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MIA40 is an oxidoreductase that catalyzes oxidative protein folding in mitochondria

Nature Structural & Molecular Biology volume 16, pages 198–206 (2009)Cite this article

Abstract

MIA40 has a key role in oxidative protein folding in the mitochondrial intermembrane space. We present the solution structure of human MIA40 and its mechanism as a catalyst of oxidative folding. MIA40 has a 66-residue folded domain made of an α-helical hairpin core stabilized by two structural disulfides and a rigid N-terminal lid, with a characteristic CPC motif that can donate its disulfide bond to substrates. The CPC active site is solvent-accessible and sits adjacent to a hydrophobic cleft. Its second cysteine (Cys55) is essential in vivo and is crucial for mixed disulfide formation with the substrate. The hydrophobic cleft functions as a substrate binding domain, and mutations of this domain are lethal in vivo and abrogate binding in vitro. MIA40 represents a thioredoxin-unrelated, minimal oxidoreductase, with a facile CPC redox active site that ensures its catalytic function in oxidative folding in mitochondria.

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Figure 1: MIA40 is functionally active in binding substrates.
Figure 2: The redox and structural properties of the CPC intramolecular disulfide bonds of human MIA40.
Figure 3: Redox potential of the CPC redox active site.
Figure 4: MIA40 is substantially unstructured at its N and C termini.
Figure 5: The solution structure of MIA402S-S.
Figure 6: Interaction of MIA40 with substrates.
Figure 7: The second cysteine, Cys55, of the active-site CPC is essential in vivo and in vitro.
Figure 8: Model for the interaction of MIA40 with its substrates.

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Protein Data Bank

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Acknowledgements

We are grateful to A. Makris (Mediterranean Agronomic Institute of Chania, Crete) for the plasmid M4801, N. Pfanner (University of Freiburg) for the porin SP6 plasmid, N. Petrakis (K.T. laboratory, Institute of Molecular Biology and Biotechnology-Foundation for Research and Technology (IMBB-FORTH)) for help with the use of the Chimera software used in Figure 5, A. Hatzi (K.T. group, IMBB-FORTH) for some help with part of the mutagenesis and T. Economou (IMBB-FORTH) and T. Pugsley (Institut Pasteur) for comments on the manuscript. This work was supported by European Network of Research Infrastructures for Providing Access and Technological Advancements in Bio-NMR Contract 026145, by the SPINE II-COMPLEXES Contract, LSHG-CT-2006-031220 “From Receptor to Gene: Structures of Complexes from Signalling Pathways Linking Immunology, Neurobiology and Cancer,” and by funds from IMBB-FORTH, the University of Crete and the European Social Fund and National Resources (to K.T.). D.P.S. was supported by a PENED grant. This work was also supported in part by the Italian MIUR-FIRB (Fondo per gli Investimenti della Ricerca di Base, Grant protocollo, MIUR-RBLA032ZM7). Molecular graphics images were produced using the UCSF Chimera package50 from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by the US National Institutes of Health grant P41 RR-01081).

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Authors and Affiliations

  1. Magnetic Resonance Center CERM, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy

    Lucia Banci, Ivano Bertini, Chiara Cefaro, Simone Ciofi-Baffoni, Angelo Gallo & Manuele Martinelli

  2. Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy

    Lucia Banci, Ivano Bertini, Chiara Cefaro, Simone Ciofi-Baffoni, Angelo Gallo & Manuele Martinelli

  3. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas (IMBB-FORTH), Heraklion 71110, Crete, Greece

    Dionisia P Sideris, Nitsa Katrakili & Kostas Tokatlidis

  4. Department of Biology, University of Crete, Heraklion 71409, Crete, Greece

    Dionisia P Sideris

  5. Department of Materials Science and Technology, University of Crete, Heraklion 71003, Crete, Greece

    Kostas Tokatlidis

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Contributions

I.B. and L.B. planned the research, discussed and guided the flow of experiments and coordinated the writing of the text, to which all the co-authors contributed; M.M. and C.C. coordinated and performed protein production and characterization; A.G. solved the MIA402S-S NMR structure; S.C.-B. planned and recorded the NMR spectra and coordinated the titration experiments; D.P.S. performed the in vivo and in vitro mutational analysis and interactions and analyzed data; N.K. provided technical support; K.T. designed experiments, analyzed data and coordinated the presentation of the data and the writing of the paper.

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Correspondence to Ivano Bertini or Kostas Tokatlidis.

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Banci, L., Bertini, I., Cefaro, C. et al. MIA40 is an oxidoreductase that catalyzes oxidative protein folding in mitochondria. Nat Struct Mol Biol 16, 198–206 (2009). https://doi.org/10.1038/nsmb.1553

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