Disease-relevant mutations alter amino acid co-evolution networks in the second nucleotide binding domain of CFTR

Autoři: Gabrianne Ivey aff001;  Robert T. Youker aff003
Působiště autorů: Kyder Christian Academy, Franklin, North Carolina, United States of America aff001;  Southwestern Community College, Sylva, North Carolina, United States of America aff002;  Department of Biology, Western Carolina University, Cullowhee, North Carolina, United States of America aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0227668


Cystic Fibrosis (CF) is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Mutations in CFTR cause impaired chloride ion transport in the epithelial tissues of patients leading to cardiopulmonary decline and pancreatic insufficiency in the most severely affected patients. CFTR is composed of twelve membrane-spanning domains, two nucleotide-binding domains (NBDs), and a regulatory domain. The most common mutation in CFTR is a deletion of phenylalanine at position 508 (ΔF508) in NBD1. Previous research has primarily concentrated on the structure and dynamics of the NBD1 domain; However numerous pathological mutations have also been found in the lesser-studied NBD2 domain. We have investigated the amino acid co-evolved network of interactions in NBD2, and the changes that occur in that network upon the introduction of CF and CF-related mutations (S1251N(T), S1235R, D1270N, N1303K(T)). Extensive coupling between the α- and β-subdomains were identified with residues in, or near Walker A, Walker B, H-loop and C-loop motifs. Alterations in the predicted residue network varied from moderate for the S1251T perturbation to more severe for N1303T. The S1235R and D1270N networks varied greatly compared to the wildtype, but these CF mutations only affect ion transport preference and do not severely disrupt CFTR function, suggesting dynamic flexibility in the network of interactions in NBD2. Our results also suggest that inappropriate interactions between the β-subdomain and Q-loop could be detrimental. We also identified mutations predicted to stabilize the NBD2 residue network upon introduction of the CF and CF-related mutations, and these predicted mutations are scored as benign by the MUTPRED2 algorithm. Our results suggest the level of disruption of the co-evolution predictions of the amino acid networks in NBD2 does not have a straightforward correlation with the severity of the CF phenotypes observed.

Klíčová slova:

Algorithms – Amino acid sequence analysis – Covariance – Cystic fibrosis – Multiple alignment calculation – Sequence alignment – Sequence analysis – Sequence motif analysis


1. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245: 1066–1073. doi: 10.1126/science.2475911 2475911

2. Lopes-Pacheco M. CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis. Front Pharmacol. 2016;7. doi: 10.3389/fphar.2016.00275 27656143

3. Kerem E, Corey M, Kerem B, Rommens J, Markiewicz D, Levison H, et al. The Relation between Genotype and Phenotype in Cystic Fibrosis—Analysis of the Most Common Mutation (ΔF508). N Engl J Med. 1990;323: 1517–1522. doi: 10.1056/NEJM199011293232203 2233932

4. Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC. In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. EMBO J. 2006;25: 4728–4739. doi: 10.1038/sj.emboj.7601373 17036051

5. Csanády L, Nairn AC, Gadsby DC. Thermodynamics of CFTR Channel Gating: A Spreading Conformational Change Initiates an Irreversible Gating Cycle. J Gen Physiol. 2006;128: 523–533. doi: 10.1085/jgp.200609558 17043148

6. The Clinical and Functional TRanslation of CFTR (CFTR2). [cited 20 Feb 2018]. Available: http://cftr2.org

7. Proctor EA, Kota P, Aleksandrov AA, He L, Riordan JR, Dokholyan NV. Rational coupled dynamics network manipulation rescues disease-relevant mutant cystic fibrosis transmembrane conductance regulator. Chem Sci. 2015;6: 1237–1246. doi: 10.1039/c4sc01320d 25685315

8. Aleksandrov AA, Kota P, Cui L, Jensen T, Alekseev AE, Reyes S, et al. Allosteric Modulation Balances Thermodynamic Stability and Restores Function of ΔF508 CFTR. J Mol Biol. 2012;419: 41–60. doi: 10.1016/j.jmb.2012.03.001 22406676

9. Chong PA, Kota P, Dokholyan NV, Forman-Kay JD. Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability. Cold Spring Harb Perspect Med. 2013;3: a009522–a009522. doi: 10.1101/cshperspect.a009522 23457292

10. He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, et al. Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ΔF508 CFTR Folding and Assembly. J Mol Biol. 2015;427: 106–120. doi: 10.1016/j.jmb.2014.07.026 25083918

11. Federici S, Iron A, Reboul MP, Desgeorges M, Claustres M, Bremont F, et al. [CFTR gene analyis in 207 patients with cystic fibrosis in southwest France: high frequency of N1303K and 1811+1.6bA>G mutations]. Arch Pediatr Organe Off Soc Francaise Pediatr. 2001;8: 150–157.

12. Feldmann D, Couderc R, Audrezet M-P, Ferec C, Bienvenu T, Desgeorges M, et al. CFTR genotypes in patients with normal or borderline sweat chloride levels. Hum Mutat. 2003;22: 340–340. doi: 10.1002/humu.9183 12955726

13. Hollenstein K, Dawson RJ, Locher KP. Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol. 2007;17: 412–418. doi: 10.1016/j.sbi.2007.07.003 17723295

14. Yuan Y-R, Blecker S, Martsinkevich O, Millen L, Thomas PJ, Hunt JF. The Crystal Structure of the MJ0796 ATP-binding Cassette: IMPLICATIONS FOR THE STRUCTURAL CONSEQUENCES OF ATP HYDROLYSIS IN THE ACTIVE SITE OF AN ABC TRANSPORTER. J Biol Chem. 2001;276: 32313–32321. doi: 10.1074/jbc.M100758200 11402022

15. Moran O. The gating of the CFTR channel. Cell Mol Life Sci. 2017;74: 85–92. doi: 10.1007/s00018-016-2390-z 27696113

16. Rapino D, Sabirzhanova I, Lopes-Pacheco M, Grover R, Guggino WB, Cebotaru L. Rescue of NBD2 Mutants N1303K and S1235R of CFTR by Small-Molecule Correctors and Transcomplementation. Brusgaard K, editor. PLOS ONE. 2015;10: e0119796. doi: 10.1371/journal.pone.0119796 25799511

17. Mendoza J, Schmidt A, Li Q, Nuvaga E, Barrett T, Bridges R, et al. Requirements for Efficient Correction of? F508 CFTR Revealed by Analyses of Evolved Sequences. Cell. 2012;148: 164–174. doi: 10.1016/j.cell.2011.11.023 22265409

18. Szollosi A, Vergani P, Csanády L. Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2. J Gen Physiol. 2010;136: 407–423. doi: 10.1085/jgp.201010434 20876359

19. Gulyás-Kovács A. Integrated Analysis of Residue Coevolution and Protein Structure in ABC Transporters. Jordan IK, editor. PLoS ONE. 2012;7: e36546. doi: 10.1371/journal.pone.0036546 22590562

20. Vernon RM, Chong PA, Lin H, Yang Z, Zhou Q, Aleksandrov AA, et al. Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations. J Biol Chem. 2017;292: 14147–14164. doi: 10.1074/jbc.M116.772335 28655774

21. Hsu Y-H, Traugh JA. Reciprocally coupled residues crucial for protein kinase Pak2 activity calculated by statistical coupling analysis. PloS One. 2010;5: e9455. doi: 10.1371/journal.pone.0009455 20209159

22. Hatley ME, Lockless SW, Gibson SK, Gilman AG, Ranganathan R. Allosteric determinants in guanine nucleotide-binding proteins. Proc Natl Acad Sci. 2003;100: 14445–14450. doi: 10.1073/pnas.1835919100 14623969

23. Kolaczkowski M, Środa-Pomianek K, Kolaczkowska A, Michalak K. A conserved interdomain communication pathway of pseudosymmetrically distributed residues affects substrate specificity of the fungal multidrug transporter Cdr1p. Biochim Biophys Acta BBA—Biomembr. 2013;1828: 479–490. doi: 10.1016/j.bbamem.2012.10.024 23122779

24. Halabi N, Rivoire O, Leibler S, Ranganathan R. Protein Sectors: Evolutionary Units of Three-Dimensional Structure. Cell. 2009;138: 774–786. doi: 10.1016/j.cell.2009.07.038 19703402

25. Cheng RR, Raghunathan M, Noel JK, Onuchic JN. Constructing sequence-dependent protein models using coevolutionary information: Constructing Sequence-Dependent Protein Models. Protein Sci. 2016;25: 111–122. doi: 10.1002/pro.2758 26223372

26. George AM (Ed.). ABC Transporters—40 Years on. 1st ed. Springer Press; 2016.

27. Osborne L, Santis G, Schwarz M, Klinger K, Dörk T, McIntosh I, et al. Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene. Hum Genet. 1992;89: 653–658. doi: 10.1007/bf00221957 1380943

28. Farhat R, Puissesseau G, El-Seedy A, Pasquet M-C, Adolphe C, Corbani S, et al. N1303K (c.3909C>G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C>T] Complex Allele in CFTR Exon 7 Aberrant Splicing. BioMed Res Int. 2015;2015: 1–8. doi: 10.1155/2015/138103 26075213

29. LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, et al. Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis. McCarty N, editor. PLoS Genet. 2014;10: e1004376. doi: 10.1371/journal.pgen.1004376 25033378

30. Abdul Wahab A, Al Thani G, Dawod ST, Kambouris M, Al Hamed M. Heterogeneity of the cystic fibrosis phenotype in a large kindred family in Qatar with cystic fibrosis mutation (I1234V). J Trop Pediatr. 2001;47: 110–112. doi: 10.1093/tropej/47.2.110 11336127

31. Banjar H. Geographic distribution of cystic fibrosis transmembrane regulator gene mutations in Saudi Arabia. East Mediterr Health J Rev Sante Mediterr Orient Al-Majallah Al-Sihhiyah Li-Sharq Al-Mutawassit. 1999;5: 1230–1235.

32. Noordhoek-van der J.J., Gulmans V.A.M., van de Graaf E.A., Hendriks J.J.E., Heijerman H.G.M., Janssens H.M., Koppelman G.H., Merkus P.J.F.M., Nuijsink M., van der Vaart H., Terheggen-Lagro S.W.J., de K.M. Winter-de Groot Staay. Dutch Cystic Fibrosis Registry Report. Dutch CF Registry; 2017 Oct. Available: https://www.ncfs.nl/bestanden/cf-registratie/report_dutch_cf_registry_2016.pdf

33. Anderson M, Welsh M. Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains. Science. 1992;257: 1701–1704. doi: 10.1126/science.1382316 1382316

34. Yu H, Burton B, Huang C-J, Worley J, Cao D, Johnson JP, et al. Ivacaftor potentiation of multiple CFTR channels with gating mutations. J Cyst Fibros. 2012;11: 237–245. doi: 10.1016/j.jcf.2011.12.005 22293084

35. Dekker JP, Fodor A, Aldrich RW, Yellen G. A perturbation-based method for calculating explicit likelihood of evolutionary co-variance in multiple sequence alignments. Bioinformatics. 2004;20: 1565–1572. doi: 10.1093/bioinformatics/bth128 14962924

36. Kass I, Horovitz A. Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations. Proteins Struct Funct Genet. 2002;48: 611–617. doi: 10.1002/prot.10180 12211028

37. Göbel U, Sander C, Schneider R, Valencia A. Correlated mutations and residue contacts in proteins. Proteins Struct Funct Genet. 1994;18: 309–317. doi: 10.1002/prot.340180402 8208723

38. Lockless SW, Ranganathan R. Evolutionarily conserved pathways of energetic connectivity in protein families. Science. 1999;286: 295–299. doi: 10.1126/science.286.5438.295 10514373

39. Dib L, Carbone A. Protein Fragments: Functional and Structural Roles of Their Coevolution Networks. Promponas VJ, editor. PLoS ONE. 2012;7: e48124. doi: 10.1371/journal.pone.0048124 23139761

40. Sullivan BJ, Durani V, Magliery TJ. Triosephosphate Isomerase by Consensus Design: Dramatic Differences in Physical Properties and Activity of Related Variants. J Mol Biol. 2011;413: 195–208. doi: 10.1016/j.jmb.2011.08.001 21839742

41. Durani V, Magliery TJ. Protein Engineering and Stabilization from Sequence Statistics. Methods in Enzymology. Elsevier; 2013. pp. 237–256. doi: 10.1016/B978-0-12-394292-0.00011–4

42. Fodor AA, Aldrich RW. Influence of conservation on calculations of amino acid covariance in multiple sequence alignments. Proteins Struct Funct Bioinforma. 2004;56: 211–221. doi: 10.1002/prot.20098 15211506

43. Sullivan BJ, Nguyen T, Durani V, Mathur D, Rojas S, Thomas M, et al. Stabilizing Proteins from Sequence Statistics: The Interplay of Conservation and Correlation in Triosephosphate Isomerase Stability. J Mol Biol. 2012;420: 384–399. doi: 10.1016/j.jmb.2012.04.025 22555051

44. Oteri F, Nadalin F, Champeimont R, Carbone A. BIS2Analyzer: a server for co-evolution analysis of conserved protein families. Nucleic Acids Res. 2017;45: W307–W314. doi: 10.1093/nar/gkx336 28472458

45. Champeimont R, Laine E, Hu S-W, Penin F, Carbone A. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins. Sci Rep. 2016;6. doi: 10.1038/srep26401 27198619

46. Pejaver V, Urresti J, Lugo-Martinez J, Pagel KA, Lin GN, Nam H-J, et al. MutPred2: inferring the molecular and phenotypic impact of amino acid variants. bioRxiv. 2017 [cited 13 Feb 2019]. doi: 10.1101/134981

47. Tang Y, Li M, Wang J, Pan Y, Wu F-X. CytoNCA: A cytoscape plugin for centrality analysis and evaluation of protein interaction networks. Biosystems. 2015;127: 67–72. doi: 10.1016/j.biosystems.2014.11.005 25451770

48. Pele J, Taddese B, Deniaud M, Garnier A, Henrion D, Abdi H, et al. Co-Variation Approaches to the Evolution of Protein Families. Adv Tech Biol Med. 2017;05. doi: 10.4172/2379-1764.1000250

49. Liu F, Zhang Z, Csanády L, Gadsby DC, Chen J. Molecular Structure of the Human CFTR Ion Channel. Cell. 2017;169: 85–95.e8. doi: 10.1016/j.cell.2017.02.024 28340353

50. Shannon P. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 2003;13: 2498–2504. doi: 10.1101/gr.1239303 14597658

51. Yang R, Scavetta R, Chang X. Interaction between the Bound Mg·ATP and the Walker A Serine Residue in NBD2 of Multidrug Resistance-Associated Protein MRP1 Plays a Crucial Role for the ATP-Dependent Leukotriene C4 Transport †. Biochemistry. 2008;47: 8456–8464. doi: 10.1021/bi8007643 18636743

52. Berger AL, Ikuma M, Welsh MJ. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain. Proc Natl Acad Sci. 2005;102: 455–460. doi: 10.1073/pnas.0408575102 15623556

53. Ramjeesingh M, Li C, Garami E, Huan L-J, Galley K, Wang Y, et al. Walker Mutations Reveal Loose Relationship between Catalytic and Channel-Gating Activities of Purified CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) †. Biochemistry. 1999;38: 1463–1468. doi: 10.1021/bi982243y 9931011

54. Douam F, Fusil F, Enguehard M, Dib L, Nadalin F, Schwaller L, et al. A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism. Pierson TC, editor. PLOS Pathog. 2018;14: e1006908. doi: 10.1371/journal.ppat.1006908 29505618

55. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14: 33–38, 27–28. doi: 10.1016/0263-7855(96)00018-5 8744570

56. John Edward Stone. Tachyon ray tracing library (built-into VMD and/or distributed with VMD). Computer Science Department, University of Missouri-Rolla. 1998. Available: http://scholarsmine.mst.edu/masters_theses/1747

57. Parente DJ, Ray JCJ, Swint-Kruse L. Amino acid positions subject to multiple coevolutionary constraints can be robustly identified by their eigenvector network centrality scores. Proteins. 2015;83: 2293–2306. doi: 10.1002/prot.24948 26503808

58. Orelle C, Alvarez FJD, Oldham ML, Orelle A, Wiley TE, Chen J, et al. Dynamics of -helical subdomain rotation in the intact maltose ATP-binding cassette transporter. Proc Natl Acad Sci. 2010;107: 20293–20298. doi: 10.1073/pnas.1006544107 21059948

59. Lewis HA, Wang C, Zhao X, Hamuro Y, Conners K, Kearins MC, et al. Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry. J Mol Biol. 2010;396: 406–430. doi: 10.1016/j.jmb.2009.11.051 19944699

60. Zhang Z, Liu F, Chen J. Molecular structure of the ATP-bound, phosphorylated human CFTR. Proc Natl Acad Sci. 2018;115: 12757–12762. doi: 10.1073/pnas.1815287115 30459277

61. René C, Paulet D, Girodon E, Costa C, Lalau G, Leclerc J, et al. p.Ser1235Arg should no longer be considered as a cystic fibrosis mutation: results from a large collaborative study. Eur J Hum Genet. 2011;19: 36–42. doi: 10.1038/ejhg.2010.137 20717170

62. Tarentino AL, Maley F. A comparison of the substrate specificities of endo-beta-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus Pneumoniae. Biochem Biophys Res Commun. 1975;67: 455–462. doi: 10.1016/0006-291x(75)90337-x 1016

63. Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, et al. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell. 2000;101: 789–800. doi: 10.1016/s0092-8674(00)80890-9 10892749

64. Kloch M, Milewski M, Nurowska E, Dworakowska B, Cutting G, Dolowy K. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing. Cell Physiol Biochem. 2010;25: 169–180. doi: 10.1159/000276549 20110677

65. Dong Q, Ernst SE, Ostedgaard LS, Shah VS, Ver Heul AR, Welsh MJ, et al. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia. J Biol Chem. 2015;290: 14140–14153. doi: 10.1074/jbc.M114.611616 25887396

66. Sato S, Ward CL, Krouse ME, Wine JJ, Kopito RR. Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation. J Biol Chem. 1996;271: 635–638. doi: 10.1074/jbc.271.2.635 8557666

67. Urbatsch IL, Gimi K, Wilke-Mounts S, Senior AE. Conserved Walker A Ser Residues in the Catalytic Sites of P-glycoprotein Are Critical for Catalysis and Involved Primarily at the Transition State Step. J Biol Chem. 2000;275: 25031–25038. doi: 10.1074/jbc.M003962200 10831598

68. Orelle C, Dalmas O, Gros P, Di Pietro A, Jault J-M. The Conserved Glutamate Residue Adjacent to the Walker-B Motif Is the Catalytic Base for ATP Hydrolysis in the ATP-binding Cassette Transporter BmrA. J Biol Chem. 2003;278: 47002–47008. doi: 10.1074/jbc.M308268200 12968023

69. Moody JE, Millen L, Binns D, Hunt JF, Thomas PJ. Cooperative, ATP-dependent Association of the Nucleotide Binding Cassettes during the Catalytic Cycle of ATP-binding Cassette Transporters. J Biol Chem. 2002;277: 21111–21114. doi: 10.1074/jbc.C200228200 11964392

70. Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. EMBO J. 2005;24: 1901–1910. doi: 10.1038/sj.emboj.7600657 15889153

71. DeStefano S, Gees M, Hwang T-C. Physiological and pharmacological characterization of the N1303K mutant CFTR. J Cyst Fibros. 2018;17: 573–581. doi: 10.1016/j.jcf.2018.05.011 29887518

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