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. 1995 Dec;69(6):2443–2448. doi: 10.1016/S0006-3495(95)80113-X

Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins.

M R Carson 1, M J Welsh 1
PMCID: PMC1236481  PMID: 8599650

Abstract

The opening and closing of the CFTR Cl- channel are regulated by ATP hydrolysis at its two nucleotide binding domains (NBDs). However, the mechanism and functional significance of ATP hydrolysis are unknown. Sequence similarity between the NBDs of CFTR and GTP-binding proteins suggested the NBDs might have a structure and perhaps a function like that of GTP-binding proteins. Based on this similarity, we predicted that the terminal residue of the LSGGQ motif in the NBDs of CFTR corresponds to a highly conserved glutamine residue in GTP-binding proteins that directly catalyzes the GTPase reaction. Mutations of this residue in NBD1 or NBD2, which were predicted to increase or decrease the rate of hydrolysis, altered the duration of channel closed and open times in a specific manner without altering ion conduction properties or ADP-dependent inhibition. These results suggest that the NBDs of CFTR, and consequently other ABC transporters, may have a structure and a function analogous to those of GTP-binding proteins. We conclude that the rates of ATP hydrolysis at NBD1 and at NBD2 determine the duration of the two states of the channel, closed and open, much as the rate of GTP hydrolysis by GTP-binding proteins determines the duration of their active state.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson M. P., Berger H. A., Rich D. P., Gregory R. J., Smith A. E., Welsh M. J. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell. 1991 Nov 15;67(4):775–784. doi: 10.1016/0092-8674(91)90072-7. [DOI] [PubMed] [Google Scholar]
  2. Anderson M. P., Gregory R. J., Thompson S., Souza D. W., Paul S., Mulligan R. C., Smith A. E., Welsh M. J. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science. 1991 Jul 12;253(5016):202–205. doi: 10.1126/science.1712984. [DOI] [PubMed] [Google Scholar]
  3. Anderson M. P., Rich D. P., Gregory R. J., Smith A. E., Welsh M. J. Generation of cAMP-activated chloride currents by expression of CFTR. Science. 1991 Feb 8;251(4994):679–682. doi: 10.1126/science.1704151. [DOI] [PubMed] [Google Scholar]
  4. Anderson M. P., Welsh M. J. Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains. Science. 1992 Sep 18;257(5077):1701–1704. doi: 10.1126/science.1382316. [DOI] [PubMed] [Google Scholar]
  5. Baukrowitz T., Hwang T. C., Nairn A. C., Gadsby D. C. Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle. Neuron. 1994 Mar;12(3):473–482. doi: 10.1016/0896-6273(94)90206-2. [DOI] [PubMed] [Google Scholar]
  6. Bear C. E., Li C. H., Kartner N., Bridges R. J., Jensen T. J., Ramjeesingh M., Riordan J. R. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell. 1992 Feb 21;68(4):809–818. doi: 10.1016/0092-8674(92)90155-6. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  8. Carson M. R., Travis S. M., Welsh M. J. The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity. J Biol Chem. 1995 Jan 27;270(4):1711–1717. doi: 10.1074/jbc.270.4.1711. [DOI] [PubMed] [Google Scholar]
  9. Carson M. R., Travis S. M., Winter M. C., Sheppard D. N., Welsh M. J. Phosphate stimulates CFTR Cl- channels. Biophys J. 1994 Nov;67(5):1867–1875. doi: 10.1016/S0006-3495(94)80668-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carson M. R., Welsh M. J. 5'-Adenylylimidodiphosphate does not activate CFTR chloride channels in cell-free patches of membrane. Am J Physiol. 1993 Jul;265(1 Pt 1):L27–L32. doi: 10.1152/ajplung.1993.265.1.L27. [DOI] [PubMed] [Google Scholar]
  11. Coleman D. E., Berghuis A. M., Lee E., Linder M. E., Gilman A. G., Sprang S. R. Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science. 1994 Sep 2;265(5177):1405–1412. doi: 10.1126/science.8073283. [DOI] [PubMed] [Google Scholar]
  12. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  13. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  16. Graziano M. P., Gilman A. G. Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha. J Biol Chem. 1989 Sep 15;264(26):15475–15482. [PubMed] [Google Scholar]
  17. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  18. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  19. Hwang T. C., Nagel G., Nairn A. C., Gadsby D. C. Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4698–4702. doi: 10.1073/pnas.91.11.4698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hyde S. C., Emsley P., Hartshorn M. J., Mimmack M. M., Gileadi U., Pearce S. R., Gallagher M. P., Gill D. R., Hubbard R. E., Higgins C. F. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990 Jul 26;346(6282):362–365. doi: 10.1038/346362a0. [DOI] [PubMed] [Google Scholar]
  21. Kartner N., Hanrahan J. W., Jensen T. J., Naismith A. L., Sun S. Z., Ackerley C. A., Reyes E. F., Tsui L. C., Rommens J. M., Bear C. E. Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. Cell. 1991 Feb 22;64(4):681–691. doi: 10.1016/0092-8674(91)90498-n. [DOI] [PubMed] [Google Scholar]
  22. Kleuss C., Raw A. S., Lee E., Sprang S. R., Gilman A. G. Mechanism of GTP hydrolysis by G-protein alpha subunits. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9828–9831. doi: 10.1073/pnas.91.21.9828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Landis C. A., Masters S. B., Spada A., Pace A. M., Bourne H. R., Vallar L. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature. 1989 Aug 31;340(6236):692–696. doi: 10.1038/340692a0. [DOI] [PubMed] [Google Scholar]
  24. Masters S. B., Miller R. T., Chi M. H., Chang F. H., Beiderman B., Lopez N. G., Bourne H. R. Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase. J Biol Chem. 1989 Sep 15;264(26):15467–15474. [PubMed] [Google Scholar]
  25. McGrath J. P., Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature. 1989 Aug 3;340(6232):400–404. doi: 10.1038/340400a0. [DOI] [PubMed] [Google Scholar]
  26. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  27. Mimmack M. L., Gallagher M. P., Pearce S. R., Hyde S. C., Booth I. R., Higgins C. F. Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8257–8261. doi: 10.1073/pnas.86.21.8257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mimura C. S., Holbrook S. R., Ames G. F. Structural model of the nucleotide-binding conserved component of periplasmic permeases. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):84–88. doi: 10.1073/pnas.88.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  31. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sondek J., Lambright D. G., Noel J. P., Hamm H. E., Sigler P. B. GTPase mechanism of Gproteins from the 1.7-A crystal structure of transducin alpha-GDP-AIF-4. Nature. 1994 Nov 17;372(6503):276–279. doi: 10.1038/372276a0. [DOI] [PubMed] [Google Scholar]
  33. Tabcharani J. A., Rommens J. M., Hou Y. X., Chang X. B., Tsui L. C., Riordan J. R., Hanrahan J. W. Multi-ion pore behaviour in the CFTR chloride channel. Nature. 1993 Nov 4;366(6450):79–82. doi: 10.1038/366079a0. [DOI] [PubMed] [Google Scholar]
  34. Teem J. L., Berger H. A., Ostedgaard L. S., Rich D. P., Tsui L. C., Welsh M. J. Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast. Cell. 1993 Apr 23;73(2):335–346. doi: 10.1016/0092-8674(93)90233-g. [DOI] [PubMed] [Google Scholar]
  35. Travis S. M., Carson M. R., Ries D. R., Welsh M. J. Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1993 Jul 25;268(21):15336–15339. [PubMed] [Google Scholar]
  36. Winter M. C., Sheppard D. N., Carson M. R., Welsh M. J. Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation. Biophys J. 1994 May;66(5):1398–1403. doi: 10.1016/S0006-3495(94)80930-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

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