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Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside


ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 Å by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six α-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a ‘pit’ to a ‘channel’ conformation.

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  1. 1

    Walker, J. E. & Runswick, M. J. The mitochondrial transport protein superfamily. J. Bioenerg. Biomembr. 25, 435–446 (1993)

  2. 2

    Torroni, A., Stepien, G., Hodge, J. A. & Wallace, D. C. Neoplastic transformation is associated with coordinate induction of nuclear and cytoplasmic oxidative phosphorylation genes. J. Biol. Chem. 265, 20589–20593 (1990)

  3. 3

    Heddi, A., Lestienne, P., Wallace, D. C. & Stepien, G. Mitochondrial DNA expression in mitochondrial myopathies and coordinated expression of nuclear genes involved in ATP production. J. Biol. Chem. 268, 12156–12163 (1993)

  4. 4

    Kaukonen, J. et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289, 782–785 (2000)

  5. 5

    Fiore, C., Arlot-Guilligay, D., Trézéguet, V., Lauquin, G. J. & Brandolin, G. Fluorometric detection of ADP/ATP carrier deficiency in human muscle. Clin. Chim. Acta 311, 125–135 (2001)

  6. 6

    Napoli, L. et al. A novel missense adenine nucleotide translocator-1 gene mutation in a Greek adPEO family. Neurology 57, 2295–2298 (2001)

  7. 7

    Craig, J. C., Mole, M., Billets, S. & El-Feraly, F. Isolation and identification of hypoglycemic agent carboxyatractylate from Xanthium strumarium. Phytochemistry 15, 1178 (1976)

  8. 8

    Candy, H. A., Pegel, K. H., Brookes, B. & Rodwell, M. The occurrence of atractyloside in Callilepsis laureola. Phytochemistry 16, 1308–1309 (1977)

  9. 9

    Bruni, A., Luciani, S. & Contessa, A. R. Inhibition by atractyloside of the binding of adenine nucleotides of rat liver mitochondria. Nature 201, 1219–1220 (1964)

  10. 10

    Duée, E. D. & Vignais, P. V. Exchange between extra- and intramitochondrial adenine nucleotides. Biochim. Biophys. Acta 107, 184–188 (1965)

  11. 11

    Pfaff, E., Klingenberg, M. & Heldt, H. W. Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria. Biochim. Biophys. Acta 104, 312–315 (1965)

  12. 12

    Klingenberg, M. Molecular aspects of the adenine nucleotide carrier from mitochondria. Arch. Biochem. Biophys. 270, 1–14 (1989)

  13. 13

    Fiore, C. et al. The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects. Biochimie 80, 137–150 (1998)

  14. 14

    Aquila, H., Misra, D., Eulitz, M. & Klingenberg, M. Complete aminoacid sequence of the ADP/ATP carrier from beef heart mitochondria. Hoppe-Seyler's J. Physiol. Chem. 363, 345–349 (1982)

  15. 15

    Brandolin, G., Dupont, Y. & Vignais, P. V. Substrate-induced modifications of the intrinsic fluorescence of the isolated adenine nucleotide carrier protein: demonstration of distinct conformational states. Biochemistry 24, 1991–1997 (1985)

  16. 16

    Gropp, T. et al. Kinetics of electrogenic transport by the ADP/ATP carrier. Biophys. J. 77, 714–726 (1999)

  17. 17

    Walker, J. E. The mitochondrial transporter family. Curr. Opin. Struct. Biol. 2, 519–526 (1992)

  18. 18

    Bogner, W., Aquila, H. & Klingenberg, M. The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate. Eur. J. Biochem. 161, 611–620 (1986)

  19. 19

    Majima, E., Koike, H., Hong, Y. M., Shinohara, Y. & Terada, H. Characterization of cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide and N-ethylmaleimide. J. Biol. Chem. 268, 22181–22187 (1993)

  20. 20

    Dianoux, A. C. et al. Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3′-O-naphthoyl-[β-32P]ADP. Identification of the binding segments by mass spectrometry. Biochemistry 39, 11477–11487 (2000)

  21. 21

    Brandolin, G., Boulay, F., Dalbon, P. & Vignais, P. V. Orientation of the N-terminal region of the membrane-bound ADP/ATP carrier protein explored by antipeptide antibodies and an arginine-specific endoprotease. Evidence that the accessibility of the N-terminal residues depends on the conformational state of the carrier. Biochemistry 28, 1093–1100 (1989)

  22. 22

    Trézéguet, V. et al. A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo. Biochim. Biophys. Acta 1457, 81–93 (2000)

  23. 23

    Hackenberg, H. & Klingenberg, M. Molecular weight and hydrodynamic parameters of the adenosine 5′-diphosphate/adenosine 5′-triphosphate carrier in TritonX-100. Biochemistry 19, 548–555 (1980)

  24. 24

    Block, M. R., Zaccai, G., Lauquin, G. J. & Vignais, P. V. Small angle neutron scattering of the mitochondrial ADP/ATP carrier protein in detergent. Biochem. Biophys. Res. Commun. 109, 471–477 (1982)

  25. 25

    Panneels, V., Schussler, U., Costagliola, S. & Sinning, I. Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2. Biochem. Biophys. Res. Commun. 300, 65–74 (2003)

  26. 26

    Nelson, D. R., Felix, C. M. & Swanson, J. M. Highly conserved charge-pair networks in the mitochondrial carrier family. J. Mol. Biol. 277, 285–308 (1998)

  27. 27

    Beyer, K. & Klingenberg, M. ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance. Biochemistry 24, 3821–3826 (1985)

  28. 28

    Vignais, P. V. Molecular and physiological aspects of adenine nucleotide transport in mitochondria. Biochim. Biophys. Acta 456, 1–38 (1976)

  29. 29

    Zeman, I. et al. Four mutations in transmembrane domains of the mitochondrial ADP/ATP carrier increase resistance to bongkrekic acid. J. Bioenerg. Biomembr. 35, 243–256 (2003)

  30. 30

    Duyckaerts, C., Sluse-Goffart, C. M., Fux, J. P., Sluse, F. E. & Liebecq, C. Kinetic mechanism of the exchanges catalysed by the adenine-nucleotide carrier. Eur. J. Biochem. 106, 1–6 (1980)

  31. 31

    Kovac, L., Lachowicz, T. M. & Slonimski, P. P. Biochemical genetics of oxidative phosphorylation. Science 158, 1564–1567 (1967)

  32. 32

    Beck, J. C., Mattoon, J. R., Hawthorne, D. C. & Sherman, F. Genetic modification of energy-conserving systems in yeast mitochondria. Proc. Natl Acad. Sci. USA 60, 186–193 (1968)

  33. 33

    Nelson, D. R., Lawson, J. E., Klingenberg, M. & Douglas, M. G. Site-directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential. J. Mol. Biol. 230, 1159–1170 (1993)

  34. 34

    Müller, V., Heidkamper, D., Nelson, D. R. & Klingenberg, M. Mutagenesis of some positive and negative residues occurring in repeat triad residues in the ADP/ATP carrier from yeast. Biochemistry 36, 16008–16018 (1997)

  35. 35

    Denessiouk, K. A. & Johnson, M. S. When fold is not important: a common structural framework for adenine and AMP binding in 12 unrelated protein families. Proteins 38, 310–326 (2000)

  36. 36

    Moodie, S. L., Mitchell, J. B. & Thornton, J. M. Protein recognition of adenylate: an example of a fuzzy recognition template. J. Mol. Biol. 263, 486–500 (1996)

  37. 37

    Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002)

  38. 38

    Abramson, J. et al. Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615 (2003)

  39. 39

    Block, M. R. & Vignais, P. V. Substrate-site interactions in the membrane-bound adenine-nucleotide carrier as disclosed by ADP and ATP analogs. Biochim. Biophys. Acta 767, 369–376 (1984)

  40. 40

    Kunji, E. R. & Harding, M. Projection structure of the atractyloside-inhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae. J. Biol. Chem. 278, 36985–36988 (2003)

  41. 41

    Holloway, P. W. A simple procedure for removal of Triton X-100 from protein samples. Anal. Biochem. 53, 304–308 (1973)

  42. 42

    Belrhali, H. et al. Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 Å resolution. Struct. Fold. Des. 7, 909–917 (1999)

  43. 43

    Harrenga, A. & Michel, H. The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction. J. Biol. Chem. 274, 33296–33299 (1999)

  44. 44

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  45. 45

    The CCP4 suite: programs for X-ray crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  46. 46

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  47. 47

    Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

  48. 48

    Navaza, J. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D 57, 1367–1372 (2001)

  49. 49

    Esnouf, R. M. Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta Crystallogr. D 55, 938–940 (1999)

  50. 50

    Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

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We thank the staff at the European Synchrotron Radiation Facilities and of the French beamline BM30A (ESRF) for synchrotron support. We also thank J. P. Rosenbusch and R. Douce for reading the manuscript, and for numerous suggestions and discussions, and P. V. Vignais for helpful comments. This work was supported by the programmes PCV (CNRS) and Emergence (Région Rhône-Alpes) and by the Région Aquitaine.

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Correspondence to Eva Pebay-Peyroula or Gérard Brandolin.

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The authors declare that they have no competing financial interests.

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Further reading

Figure 1: Architecture of the ADP/ATP carrier.
Figure 2: Section through the carrier.
Figure 3: The threefold repeat of the ADP/ATP carrier.
Figure 4: Electrostatic potential surface.
Figure 5: The binding of CATR.
Figure 6: The closed conformation of the carrier viewed from the matrix.


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