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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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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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.).
The authors declare no competing interests.
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Supplementary Table 1, Supplementary Figs. 1–14 and Supplementary Video legends.
HS-AFM movies showing closed conformations of the reduced form of PDI.
HS-AFM movies showing conformational dynamics of oxidized PDI.
HS-AFM movies showing transient dimerization of PDI in the presence of reduced and denatured BPTI.
High-speed AFM movies showing long-lived and transformable PDI dimers in the presence of reduced and denatured RNase A
HS-AFM movies showing two PDI dimers bound to Cys-blocked plasminogen.
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Current Opinion in Chemical Biology (2019)
Biochimica et Biophysica Acta (BBA) - General Subjects (2019)