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Structure of the cystic fibrosis transmembrane conductance regulator in the inward-facing conformation revealed by single particle electron microscopy

1 Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, UK;
2 University College in Qunfudah, The University of Umm-Alqura, Kingdom of Saudi Arabia;
3 Bioinformatics Institute, 30 Biopolis Street, #07-01 Matrix, 138671 Singapore;
4 Department of Biochemsitry and Biophysics, University of North Carolina, Chapel Hill, 6107 Thurston-Bowles, Campus Box 7248, Chapel Hill, NC 27599, USA

Special Issues: Structural analysis of macromolecules using Cryo electron microscopy

The most common inherited disease in European populations is cystic fibrosis. Mutations in the gene lead to loss of function of the cystic fibrosis transmembrane conductance regulator protein (CFTR). CFTR is a member of the ATP-binding cassette family of membrane proteins that mostly act as active transporters using ATP to move substances across membranes. These proteins undergo large conformational changes during the transport cycle, consistent with an inward-facing to outward-facing translocation mechanism that was originally proposed by Jardetzky. CFTR is the only member of this family of proteins that functions as an ion channel, and in this case ATP and phosphorylation of a regulatory domain controls the opening of the channel. In this article we describe the inward-facing conformation of the protein and show it can be modulated by the presence of a purified recombinant NHERF1-PDZ1 domain that binds with high affinity to the CFTR C-terminal PDZ motif (-QDTRL). ATP hydrolysis activity of CFTR can also be modulated by glutathione, which we postulate may bind to the inward-facing conformation of the protein. A homology model for CFTR, based on a mitochondrial ABC transporter of glutathione in the inward-facing configuration has been generated. The map and the model are discussed with respect to the biology of the channel and the specific relationship between glutathione levels in the cell and CFTR. Finally, disease-causing mutations are mapped within the model and discussed in terms of their likely physiological effects.
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Keywords cystic fibrosis; CFTR; electron microscopy; ABCC7

Citation: Ateeq Al-Zahrani, Natasha Cant, Vassilis Kargas, Tracy Rimington, Luba Aleksandrov, John R. Riordan, Robert C. Ford. Structure of the cystic fibrosis transmembrane conductance regulator in the inward-facing conformation revealed by single particle electron microscopy. AIMS Biophysics, 2015, 2(2): 131-152. doi: 10.3934/biophy.2015.2.131

References

  • 1. Cutting GR (2005) Modifier genetics: cystic fibrosis. Annu Rev Genomics Hum Genet 6: 237-260.    
  • 2. Higgins CF (1992) ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8: 67-113.    
  • 3. Riordan JR, Rommens JM, Kerem B, et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073.    
  • 4. Gray MA, Winpenny JP, Verdon B, et al. (1995) Chloride channels and cystic fibrosis of the pancreas. Biosci Rep 15: 531-541.    
  • 5. McCarty NA (2000) Permeation through the CFTR chloride channel. J Exp Biol 203: 1947-1962.
  • 6. Vankeerberghen A, Cuppens H, Cassiman JJ (2002) The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions. J Cyst Fibros 1: 13-29.    
  • 7. Riordan JR (2008) CFTR function and prospects for therapy. Annu Rev Biochem 77: 701-726.    
  • 8. Loo MA, Jensen TJ, Cui L, et al. (1998) Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome. EMBO J 17: 6879-6887.    
  • 9. Lukacs GL, Chang XB, Bear C, et al. (1993) The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells. J Biol Chem 268: 21592-21598.
  • 10. Lukacs GL, Segal G, Kartner N, et al. (1997) Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem J 328 ( Pt 2): 353-361.
  • 11. O'Ryan L, Rimington T, Cant N, et al. (2012) Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae. J Vis Exp.
  • 12. Huang P, Liu Q, Scarborough GA (1998) Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator. Anal Biochem 259: 89-97.    
  • 13. Huang P, Stroffekova K, Cuppoletti J, et al. (1996) Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast. Biochim Biophys Acta 1281: 80-90.    
  • 14. Ketchum CJ, Rajendrakumar GV, Maloney PC (2004) Characterization of the adenosinetriphosphatase and transport activities of purified cystic fibrosis transmembrane conductance regulator. Biochemistry 43: 1045-1053.    
  • 15. Krueger-Koplin RD, Sorgen PL, Krueger-Koplin ST, et al. (2004) An evaluation of detergents for NMR structural studies of membrane proteins. J Biomol NMR 28: 43-57.    
  • 16. Yang Z, Wang C, Zhou Q, et al. (2014) Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains. Protein Sci Public Protein Soci 23: 769-789.    
  • 17. Matar-Merheb R, Rhimi M, Leydier A, et al. (2011) Structuring detergents for extracting and stabilizing functional membrane proteins. PloS one 6: e18036.    
  • 18. Galian C, Manon F, Dezi M, et al. (2011) Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. PloS one 6: e19677.    
  • 19. Vinothkumar KR, Henderson R (2010) Structures of membrane proteins. Q Rev Biophys 43: 65-158.
  • 20. Guggino WB (2004) The cystic fibrosis transmembrane regulator forms macromolecular complexes with PDZ domain scaffold proteins. Proc Am Thorac Soc 1: 28-32.    
  • 21. Karthikeyan S, Leung T, Birrane G, et al. (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.    
  • 22. Karthikeyan S, Leung T, Ladias JA (2002) Structural determinants of the Na+/H+ exchanger regulatory factor interaction with the beta 2 adrenergic and platelet-derived growth factor receptors. J Biol Chem 277: 18973-18978.    
  • 23. Li C, Naren AP (2011) Analysis of CFTR interactome in the macromolecular complexes. Methods Mol Biol 741: 255-270.    
  • 24. Li C, Roy K, Dandridge K, et al. (2004) Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane. J Biol Chem 279: 24673-24684.    
  • 25. Wang S, Yue H, Derin RB, et al. (2000) Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell 103: 169-179.    
  • 26. Bozoky Z, Krzeminski M, Muhandiram R, et al. (2013) Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions. Proc Natl Acad Sci U S A.
  • 27. Guerra L, Favia M, Fanelli T, et al. (2004) Stimulation of Xenopus P2Y1 receptor activates CFTR in A6 cells. Pflugers Arch 449: 66-75.    
  • 28. Bossard F, Robay A, Toumaniantz G, et al. (2007) NHE-RF1 protein rescues DeltaF508-CFTR function. Am J Physiol Lung Cell Mol Physiol 292: L1085-1094.
  • 29. Zhang L, Aleksandrov LA, Riordan JR, et al. (2011) Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling. Biochim Biophys Acta 1808: 399-404.
  • 30. Zhang L, Aleksandrov LA, Zhao ZF, et al. (2009) Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding. J Struct Biol 167: 242-251.    
  • 31. Rosenberg MF, O'Ryan LP, Hughes G, et al. (2011) The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate. J Biol Chem 286: 42647-42654.    
  • 32. Dawson RJ, Locher KP (2006) Structure of a bacterial multidrug ABC transporter. Nature 443: 180-185.    
  • 33. Ward A, Reyes CL, Yu J, et al. (2007) Flexibility in the ABC transporter MsbA: Alternating access with a twist. Proc Natl Acad Sci U S A 104: 19005-19010.    
  • 34. Hildebrandt E, Zhang Q, Cant N, et al. (2014) A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR). Biochim Biophys Acta 1838: 2825-2837.
  • 35. Al-Zahrani A (2014) Structural biology of Cystic Fibrosis Transmembrane Conductance Regulator, an ATP-binding cassette protein of medical importance. PhD Thesis, University of Manchester.
  • 36. Pollock N, Cant N, Rimington T, et al. (2014) Purification of the cystic fibrosis transmembrane conductance regulator protein expressed in Saccharomyces cerevisiae. J Vis Exp: JoVE.
  • 37. Chifflet S, Torriglia A, Chiesa R, et al. (1988) A method for the determination of inorganic-phosphate in the presence of labile organic phosphate and high-concentrations of protein- application to lens ATPases. Anal Biochem 168: 1-4.    
  • 38. Rothnie A, Theron D, Soceneantu L, et al. (2001) The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence. Eur Biophys J Biophys Lett 30: 430-442.    
  • 39. Cant N, Pollock N, Rimington T, et al. (2014) Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae. J Vis Exp 87.
  • 40. Schultz BD, Bridges RJ, Frizzell RA (1996) Lack of conventional ATPase properties in CFTR chloride channel gating. J Membr Biol 151: 63-75.    
  • 41. Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128: 82-97.    
  • 42. Zhang L, Aleksandrov LA, Riordan JR, et al. (2011) Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling. Biochim Biophys Acta 1808: 399-404.    
  • 43. Zhang L, Aleksandrov LA, Zhao Z, et al. (2009) Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding. J Struct Biol.
  • 44. Henderson R, Sali A, Baker ML, et al. (2012) Outcome of the first electron microscopy validation task force meeting. Structure 20: 205-214.    
  • 45. Srinivasan V, Pierik AJ, Lill R (2014) Crystal structures of nucleotide-free and glutathione-bound mitochondrial ABC transporter Atm1. Science 343: 1137-1140.    
  • 46. Claude JB, Suhre K, Notredame C, et al. (2004) CaspR: a web server for automated molecular replacement using homology modelling. Nucleic Acids Res 32: W606-609.    
  • 47. Eswar N, Webb B, Marti-Renom MA, et al. (2007) Comparative protein structure modeling using MODELLER. Current protocols in protein science/editorial board, John E Coligan Chapter 2: Unit 2 9.
  • 48. Laskowski RA, Rullmannn JA, MacArthur MW, et al. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8: 477-486.
  • 49. Yang Z, Lasker K, Schneidman-Duhovny D, et al. (2012) UCSF Chimera, MODELLER, and IMP: an integrated modeling system. J Struct Biol 179: 269-278.
  • 50. Zhang L, Aleksandrov LA, Riordan JR, et al. (2010) Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling. Biochim Biophys Acta.
  • 51. Zhang L, Aleksandrov LA, Zhao Z, et al. (2009) Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding. J Struct Biol 167: 242-251.    
  • 52. Eckford PD, Li C, Ramjeesingh M, et al. (2012) Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner. J Biol Chem 287: 36639-36649.    
  • 53. Venerando A, Franchin C, Cant N, et al. (2013) Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation. PloS one 8: e74232.    
  • 54. Kogan I, Ramjeesingh M, Huan LJ, et al. (2001) Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity. J Biol Chem 276: 11575-11581.    
  • 55. Aleksandrov AA, Kota P, Cui L, et al. (2012) Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR. J Mol Biol 419: 41-60.    
  • 56. Alexandrov AI, Mileni M, Chien EY, et al. (2008) Microscale fluorescent thermal stability assay for membrane proteins. Structure 16: 351-359.    
  • 57. Lysko KA, Carlson R, Taverna R, et al. (1981) Protein involvement in structural transition of erythrocyte ghosts. Use of thermal gel analysis to detect protein aggregation. Biochemistry 20: 5570-5576.
  • 58. Soler G, Mattingly JR, Martinez-Carrion M (1984) Effects of heating on the ion-gating function and structural domains of the acetylcholine receptor. Biochemistry 23: 4630-4636.    
  • 59. Al Zahrani A (2014) PhD Thesis, University of Manchester.
  • 60. Karthikeyan S, Leung T, Birrane G, et al. (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.
  • 61. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372: 774-797.
  • 62. Karthikeyan S, Leung T, Ladias JA (2002) Structural determinants of the Na+/H+ exchanger regulatory factor interaction with the beta 2 adrenergic and platelet-derived growth factor receptors. J Biol Chem 277: 18973-18978.
  • 63. Karthikeyan S, Leung T, Ladias JA (2001) Structural basis of the Na+/H+ exchanger regulatory factor PDZ1 interaction with the carboxyl-terminal region of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 276: 19683-19686.
  • 64. Zhang L, Aleksandrov LA, Zhao Z, et al. (2009) Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding. J Struct Biol 167: 242-251.
  • 65. Gadsby DC, Vergani P, Csanady L (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440: 477-483.    
  • 66. Ma T, Thiagarajah JR, Yang H, et al. (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110: 1651-1658.    
  • 67. Verkman AS (1990) Development and biological applications of chloride-sensitive fluorescent indicators. Am J Physiol 259: C375-388.
  • 68. Wellhauser L, Kim Chiaw P, Pasyk S, et al. (2009) A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability. Mol Pharmacol 75: 1430-1438.    
  • 69. Laverty G, Anttila A, Carty J, et al. (2012) CFTR mediated chloride secretion in the avian renal proximal tubule. Comp Biochem Physiol A Mol Integr Physiol 161: 53-60.
  • 70. Cole SP (2014) Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future. Ann Rev Pharmacol Toxicol 54: 95-117.
  • 71. Paumi CM, Chuk M, Snider J, et al. (2009) ABC transporters in Saccharomyces cerevisiae and their interactors: new technology advances the biology of the ABCC (MRP) subfamily. Microbiol Mol Biol Rev 73: 577-593.    
  • 72. Maher P (2005) The effects of stress and aging on glutathione metabolism. Ageing Res Rev 4: 288-314.    
  • 73. Kogan I, Ramjeesingh M, Li C, et al. (2003) CFTR directly mediates nucleotide-regulated glutathione flux. Embo J 22: 1981-1989.    
  • 74. Roum JH, Buhl R, McElvaney NG, et al. (1993) Systemic deficiency of glutathione in cystic fibrosis. J Appl Physiol 75: 2419-2424.
  • 75. Gao L, Kim KJ, Yankaskas JR, et al. (1999) Abnormal glutathione transport in cystic fibrosis airway epithelia. Am J Physiol 277: L113-118.
  • 76. Marson FA, Bertuzzo CS, Ribeiro AF, et al. (2014) Polymorphisms in the glutathione pathway modulate cystic fibrosis severity: a cross-sectional study. BMC Med Genet 15: 27.
  • 77. Rubera I, Duranton C, Melis N, et al. (2013) Role of CFTR in oxidative stress and suicidal death of renal cells during cisplatin-induced nephrotoxicity. Cell Death Disease 4: e817.
  • 78. Cui G, Song B, Turki HW, et al. (2012) Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers. Pflugers Arch: Eur J Physiol 463: 405-418.    
  • 79. Bozoky Z, Krzeminski M, Muhandiram R, et al. (2013) Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions. Proc Natl Acad Sci U S A 110: E4427-4436.    

 

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