Biochemical and Biophysical Approaches to Probe CFTR Structure

  • André SchmidtEmail author
  • Juan L. Mendoza
  • Philip J. Thomas
Part of the Methods in Molecular Biology book series (MIMB, volume 741)


The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain integral membrane protein central to epithelial fluid secretion (see Chapter 21). Its activity is defective in the recessive genetic disease cystic fibrosis (CF). The most common CF-causing mutation is F508del in the first nucleotide binding domain (NBD1) of CFTR. This mutation is found on at least one allele of more than 90% of all CF patients. It is known to interfere with the trafficking/maturation of CFTR through the secretory pathway, leading to a loss-of-function at the plasma membrane. Notably, correction of the trafficking defect by addition of intragenic second-site suppressor mutations, or the alteration of bulk solvent conditions, such as by reducing the temperature or adding osmolytes, leads to appearance of functional channels at the membrane – thus, the rescued F508del-CFTR retains measurable function. High-resolution structural models of NBD1 from X-ray crystallographic data indicate that F508 is exposed on the surface of the domain in a position predicted by homologous ABC transporter structures to lie at the interface with the intracellular loops (ICLs) connecting the transmembrane spans. Determining the relative impact of the F508del mutation directly on NBD1 folding or on steps of domain assembly or both domain folding and assembly requires methods for evaluating the structure and stability of the isolated domain.

Key words

CFTR structure CFTR folding stability NBD1 spectroscopy 


  1. 1.
    Kleizen, B., and Braakman, I. (2004) Protein folding and quality control in the endoplasmic reticulum. Curr. Opin. Cell Biol. 16, 343–349.PubMedCrossRefGoogle Scholar
  2. 2.
    Zhang, F., Kartner, N., and Lukacs, G. L. (1998) Limited proteolysis as a probe for arrested conformational maturation of delta F508 CFTR. Nat. Struct. Biol. 5, 180–183.PubMedCrossRefGoogle Scholar
  3. 3.
    Amaral, M. D. (2006) Therapy through chaperones: sense or antisense? Cystic fibrosis as a model disease. J. Inherit. Metab. Dis. 29, 477–487.PubMedCrossRefGoogle Scholar
  4. 4.
    Wigley, W. C., Corboy, M. J., Cutler, T. D., Thibodeau, P. H., Oldan, J., Lee, M. G., et al. (2002) A protein sequence that can encode native structure by disfavoring alternate conformations. Nat. Struct. Biol. 9, 381–388.PubMedGoogle Scholar
  5. 5.
    Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83, 129–135.PubMedCrossRefGoogle Scholar
  6. 6.
    Ward, C. L., Omura, S., and Kopito, R. R. (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83, 121–127.PubMedCrossRefGoogle Scholar
  7. 7.
    Mendoza, J. L., and Thomas, P. J. (2007) Building an understanding of cystic fibrosis on the foundation of ABC transporter structures. J. Bioenerg. Biomembr. 39, 499–505.PubMedCrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., et al. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827–834.PubMedCrossRefGoogle Scholar
  10. 10.
    Yang, Y., Janich, S., Cohn, J. A., and Wilson, J. M. (1993) The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc. Natl. Acad. Sci. USA 90, 9480–9484.PubMedCrossRefGoogle Scholar
  11. 11.
    Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and Welsh, M. J. (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358, 761–764.PubMedCrossRefGoogle Scholar
  12. 12.
    Brown, C. R., Hong-Brown, L. Q., Biwersi, J., Verkman, A. S., and Welch, W. J. (1996) Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones 1, 117–125.PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang, X. M., Wang, X. T., Yue, H., Leung, S. W., Thibodeau, P. H., Thomas, P. J., et al. (2003) Organic solutes rescue the functional defect in delta F508 cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 278, 51232–51242.PubMedCrossRefGoogle Scholar
  14. 14.
    Meacham, G. C., Lu, Z., King, S., Sorscher, E., Tousson, A., and Cyr, D. M. (1999) The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis. EMBO J. 18, 1492–1505.PubMedCrossRefGoogle Scholar
  15. 15.
    Wang, X., Venable, J., LaPointe, P., Hutt, D. M., Koulov, A. V., Coppinger, J., et al. (2006) Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127, 803–815.PubMedCrossRefGoogle Scholar
  16. 16.
    Younger, J. M., Ren, H. Y., Chen, L., Fan, C. Y., Fields, A., Patterson, C., et al. (2004) A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase. J. Biol. Chem. 167, 1075–1085.Google Scholar
  17. 17.
    Pissarra, L. S., Farinha, C. M., Xu, Z., Schmidt, A., Thibodeau, P. H., Cai, Z., et al. (2008) Solubilizing mutations used to crystallize one CFTR domain attenuate the trafficking and channel defects caused by the major cystic fibrosis mutation. Chem. Biol. 15, 62–69.PubMedCrossRefGoogle Scholar
  18. 18.
    Teem, J. L., Berger, H. A., Ostedgaard, L. S., Rich, D. P., Tsui, L. C., and Welsh, M. J. (1993) Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast. Cell 73, 335–346.PubMedCrossRefGoogle Scholar
  19. 19.
    Teem, J. L., Carson, M. R., and Welsh, M. J. (1996) Mutation of R555 in CFTR-delta F508 enhances function and partially corrects defective processing. Recept. Channels 4, 63–72.PubMedGoogle Scholar
  20. 20.
    Dork, T., Wulbrand, U., Richter, T., Neumann, T., Wolfes, H., Wulf, B., et al. (1991) Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene. Hum. Genet. 87, 441–446.PubMedCrossRefGoogle Scholar
  21. 21.
    Dork, T., Wulbrand, U., Steinkamp, G., and Tummler, B. (1992) Mild course of cystic fibrosis associated with heterozygosity for infrequent mutations in the first nucleotide-binding fold of CFTR. Acta Paediatr. 81, 82–83.PubMedCrossRefGoogle Scholar
  22. 22.
    Roxo-Rosa, M., Xu, Z., Schmidt, A., Neto, M., Cai, Z., Soares, C. M., et al. (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Proc. Natl. Acad. Sci. USA 103, 17891–17896.PubMedCrossRefGoogle Scholar
  23. 23.
    Farinha, C. M., and Amaral, M. D. (2005) Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin. Mol. Cell. Biol. 25, 5242–5252.PubMedCrossRefGoogle Scholar
  24. 24.
    Rosser, M. F., Grove, D. E., Chen, L., and Cyr, D. M. (2008) Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2. Mol. Biol. Cell 19, 4570–4579.PubMedCrossRefGoogle Scholar
  25. 25.
    Gnann, A., Riordan, J. R., and Wolf, D. H. (2004) Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast. Mol. Biol. Cell 15, 4125–4135.PubMedCrossRefGoogle Scholar
  26. 26.
    Younger, J. M., Chen, L., Ren, H. Y., Rosser, M. F., Turnbull, E. L., Fan, C. Y., et al. (2006) Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 126, 571–582.PubMedCrossRefGoogle Scholar
  27. 27.
    Schmidt, B. Z., Watts, R. J., Aridor, M., and Frizzell, R. A. (2009) Cysteine string protein promotes proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) by increasing its interaction with the C terminus of Hsp70-interacting protein and promoting CFTR ubiquitylation. J. Biol. Chem. 284, 4168–4178.PubMedCrossRefGoogle Scholar
  28. 28.
    Du, K., Sharma, M., and Lukacs, G. L. (2005) The DeltaF508 cystic fibrosis mutation impairs domain-domain interactions and arrests post-translational folding of CFTR. Nat. Struct. Biol. 12, 17–25.CrossRefGoogle Scholar
  29. 29.
    Cui, L., Aleksandrov, L., Chang, X. B., Hou, Y. X., He, L., Hegedus, T., et al. (2007) Domain interdependence in the biosynthetic assembly of CFTR. J. Mol. Biol. 365, 981–994.PubMedCrossRefGoogle Scholar
  30. 30.
    Kleizen, B., van Vlijmen, T., de Jonge, H. R., and Braakman, I. (2005) Folding of CFTR is predominantly cotranslational. Mol. Cell 20, 277–287.PubMedCrossRefGoogle Scholar
  31. 31.
    Atwell, S., Brouillette, C. G., Conners, K., Emtage, S., Gheyi, T., Guggino, W. B., et al. (2010) Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant. Protein Eng. Des. Sel. 23, 375–384.PubMedCrossRefGoogle Scholar
  32. 32.
    Lewis, H. A., Wang, C., Zhao, X., Hamuro, Y., Conners, K., Kearins, M. C., et al. (2010) Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry. J. Mol. Biol. 396, 406–430.PubMedCrossRefGoogle Scholar
  33. 33.
    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
  34. 34.
    Thibodeau, P. H., Brautigam, C. A., Machius, M., and Thomas, P. J. (2005) Side chain and backbone contributions of Phe508 to CFTR folding. Nat. Struct. Mol. Biol. 12, 10–16.PubMedCrossRefGoogle Scholar
  35. 35.
    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
  36. 36.
    Dawson, R. J., and Locher, K. P. (2006) Structure of a bacterial multidrug ABC transporter. Nature 443, 180–185.PubMedCrossRefGoogle Scholar
  37. 37.
    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
  38. 38.
    Reyes, C. L., and Chang, G. (2005) Lipopolysaccharide stabilizes the crystal packing of the ABC transporter MsbA. Acta Crystallogr. 61, 655–658.Google Scholar
  39. 39.
    Kanelis, V., Hudson, R. P., Thibodeau, P. H., Thomas, P. J., and Forman-Kay, J. D. (2010) NMR evidence for differential phosphorylation-dependent interactions in WT and DeltaF508 CFTR. EMBO J. 29, 263–277.PubMedCrossRefGoogle Scholar
  40. 40.
    Hartman, J., Huang, Z., Rado, T. A., Peng, S., Jilling, T., Muccio, D. D., et al. (1992) Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product. J. Biol. Chem. 267, 6455–6458.PubMedGoogle Scholar
  41. 41.
    Ko, Y. H., Thomas, P. J., Delannoy, M. R., and Pedersen, P. L. (1993) The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein. J. Biol. Chem. 268, 24330–24338.PubMedGoogle Scholar
  42. 42.
    Neville, D. C., Rozanas, C. R., Tulk, B. M., Townsend, R. R., and Verkman, A. S. (1998) Expression and characterization of the NBD1-R domain region of CFTR: evidence for subunit-subunit interactions. Biochemistry 37, 2401–2409.PubMedCrossRefGoogle Scholar
  43. 43.
    Qu, B. H., and Thomas, P. J. (1996) Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway. J. Biol. Chem. 271, 7261–7264.PubMedCrossRefGoogle Scholar
  44. 44.
    Yike, I., Ye, J., Zhang, Y., Manavalan, P., Gerken, T. A., and Dearborn, D. G. (1996) A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms. Protein Sci. 5, 89–97.PubMedCrossRefGoogle Scholar
  45. 45.
    Chan, K. W., Csanady, L., Seto-Young, D., Nairn, A. C., and Gadsby, D. C. (2000) Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator’s NH(2)-terminal nucleotide binding domain. J. General Phys. 116, 163–180.CrossRefGoogle Scholar
  46. 46.
    Gibson, A. L., Wagner, L. M., Collins, F. S., and Oxender, D. L. (1991) A bacterial system for investigating transport effects of cystic fibrosis-associated mutations. Science 254, 109–111.PubMedCrossRefGoogle Scholar
  47. 47.
    Mossessova, E., and Lima, C. D. (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876.PubMedCrossRefGoogle Scholar
  48. 48.
    Hutt, D. M., Herman, D., Rodrigues, A. P., Noel, S., Pilewski, J. M., Matteson, J., et al. (2010) Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis. Nat. Chem. Biol. 6, 25–33.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • André Schmidt
    • 1
    Email author
  • Juan L. Mendoza
    • 1
  • Philip J. Thomas
    • 1
  1. 1.Department of PhysiologyUniversity of Texas Southwestern Medical CenterDallasUSA

Personalised recommendations