CFTR Three-Dimensional Structure

  • Robert C. FordEmail author
  • James Birtley
  • Mark F. Rosenberg
  • Liang Zhang
Part of the Methods in Molecular Biology book series (MIMB, volume 741)


CFTR is a member of the ATP-binding cassette family of membrane proteins. This is one of the best characterised membrane protein families in terms of structure and function. CFTR operates as an ion channel, unlike nearly all other family members which are active transporters. Here, we discuss methods that have allowed such data to be obtained for CFTR.

Key words

CFTR cystic fibrosis structure ion channel membrane protein electron microscopy electron crystallography 



We are extremely grateful to our collaborators at the University of North Carolina (Chapel Hill) led by Professor John Riordan. Without their advice, information, resources and protein, we would not have delved into the CFTR structural world. The authors acknowledge the financial support of the Cystic Fibrosis Foundation (USA).


  1. 1.
    Rees, D. C., Johnson, E., and Lewinson, O. (2009) ABC transporters: The power to change. Nat. Rev. Mol. Cell Biol. 10, 218–227.PubMedCrossRefGoogle Scholar
  2. 2.
    Riordan, J. R. (2008) CFTR function and prospects for therapy. Annu. Rev. Biochem. 77, 701–726.PubMedCrossRefGoogle Scholar
  3. 3.
    Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., et al. (1989) Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066–1073.PubMedCrossRefGoogle Scholar
  4. 4.
    Kos, V., and Ford, R. C. (2009) The ATP-binding cassette family: A structural perspective. Cell. Mol. Life Sci. 66, 3111–3126.PubMedCrossRefGoogle Scholar
  5. 5.
    Aller, S. G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., et al. (2009) Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718–1722.PubMedCrossRefGoogle Scholar
  6. 6.
    Dawson, R. J., and Locher, K. P. (2006) Structure of a bacterial multidrug ABC transporter. Nature 443, 180–185.PubMedCrossRefGoogle Scholar
  7. 7.
    Dawson, R. J., and Locher, K. P. (2007) Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP. FEBS Lett. 581, 935–938.PubMedCrossRefGoogle Scholar
  8. 8.
    Hollenstein, K., Frei, D. C., and Locher, K. P. (2007) Structure of an ABC transporter in complex with its binding protein. Nature 446, 213–216.PubMedCrossRefGoogle Scholar
  9. 9.
    Locher, K. P. (2004) Structure and mechanism of ABC transporters. Curr. Opin. Struct. Biol. 14, 426–431.PubMedCrossRefGoogle Scholar
  10. 10.
    Locher, K. P., Lee, A. T., and Rees, D. C. (2002) The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism. Science 296, 1091–1098.PubMedCrossRefGoogle Scholar
  11. 11.
    McDevitt, C. A., Shintre, C. A., Grossmann, J. G., Pollock, N. L., Prince, S. M., Callaghan, R., et al. (2008) Structural insights into P-glycoprotein (ABCB1) by small angle X-ray scattering and electron crystallography. FEBS Lett. 582, 2950–2956.PubMedCrossRefGoogle Scholar
  12. 12.
    Oldham, M. L., Davidson, A. L., and Chen, J. (2008) Structural insights into ABC transporter mechanism. Curr. Opin. Struct. Biol. 18, 726–733.PubMedCrossRefGoogle Scholar
  13. 13.
    Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515–521.PubMedCrossRefGoogle Scholar
  14. 14.
    Pinkett, H. W., Lee, A. T., Lum, P., Locher, K. P., and Rees, D. C. (2007) An inward-facing conformation of a putative metal-chelate-type ABC transporter. Science 315, 373–377.PubMedCrossRefGoogle Scholar
  15. 15.
    Ward, A., Reyes, C. L., Yu, J., Roth, C. B., and Chang, G. (2007) Flexibility in the ABC transporter MsbA: Alternating access with a twist. Proc. Natl. Acad. Sci. USA 104, 19005–19010.PubMedCrossRefGoogle Scholar
  16. 16.
    Kadaba, N. S., Kaiser, J. T., Johnson, E., Lee, A., and Rees, D. C. (2008) The high-affinity E. coli methionine ABC transporter: Structure and allosteric regulation. Science 321, 250–253.PubMedCrossRefGoogle Scholar
  17. 17.
    Holzenburg, A., Wilson, F. H., Finbow, M. E., and Ford, R. C. (1992) Structural investigations of membrane proteins: The versatility of electron microscopy. Biochem. Soc. Trans. 20, 591–597.PubMedGoogle Scholar
  18. 18.
    Bremer, A., Henn, C., Engel, A., Baumeister, W., and Aebi, U. (1992) Has negative staining still a place in biomacromolecular electron microscopy? Ultramicroscopy 46, 85–111.PubMedCrossRefGoogle Scholar
  19. 19.
    Brenner, S., and Horne, R. W. (1959) A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103–110.PubMedCrossRefGoogle Scholar
  20. 20.
    Harris, J. R., and Holzenburg, A. (1995) Human erythrocyte catalase: 2-D crystal nucleation and production of multiple crystal forms. J. Struct. Biol. 115, 102–112.PubMedCrossRefGoogle Scholar
  21. 21.
    Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W., et al. (1988) Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228.PubMedCrossRefGoogle Scholar
  22. 22.
    Henderson, R. (1995) The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171–193.PubMedCrossRefGoogle Scholar
  23. 23.
    Henderson, R. (2004) Realizing the potential of electron cryo-microscopy. Q. Rev. Biophys. 37, 3–13.PubMedCrossRefGoogle Scholar
  24. 24.
    Knapek, E., and Dubochet, J. (1980) Beam damage to organic material is considerably reduced in cryo-electron microscopy. J. Mol. Biol. 141, 147–161.PubMedCrossRefGoogle Scholar
  25. 25.
    Auer, M., Scarborough, G. A., and Kuhlbrandt, W. (1999) Surface crystallisation of the plasma membrane H+-ATPase on a carbon support film for electron crystallography. J. Mol. Biol. 287, 961–968.PubMedCrossRefGoogle Scholar
  26. 26.
    Auer, M., Scarborough, G. A., and Kuhlbrandt, W. (1998) Three-dimensional map of the plasma membrane H+-ATPase in the open conformation. Nature 392, 840–843.PubMedCrossRefGoogle Scholar
  27. 27.
    Auer, M., Madden, D. R., Kuhlbrandt, W., and Scarborough, G. A. (1998) Structure of the neurospora plasma membrane H+-ATPase at 8 angstrom resolution. Biophys. J. 74, A43–A43.Google Scholar
  28. 28.
    Rosenberg, M. F., Callaghan, R., Modok, S., Higgins, C. F., and Ford, R. C. (2005) Three-dimensional structure of P-glycoprotein – The transmembrane regions adopt an asymmetric configuration in the nucleotide-bound state. J. Biol. Chem. 280, 2857–2862.PubMedCrossRefGoogle Scholar
  29. 29.
    Rosenberg, M. F., Kamis, A. B., Aleksandrov, L. A., Ford, R. C., and Riordan, J. R. (2004) Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR). J. Biol. Chem. 279, 39051–39057.PubMedCrossRefGoogle Scholar
  30. 30.
    Lewis, H. A., Buchanan, S. G., Burley, S. K., Conners, K., Dickey, M., Dorwart, M. et al (2004) Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator. EMBO J. 23, 282–293.PubMedCrossRefGoogle Scholar
  31. 31.
    Lewis, H. A., Zhao, X., Wang, C., Sauder, J. M., Rooney, I., Noland, B. W., et al. (2005) Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. J. Biol. Chem. 280, 1346–1353.PubMedCrossRefGoogle Scholar
  32. 32.
    Karthikeyan, S., Leung, T., Birrane, G., Webster, G., and Ladias, J. A. (2001) Crystal structure of the PDZ1 domain of human Na(+)/H(+) exchanger regulatory factor provides insights into the mechanism of carboxyl-terminal leucine recognition by class I PDZ domains. J. Mol. Biol. 308, 963–973.PubMedCrossRefGoogle Scholar
  33. 33.
    Baker, J. M., Hudson, R. P., Kanelis, V., Choy, W. Y., Thibodeau, P. H., Thomas, P. J., et al. (2007) CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices. Nat. Struct. Mol. Biol. 14, 738–745.PubMedCrossRefGoogle Scholar
  34. 34.
    Cormet-Boyaka, E., Jablonsky, M., Naren, A. P., Jackson, P. L., Muccio, D. D., and Kirk, K. L. (2004) Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation. Proc. Natl. Acad. Sci. USA 101, 8221–8226.PubMedCrossRefGoogle Scholar
  35. 35.
    Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., et al. (2007) GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273.PubMedCrossRefGoogle Scholar
  36. 36.
    Ostermeier, C., Iwata, S., Ludwig, B., and Michel, H. (1995) Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase. Nat. Struct. Biol. 2, 842–846.PubMedCrossRefGoogle Scholar
  37. 37.
    Tate, C. G., and Schertler, G. F. (2009) Engineering G protein-coupled receptors to facilitate their structure determination. Curr. Opin. Struct. Biol. 19, 386–395.PubMedCrossRefGoogle Scholar
  38. 38.
    Drew, D., Newstead, S., Sonoda, Y., Kim, H., von Heijne, G., and Iwata, S. (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat. Protoc. 3, 784–798.PubMedCrossRefGoogle Scholar
  39. 39.
    Chloupkova, M., Pickert, A., Lee, J. Y., Souza, S., Trinh, Y. T., Connelly, S. M., et al. (2007) Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. Biochemistry 46, 7992–8003.PubMedCrossRefGoogle Scholar
  40. 40.
    Eifler, N., Duckely, M., Sumanovski, L. T., Egan, T. M., Oksche, A., Konopka, J. B., et al. (2007) Functional expression of mammalian receptors and membrane channels in different cells. J. Struct. Biol. 159, 179–193.PubMedCrossRefGoogle Scholar
  41. 41.
    Ludtke, S. J., Baldwin, P. R., and Chiu, W. (1999) EMAN: Semi-automated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97.PubMedCrossRefGoogle Scholar
  42. 42.
    Ludtke, S. J., Jakana, J., Song, J. L., Chuang, D. T., and Chiu, W. (2001) A 11.5 A single particle reconstruction of GroEL using EMAN. J. Mol. Biol. 314, 253–262.PubMedCrossRefGoogle Scholar
  43. 43.
    Ludtke, S. J., Chen, D. H., Song, J. L., Chuang, D. T., and Chiu, W. (2004) Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy. Structure 12, 1129–1136.PubMedCrossRefGoogle Scholar
  44. 44.
    Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., et al. (2004) UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.PubMedCrossRefGoogle Scholar
  45. 45.
    Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., et al. (1996) SPIDER and WEB: Processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199.PubMedCrossRefGoogle Scholar
  46. 46.
    Crowther, R. A., Henderson, R., and Smith, J. M. (1996) MRC image processing programs. J. Struct. Biol. 116, 9–16.PubMedCrossRefGoogle Scholar
  47. 47.
    Zeng, X., Gipson, B., Zheng, Z. Y., Renault, L., and Stahlberg, H. (2007) Automatic lattice determination for two-dimensional crystal images. J. Struct. Biol. 160, 353–361.PubMedCrossRefGoogle Scholar
  48. 48.
    Philippsen, A., Schenk, A. D., Stahlberg, H., and Engel, A. (2003) Iplt-image processing library and toolkit for the electron microscopy community. J. Struct. Biol. 144, 4–12.PubMedCrossRefGoogle Scholar
  49. 49.
    Gipson, B., Zeng, X., Zhang, Z. Y., and Stahlberg, H. (2007) 2dx – User-friendly image processing for 2D crystals. J. Struct. Biol. 157, 64–72.PubMedCrossRefGoogle Scholar
  50. 50.
    Gipson, B., Zeng, X., and Stahlberg, H. (2007) 2dx_merge: Data management and merging for 2D crystal images. J. Struct. Biol. 160, 375–384.PubMedCrossRefGoogle Scholar
  51. 51.
    Kunji, E. R. S., von Gronau, S., Oesterhelt, D., and Henderson, R. (2000) The three-dimensional structure of halorhodopsin to 5 angstrom by electron crystallography: A new unbending procedure for two-dimensional crystals by using a global reference structure. Proc. Natl. Acad. Sci. USA 97, 4637–4642.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Robert C. Ford
    • 1
    Email author
  • James Birtley
    • 1
  • Mark F. Rosenberg
    • 1
  • Liang Zhang
    • 2
  1. 1.Faculty of Life SciencesManchester Interdisciplinary Biocentre, The University of ManchesterManchesterUK
  2. 2.Department of Cell Biology and PhysiologyUniversity of PittsburghPittsburghUSA

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