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Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Abstract

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane1,2. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential2. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix2. This leakage, or ‘uncoupling’ of the proton electrochemical potential, is mediated primarily by uncoupling proteins2. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination3, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure4), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1–4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

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Figure 1: NMR spectra, GDP binding and outline of RDC-based molecular fragment assignment.
Figure 2: Operations involved in RDC-based structural segment building.
Figure 3: Solution structure of UCP2 and region of GDP binding.
Figure 4: Comparison of UCP2 and ANT1.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structure of UCP2 has been deposited in the Protein Data Bank under accession number 2LCK.

References

  1. Palmieri, F. et al. Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins. Biochim. Biophys. Acta 1757, 1249–1262 (2006)

    Article  CAS  Google Scholar 

  2. Krauss, S., Zhang, C. Y. & Lowell, B. B. The mitochondrial uncoupling-protein homologues. Nature Rev. Mol. Cell Biol. 6, 248–261 (2005)

    Article  CAS  Google Scholar 

  3. Tate, C. G. & Stevens, R. C. Growth and excitement in membrane protein structural biology. Curr. Opin. Struct. Biol. 20, 399–400 (2010)

    Article  CAS  Google Scholar 

  4. Pebay-Peyroula, E. et al. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426, 39–44 (2003)

    Article  ADS  CAS  Google Scholar 

  5. Mitchell, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191, 144–148 (1961)

    Article  ADS  CAS  Google Scholar 

  6. Aquila, H., Link, T. A. & Klingenberg, M. The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane. EMBO J. 4, 2369–2376 (1985)

    Article  CAS  Google Scholar 

  7. Zhang, C. Y. et al. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Cell 105, 745–755 (2001)

    Article  CAS  Google Scholar 

  8. Diao, J. et al. UCP2 is highly expressed in pancreatic alpha-cells and influences secretion and survival. Proc. Natl Acad. Sci. USA 105, 12057–12062 (2008)

    Article  ADS  CAS  Google Scholar 

  9. Andrews, Z. B. et al. UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals. Nature 454, 846–851 (2008)

    Article  ADS  CAS  Google Scholar 

  10. Harper, M. E. et al. Characterization of a novel metabolic strategy used by drug-resistant tumor cells. FASEB J. 16, 1550–1557 (2002)

    Article  CAS  Google Scholar 

  11. Samudio, I., Fiegl, M. & Andreeff, M. Mitochondrial uncoupling and the Warburg effect: molecular basis for the reprogramming of cancer cell metabolism. Cancer Res. 69, 2163–2166 (2009)

    Article  CAS  Google Scholar 

  12. Schnell, J. R. & Chou, J. J. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591–595 (2008)

    Article  ADS  CAS  Google Scholar 

  13. Hiller, S. et al. Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321, 1206–1210 (2008)

    Article  ADS  CAS  Google Scholar 

  14. Wang, J., Pielak, R. M., McClintock, M. A. & Chou, J. J. Solution structure and functional analysis of the influenza B proton channel. Nature Struct. Mol. Biol. 16, 1267–1271 (2009)

    Article  CAS  Google Scholar 

  15. Zhou, Y. et al. NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol. Cell 31, 896–908 (2008)

    Article  CAS  Google Scholar 

  16. Van Horn, W. D. et al. Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324, 1726–1729 (2009)

    Article  ADS  CAS  Google Scholar 

  17. Gautier, A., Mott, H. R., Bostock, M. J., Kirkpatrick, J. P. & Nietlispach, D. Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nature Struct. Mol. Biol. 17, 768–774 (2010)

    Article  CAS  Google Scholar 

  18. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997)

    Article  ADS  CAS  Google Scholar 

  19. Douglas, S. M., Chou, J. J. & Shih, W. M. DNA-nanotube-induced alignment of membrane proteins for NMR structure determination. Proc. Natl Acad. Sci. USA 104, 6644–6648 (2007)

    Article  ADS  CAS  Google Scholar 

  20. Jones, T. A. & Thirup, S. Using known substructures in protein model building and crystallography. EMBO J. 5, 819–822 (1986)

    Article  CAS  Google Scholar 

  21. Delaglio, F., Kontaxis, G. & Bax, A. Protein structure determination using molecular fragment replacement and NMR dipolar couplings. J. Am. Chem. Soc. 122, 2142–2143 (2000)

    Article  CAS  Google Scholar 

  22. Shen, Y. et al. Consistent blind protein structure generation from NMR chemical shift data. Proc. Natl Acad. Sci. USA 105, 4685–4690 (2008)

    Article  ADS  CAS  Google Scholar 

  23. Raman, S. et al. NMR structure determination for larger proteins using backbone-only data. Science 327, 1014–1018 (2010)

    Article  ADS  CAS  Google Scholar 

  24. Losonczi, J. A., Andrec, M., Fischer, M. W. F. & Prestegard, J. H. Order matrix analysis of residual dipolar couplings using singular value decomposition. J. Magn. Reson. 138, 334–342 (1999)

    Article  ADS  CAS  Google Scholar 

  25. Cornilescu, G., Marquardt, J. L., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837 (1998)

    Article  CAS  Google Scholar 

  26. Battiste, J. L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39, 5355–5365 (2000)

    Article  CAS  Google Scholar 

  27. Wang, Y. & Tajkhorshid, E. Electrostatic funneling of substrate in mitochondrial inner membrane carriers. Proc. Natl Acad. Sci. USA 105, 9598–9603 (2008)

    Article  ADS  CAS  Google Scholar 

  28. Dehez, F., Pebay-Peyroula, E. & Chipot, C. Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel. J. Am. Chem. Soc. 130, 12725–12733 (2008)

    Article  CAS  Google Scholar 

  29. Kunji, E. R. & Robinson, A. J. Coupling of proton and substrate translocation in the transport cycle of mitochondrial carriers. Curr. Opin. Struct. Biol. 20, 440–447 (2010)

    Article  CAS  Google Scholar 

  30. Schwieters, C. D., Kuszewski, J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 66–74 (2002)

    Google Scholar 

  31. Salzmann, M., Wider, G., Pervushin, K. & Wuthrich, K. Improved sensitivity and coherence selection for [N-15,H-1]-TROSY elements in triple resonance experiments. J. Biomol. NMR 15, 181–184 (1999)

    Article  CAS  Google Scholar 

  32. Kay, L. E., Torchia, D. A. & Bax, A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979 (1989)

    Article  CAS  Google Scholar 

  33. Kontaxis, G., Clore, G. & Bax, A. Evaluation of cross-correlation effects and measurement of one-bond couplings in proteins with short transverse relaxation times. J. Magn. Reson. 143, 184–196 (2000)

    Article  ADS  CAS  Google Scholar 

  34. Jaroniec, C. P., Ulmer, T. S. & Bax, A. Quantitative J correlation methods for the accurate measurement of 13C′-13Cα dipolar couplings in proteins. J. Biomol. NMR 30, 181–194 (2004)

    Article  CAS  Google Scholar 

  35. Chou, J. J., Delaglio, F. & Bax, A. Measurement of one-bond 15N-13C′ dipolar couplings in medium sized proteins. J. Biomol. NMR 18, 101–105 (2000)

    Article  CAS  Google Scholar 

  36. Zweckstetter, M. & Bax, A. Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J. Am. Chem. Soc. 122, 3791–3792 (2000)

    Article  CAS  Google Scholar 

  37. Schwieters, C. D., Kuszewski, J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 66–74 (2002)

    Google Scholar 

  38. Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improvements and extensions in the conformational database potential for the refinement of NMR and X-ray structures of proteins and nucleic acids. J. Magn. Reson. 125, 171–177 (1997)

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

We thank K. Oxenoid and R. Sounier for discussions, M. McClintock for help with DNA nanotube preparation, I. Stokes-Rees and P. Sliz for help with computations, and N. Voigt for help with figures. The work was supported by NIH grants 1U54GM094608 (to J.J.C.) and 1DP2OD004641 (to W.M.S.). S.C.H. is an Investigator at the Howard Hughes Medical Institute.

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M.J.B., W.M.S., S.C.H. and J.J.C. designed the study; M.J.B. prepared NMR samples; M.J.B. and W.M.S. prepared DNA nanotubes; M.J.B. and J.J.C. designed experiments, collected and analysed NMR data, and determined the structure; M.J.B. and J.J.C. wrote the paper; and all authors contributed to editing the manuscript.

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Correspondence to James J. Chou.

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The authors declare no competing financial interests.

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Berardi, M., Shih, W., Harrison, S. et al. Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching. Nature 476, 109–113 (2011). https://doi.org/10.1038/nature10257

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