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Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding

Abstract

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.

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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.

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Acknowledgements

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.).

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Authors

Contributions

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.

Corresponding authors

Correspondence to Masaki Okumura or Teru Ogura or Kenji Inaba.

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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.

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Okumura, M., Noi, K., Kanemura, S. et al. Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding. Nat Chem Biol 15, 499–509 (2019). https://doi.org/10.1038/s41589-019-0268-8

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