS-AFM movies showing two PDI dimers bound to Cys-blocked plasminogen.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading