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Purification, Characterization, and Expression of CFTR Nucleotide-Binding Domains

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Abstract

The nucleotide binding domains (NBDs) within CFTR were initially predicted to lie in the cell cytoplasm, and to gate anion permeability through a pore that was present in membrane spanning α helices of the overall polypeptide. Our studies designed to characterize CFTR suggest several important features of the isolated nucleotide binding domain. NBD-1 appears to bind nucleotides with similar affinity to the full-length CFTR protein. In solution, the domain contains a high β sheet content and self-associates into ordered polymers with molecular mass greater than 300,000 Daltons. The domain is very lipophilic, disrupts liposomes, and readily enters the planar lipid bilayer. Clinically important mutations in the domain may disrupt the nucleotide binding capabilities of the protein, either through a direct effect on the nucleotide binding site, or through effects that influence the overall folding of the domain in vitro. Finally, after expression in human epithelial cells (including epithelial cells from a CF patient), the first nucleotide binding domain targets the plasma membrane even in the absence of other constituents of full-length CFTR and mediates anion permeability in these cells.

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REFERENCES

  1. J. R. Riordan, J. M. Rommens, B. T. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J. L. Chou, M. L. Drumm, M. C. Iannuzzi, F. S. Collins, and L. C. Tsui, “Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA,” Science 245, 1066–1073 (1989).

    Google Scholar 

  2. S. C. Hyde, P. Emsley, M. J. Martshorn, M. M. Mimmack, U. Gileadi, S. R. Pearce, M. P. Gallagher, D. R. Gill, R. E. Hubbard, and C. F. Higgins, “Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance, and bacterial transport,” Science 346, 362–365 (1990).

    Google Scholar 

  3. C. S. Mimura, S. R. Holdbrook, and G. F. I. Ames. “Structural model of the nucleotide binding conserved component of periplasmic permeases,” Proc. Natl. Acad. Sci. USA 88, 84–88 (1991).

    Google Scholar 

  4. J. Hartman, Z. Huang, T. A. Rado, S. Peng, T. Jilling, D. D. Muccio, and E. J. Sorscher, “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 (1992).

    Google Scholar 

  5. N. Arispe, E. Rojas, J. Hartman, E. J. Sorscher, and H. Pollard. “Intrinsic anion channel activity of the recombinant first nucleotide binding fold domain of the cystic fibrosis transmembrane regulator protein,” Proc. Natl. Acad. Sci. USA 89, 1539–1543 (1992).

    Google Scholar 

  6. J. Logan, D. Hiestand, P. Daram, Z. Huang, D. D. Muccio, B. Haley, W. Cook, and E. J. Sorscher, “CFTR mutations which disrupt nucleotide binding,” J. Clin. Invest. 94, 228–236 (1994).

    Google Scholar 

  7. B. E. Cohen, G. Lee, K. A. Jacobson, Y.-C. Kim, Z. Huang, E. J. Sorscher, and H. B. Pollard, “CPX (1,3-dipropyl-8-cyclo-pentylxanthine) and other alkylxanthines differentially bind to the wild type and ΔF508 mutant first nucleotide-binding fold (NBF-1) domains of the cystic fibrosis transmembrane conductance regulator,” Biochemistry 36, 6455–6461 (1997).

    Google Scholar 

  8. S. Peng, M. Sommerfelt, J. Logan, Z. Huang, T. Jilling, K. Kirk, E. Hunter, and E. J. Sorscher, “One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system,” Protein Express. Purif. 4, 95–100 (1993).

    Google Scholar 

  9. S. Peng, M. A. Sommerfelt, G. Berta, A. K. Berry, K. Kirk, E. Hunter, and E. J. Sorscher, “Rapid purification of recombinant baculovirus using fluorescence-activated cell sorting,” Biotechniques 14, 274–277 (1993).

    Google Scholar 

  10. J. Hartman, R. A. Frizzell, T. A. Rado, D. J. Benos, and E. J. Sorscher, “Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase,” Biotechnol. Bioeng. 39, 828–832 (1992).

    Google Scholar 

  11. J. P. Clancy, J. S. Hong, Z. Bebok, S. A. King, S. Demolombe, M. D. DuVall, D. M. Bedwell, and E. J. Sorscher, “Expression of R553X, G542X, and CFTR nucleotide-binding domain-1 causes increased anion permeability in epithelial cells,” submitted.

  12. S. A. King, J. P. Clancy, P. E. Prevelige, Jr., Z. Huang, J. S. Hong, M. J. Jablonski, J. J. Lebowitz, and E. J. Sorscher, “ΔF508 decreases the self-association of the first nucleotide-binding domain of CFTR,” Ped. Pulm. Suppl. 13, 37 (1996).

    Google Scholar 

  13. E. A. Pasyk, and J. K. Foskett, “Mutant (ΔF508) cystic fibrosis transmembrane conductance regulator Cl− channel is functional when retained in endoplasmic reticulum of mammalian cells,” J. Biol. Chem. 270, 12347–12350 (1995).

    Google Scholar 

  14. C. E. Bear, C. Li, N. Kartner, R. J. Bridges, T. J. Jensen, M. Ramjeesingh, and J. R. Riordan, “Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR),” Cell 68, 809–818 (1992).

    Google Scholar 

  15. Z. Bebok, C. J. Venglarik, Z. Panczel, T. Jilling, K. L. Kirk, and E. J. Sorscher, “Epithelial polarity and trans-epithelial transport through ΔF508 CFTR,” submitted.

  16. B. H. Qu, and P. J. Thomas, “Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway,” J. Biol. Chem. 271, 7261–7264 (1996).

    Google Scholar 

  17. P. J. Thomas, Y. H. Ko, and P. L. Pedersen, “Altered protein folding may be the molecular basis of most cases of cystic fibrosis,” FEBS Lett. 312, 7–9 (1992).

    Google Scholar 

  18. J. Logan, D. Hiestand, J. Hartman, Z. Huang, D. D. Muccio, B. Haley, W. J. Cook, and E. J. Sorscher, “CFTR mutations which disrupt nucleotide binding,” Ped. Pulm. Suppl. 9, 204 (1993).

    Google Scholar 

  19. S. Bar-Noy, P. McPhie, G. Lee, Z. Huang, E. J. Sorscher, O. Eidelman, and H. B. Pollard, “ΔF508 mutation in CFTR modifies phospholipid interactions with NBF-1 domain: Implications for the pathogenesis of cystic fibrosis,” Ped. Pulmon. Suppl. 13, 17a (1996).

    Google Scholar 

  20. G. Lee, B. E. Cohen, S. Bar-Noy, O. Eidelman, K. A. Jacobson, and H. B. Pollard, “CPX binds to 30-residue peptide domain lα (CFTR [477-508]) from first nucleotide binding fold, and potentiates peptide-membrane interactions,” Ped. Pulmon. Suppl. 12, 23a (1995).

    Google Scholar 

  21. V. Baichwal, D. Liu, and G. F. L. Ames, “The ATP-binding component of prokaryotic traffic ATPase is exposed to the periplasmic (external) surface,” Proc. Natl. Acad. Sci. USA 90, 620–624 (1993).

    Google Scholar 

  22. E. Schneider, S. Hunke, and S. Tebbe, “The MalK protein of the ATP-binding cassette transporter for maltose of Escherichia coli is accessible to protease digestion from the periplasmic side of the membrane,” J. Bacteriol. 177(18), 5364–5367 (1995).

    Google Scholar 

  23. R. Dudler, C. Schmidhauser, R. W. Parish, R. E. H. Wettenhall, and T. Schmidt, “A mycoplasma high-affinity transport system and the in vivo invasiveness of mouse sarcoma cells,” EMBO J. 7, 3963–3970 (1988).

    Google Scholar 

  24. I. D. Hiles, M. P. Gallagher, D. J. Jamieson, and C. F. Higgins. “Molecular characterization of the oligopeptide permease of Salmonella typhimurium,” J. Mol. Biol. 195, 125–142 (1987).

    Google Scholar 

  25. C. A. Doige and G. F. Ames, “ATP-dependent transport systems in bacteria and humans: Relevance to cystic fibrosis and multidrug resistance,” Annu. Rev. Microbiol. 47, 291–319 (1993).

    Google Scholar 

  26. Y. H. Ko, M. R. Delannoy, and P. L. Pedersen, “Cytosolic localization of NBF1 of CFTR rather than an association with the lipid bilayer,” Ped. Pulm. Suppl. 12, 16 (1995).

    Google Scholar 

  27. D. B. Gruis, K. E. Franke, and E. M. Price, “Extracellular labeling of recombinant NBD-1,” Ped. Pulmon. Suppl. 12, 6a (1995).

    Google Scholar 

  28. M. P. Anderson, R. J. Gregory, S. Thompson, D. W. Souza, S. Paul, R. C. Mulligan, A. E. Smith, and M. J. Welsh, “Demonstration that CFTR is a chloride channel by alteration of its anion selectivity,” Science 253, 202–205 (1991).

    Google Scholar 

  29. T. P. Carrol, M. M. Morales, S. B. Fulmer, S. S. Allen, T. R. Flotte, G. R. Cutting, and W. B. Guggino, “Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator,” J. Biol. Chem. 270, 11941–11946 (1995).

    Google Scholar 

  30. D. N. Sheppard, L. S. Ostedgaard, D. P. Rich, and M. J. Welsh, “The amino-terminal portion of CFTR forms a regulated Cl− channel,” Cell 76, 1091–1098 (1994).

    Google Scholar 

  31. M. Cheung and M. H. Akabas, “Identification of cystic fibrosis transmembrane conductance regulatory channel-lining residues in and flanking the M6 membrane-spanning segment,” Biophys. J. 70, 2688–2695 (1996).

    Google Scholar 

  32. J. Marshall, J. S. Fang, L. S. Ostedgaard, C. R. O'Riordan, D. Ferrara, J. F. Amara, H. Hoppe IV, R. K. Scheule, M. J. Welsh, A. E. Smith, and S. H. Cheng, “Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro,” J. Biol. Chem. 269, 2987–2995 (1994).

    Google Scholar 

  33. S. Sato, C. L. Ward, M. E. Krouse, J. J. Wine, and R. R. Kopito, “Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation,” J. Biol. Chem. 271, 635–638 (1996).

    Google Scholar 

  34. C. R. Brown, L. Q. Hong-Brown, J. Biwersi, A. S. Verkman, and W. J. Welch, “Chemical chaperones correct the mutant phenotype of the ΔF508 cystic fibrosis transmembrane conductance regulator protein,” Cell Stress Chaperones 1, 117–125 (1996).

    Google Scholar 

  35. P. J. Thomas, as presented at the Williamsburg CF Meetings, Williamsburg, Virginia, 1995.

  36. B. Moss, O. Elroy-Stein, T. Mizukami, W. A. Alexander, and T. R. Fuerst, “New mammalian expression vectors,” Nature 348, 91–92 (1990).

    Google Scholar 

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Authors and Affiliations

  1. Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama

    J. P. Clancy, Zs. Bebök & E. J. Sorscher

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Clancy, J.P., Bebök, Z. & Sorscher, E.J. Purification, Characterization, and Expression of CFTR Nucleotide-Binding Domains. J Bioenerg Biomembr 29, 475–482 (1997). https://doi.org/10.1023/A:1022487024031

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