ABCMdb

ABCC7 p.Phe508Arg

ClinVar:	c.1523T>C , p.Phe508Ser ? , not provided c.1523T>G , p.Phe508Cys N , Benign
CF databases:	c.1521_1523delCTT , p.Phe508del D , CF-causing c.1523T>C , p.Phe508Ser (CFTR1) D , This mutation was found in a patient with CBAVD. c.1523T>G , p.Phe508Cys (CFTR1) ? ,
Predicted by SNAP2:	A: D (95%), C: D (75%), D: D (95%), E: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Side chain and backbone contributions of Phe508 to... Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26. Thibodeau PH, Brautigam CA, Machius M, Thomas PJ
Side chain and backbone contributions of Phe508 to CFTR folding.
Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26., [PMID:15619636]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

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18 genet.sickkids.on.ca/cftr), and F508R, a maturation-deficient mutation5, were determined. Login to comment
33 The F508A,F508M,F508P,F508D,F508Q,F508R and F508S mutant proteins were more similar to the wild type than the ∆F508 protein in their temperature-dependence of refolding (Fig. 1b,c). Login to comment
43 The missense mutant proteins F508A, F508M,F508P,F508D,F508Q,F508R and F508S had similar ∆Gunfolding and m-values, 3.4-3.8 kcal mol-1 and 1.5-1.7 kcal mol-1 M-1 denaturant, respectively, highlighting the fact that changes in the bulk or chemical properties of the substituted side chain had little effect on the native-state stabilities of these domains as measured by denaturation with GuHCl (Table 1). Login to comment
46 How does the isolated NBD accommodate such Temperature (ºC) 4 10 16 22 Fractionalyield 0.0 0.5 1.0 Temperature (ºC) 4 10 16 22 Temperature (ºC) 4 10 16 22 Wild type ∆F508 Wild type ∆F508 ̄ F508A ̄ F508M F508P F508W ͷ F508W W496F Wild type ∆F508 F508Q F508R F508D F508S a b c Figure 1 NBD1 folding efficiency as a function of folding temperature. Login to comment
53 Table 1 Stability of wild-type and mutant NBD proteins Protein ∆Gunfolding ∆∆Gunfolding m-value (kcal mol-1) (kcal mol-1) (kcal mol-1 M-1) Wild type 3.7 ± 0.1 0 1.7 ∆F508 3.6 ± 0.1 0.1 1.7 F508A 3.6 ± 0.2 0.1 1.6 F508M 3.5 ± 0.1 0.1 1.6 F508P 3.5 ± 0.3 0.2 1.6 F508D 3.6 ± 0.1 0.1 1.6 F508Q 3.5 ± 0.2 0.2 1.6 F508R 3.4 ± 0.3 0.3 1.6 F508S 3.8 ± 0.2 -0.1 1.6 considerable changes in amino acid character at position 508 when this position is critical to the proper biogenesis of the full-length protein, and what are the underlying structural changes associated with these substitutions? Login to comment
54 NBD1 structure To directly assess the structural changes associated with the substitution of residues at position 508, crystal structures of two missense-mutant proteins were determined for the highly similar murine NBD1: F508S, a previously identified non-CF-causing variant, and F508R,a previously described maturation- deficientmutation.Theproteinswereexpressed and purified essentially as described for the wild-type protein and crystallized under conditions similar to the wild-type protein in the presence of Mg2+ andATP with sodium acetate as the precipitant26. Login to comment
55 Tetragonal bipyramidal crystals grew for the F508R proteins, whereas the F508S protein spontaneously crystallized as large tetragonal plates. Login to comment
56 The F508S and F508R crystals diffracted to 2.7 and 3.1 Å,respectively and structures were determined with final R/Rfree valuesof 0.207/0.262and0.254/0.266, respectively (Table 2). Login to comment
59 All of the major structural elements are conserved and the r.m.s. deviations between the Cα atoms of the wild type and F508S or F508R structures were <0.33 Å (Fig.2a)26. Login to comment
63 This rotation is observed in only one of two monomers in the asymmetric unit when the phenylalanine is replaced by the larger arginine side chain in the F508R structure (Fig.2c).In both mutants,like the wild type,the side chain of the residue at position 508 is largely surface-exposed and accessible (Fig.2d). Login to comment
67 The conformation of ATP in the wild-type26 and F508S structures is very similar, with a noncanonical interaction between the NBD and ATP and unusual torsional angles in the ATP molecule (SupplementaryFig.1online).In the F508R structure,the two monomers that occur in the asymmetric unit contain ATP in different conformations. Login to comment
71 Examination of the calculated molecular surface of the wild-type, F508S and F508R proteins is revealing. Login to comment
73 Quantification of the surface-accessibility of position 508 reveals that the wild-type and F508S side chains are very similar at 8.5 and 9.6 Å2 respectively.The F508R protein has greater average accessible surface area at position 508, with a value of 16.8 Å2. Login to comment
78 Wild type, green; F508S variant, orange; F508R variant, blue. Login to comment
79 The r.m.s. deviations between the wild-type and F508S or F508R structures are ~0.3 Å. Login to comment
85 (c) The 2Fo - Fc map (contoured at 1 σ) calculated from the F508R data at a resolution of 3.1 Å superposed on the final F508R model. Login to comment
92 The known polymorphism F508C and the non-CF-causing variant F508S both showed measurable quantities of band C at steady-state levels, as would be expected for non-CF-causingsubstitutions.Thehydrophobicaminoacidsubstitutions F508I,F508W and F508Y did not produce substantial steady-state levels of band C as measured by western blotting, nor did the ionizable amino acid substitutions F508D, F508E, F508K, F508H or F508R. Login to comment
103 Most notably, the crystal structures of F508S and F508R both indicate that substitutions for Phe508 do not substantially impact the structure of NBD1,providing further evidence for the high tolerance for substitution at this position in the isolated domain. Login to comment
113 W ild type ∆∆F508 F508 F508D F508K F508E F508R F508H F508S F508T F508N F508Q C B Charged Polar F508A F508C F508I F508L ∆F508 F508 W ild type C B F508W F508Y F508G F508P Hydrophobic F508M F508V ̅̆ ̆ ̅ Figure 3 Maturation of full-length CFTR mutants. Login to comment
178 The structure of F508R was determined using the molecular replacement protocols available in CNS version 1.1 (ref. 48). Login to comment
186 The atomic coordinates and structure factors for the F508S and F508R NBD1 structures have been deposited in the Protein Data Bank (accession codes 1XF9 and 1XFA, respectively). Login to comment
188 Table 2 Data collection and refinement statistics F508S F508R Data collection Space group P4212 I4122 Cell dimensions (Å) a 170.45 139.99 b 170.45 139.99 c 109.07 278.72 Resolution (Å) 40.4-2.7 (2.75-2.7) 44.3-3.1 (3.15-3.1) Rsym 0.078 (0.431) 0.071 (0.987) I / σI 22.9 (2.3) 34.8 (2.5) Completeness (%) 98.8 (95.6) 99.9 (100) Redundancy 7.7 (3.3) 11.9 (10.7) Refinement Resolution (Å) 40.4-2.7 44.3-3.1 No. Login to comment

[hide] Building an understanding of cystic fibrosis on th... J Bioenerg Biomembr. 2007 Dec;39(5-6):499-505. Mendoza JL, Thomas PJ
Building an understanding of cystic fibrosis on the foundation of ABC transporter structures.
J Bioenerg Biomembr. 2007 Dec;39(5-6):499-505., [PMID:18080175]

Abstract [show]
Cystic fibrosis (CF) is a fatal disease affecting the lungs and digestive system by impairment of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). While over 1000 mutations in CFTR have been associated with CF, the majority of cases are linked to the deletion of phenylalanine 508 (delta F508). F508 is located in the first nucleotide binding domain (NBD1) of CFTR. This mutation is sufficient to impair the trafficking of CFTR to the plasma membrane and, thus, its function. As an ABC transporter, recent structural data from the family provide a framework on which to consider the effect of the delta F508 mutation on CFTR. There are fifty-seven known structures of ABC transporters and domains thereof. Only six of these structures are of the intact transporters. In addition, modern bioinformatic tools provide a wealth of sequence and structural information on the family. We will review the structural information from the RCSB structure repository and sequence databases of the ABC transporters. The available structural information was used to construct a model for CFTR based on the ABC transporter homologue, Sav1866, and provide a context for understanding the molecular pathology of Cystic Fibrosis.

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67 In contrast to the large number of sequences available for the NBD align- Table 1 ABC transporter structures in the research collaboratory for structural bioinformatics (RCSB) database Release Date PDB Protein Resolution Species NBD Structures Jun-05 1Z47 CysA 1.9 Alicyclobacillus acidocaldarius Sep-03 1OXX GlcV G144A 1.45 Sulfolobus solfataricus Jun-03 1OXT GlcV nucleotide free 2.1 Sulfolobus solfataricus Jun-03 1OXU GlcV w/ ADP 2.1 Sulfolobus solfataricus Jun-03 1OXV GlcV w/ AMP.PNP 2.1 Sulfolobus solfataricus Jun-03 1OXS GlcV w/ iodide ions 1.65 Sulfolobus solfataricus Nov-99 1B0U HisP w/ ATP 1.5 Salmonella typimurium Jun-03 1MT0 HlyB (467-707) 2.6 Escherichia coli Aug-06 2FFB HlyB E631Q w/ ADP 1.9 Escherichia coli Aug-06 2FGK HlyB E631Q w/ ATP 2.7 Escherichia coli Aug-06 2FFA HlyB H662A w/ ADP 1.7 Escherichia coli Aug-06 2FGJ HlyB H662A w/ ATP 2.6 Escherichia coli Jun-05 1XEF HlyB w/ ATP 2.5 Escherichia coli Aug-06 2FF7 HlyB w/ADP 1.6 Escherichia coli Dec-03 1MV5 LmrA w/ ADP, ATP 3.1 Lactococcus lactis Dec-00 1G29 MalK 1.9 Thermococcus litoralis Sep-03 1Q1E MalK 2.9 Escherichia coli Dec-04 1VCI MalK w/ ATP 2.9 Pyrococcus horikoshii Aug-07 2QRR metN 1.71 Vibrio parahaemolyticus Aug-07 2QSW metN C-terminal domain 1.5 Enterococcus faecalis Jul-02 1L2T MJ0796 E171Q Dimeric Structure 1.9 Methanococcus jannaschii Jul-01 1F3O MJ0796 w/ ADP 2.7 Methanococcus jannaschii Nov-01 1GAJ MJ1267 2.5 Methanococcus jannaschii Jul-01 1G6H MJ1267 w/ ADP 1.6 Methanococcus jannaschii Feb-03 1G9X MJ1267 w/ ADP 2.6 Methanococcus jannaschii May-06 2CBZ MRP1-NBD1 1.5 Homo sapiens Nov-04 1V43 Multi-sugar transporter 2.2 Pyrococcus horikoshii Aug-04 1SGW Putative 1.7 Pyrococcus furiosus Apr-07 2P0S Putative 1.6 Porphyromonas gingivalis Sep-07 2IHY Putative 1.9 Staphylococcus aureus Jan-06 2D3W SufC 2.5 Escherichia coli Oct-06 2IXE Tap1 D645N w/ ATP 2 Rattus norvegicus Oct-06 2IXF Tap1 D645Q, Q678H w/ ATP 2 Rattus norvegicus Oct-06 2IXG Tap1 S621A, G622V, D645N w/ ATP 2.7 Homo sapiens Sep-01 1JJ7 Tap1 w/ ADP 2.4 Homo sapiens Aug-02 1JI0 Thermatoga w/ ATP 2 Sulfolobus solfataricus Nov-04 1VPL TM0544 2.1 Thermotoga maritima CFTR NBD Structures Nov-04 1XMJ Human CFTR dF508 NBD1 2.3 Homo sapiens Nov-05 2BBT Human CFTR dF508 NBD1 w/ two solublizing mutations 2.3 Homo sapiens Nov-05 2BBS Human CFTR dF508 NBD1 w/ 3M 2.05 Homo sapiens Nov-05 2BBO Human CFTR NBD1 2.55 Homo sapiens Nov-04 1XMI Human CFTR NBD1-F508A w/ ATP 2.25 Homo sapiens Dec-04 1XFA Murine CFTR-F508R 3.1 Mus musculus Dec-04 1XF9 Murine CFTR-F508S 2.7 Mus musculus Dec-03 1R0W Murine CFTR NBD1 2.2 Mus musculus Dec-03 1R0Z Murine CFTR NBD1 - phosphorylated w/ ATP 2.35 Mus musculus J Bioenerg Biomembr (2007) 39:499-505 501501 ments in Pfam, only 6,419 transmembrane sequences are in the MSA of ABC transporter TMDs, PF00664 (Sonnhammer et al. 1997). Login to comment

[hide] CFTR function and prospects for therapy. Annu Rev Biochem. 2008;77:701-26. Riordan JR
CFTR function and prospects for therapy.
Annu Rev Biochem. 2008;77:701-26., [PMID:18304008]

Abstract [show]
Mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel cause cystic fibrosis (CF). The multidomain integral membrane glycoprotein, a member of the adenine nucleotide-binding cassette (ABC) transporter family, conserved in metazoan salt-transporting tissues, is required to control ion and fluid homeostasis on epithelial surfaces. This review considers different therapeutic strategies that have arisen from knowledge of CFTR structure and function as well as its biosynthetic processing, intracellular trafficking, and turnover.

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418 However, this 710 Riordan was also found to be the case with the isolated domains containing either F508S or F508R substitutions (110). Login to comment

[hide] Structure and dynamics of NBD1 from CFTR character... J Mol Biol. 2010 Feb 19;396(2):406-30. Epub 2009 Nov 26. Lewis HA, Wang C, Zhao X, Hamuro Y, Conners K, Kearins MC, Lu F, Sauder JM, Molnar KS, Coales SJ, Maloney PC, Guggino WB, Wetmore DR, Weber PC, Hunt JF
Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.
J Mol Biol. 2010 Feb 19;396(2):406-30. Epub 2009 Nov 26., 2010-02-19 [PMID:19944699]

Abstract [show]
The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

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132 Bright green and magenta represent human F508 and F508A structures, respectively, shades of red/orange represent human ΔF508 human structures, and shades of blue/cyan represent murine structures (F508, F508S, or F508R). Login to comment

[hide] The cystic fibrosis-causing mutation deltaF508 aff... J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28. Thibodeau PH, Richardson JM 3rd, Wang W, Millen L, Watson J, Mendoza JL, Du K, Fischman S, Senderowitz H, Lukacs GL, Kirk K, Thomas PJ
The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.
J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28., 2010-11-12 [PMID:20667826]

Abstract [show]
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the DeltaF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the DeltaF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that DeltaF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

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254 Consistent with this, structures of NBD1 F508S and F508R and trafficking of F508S and F508R full-length CFTR demonstrate that the severity of physicochemical alterations at the 508 position correlate with protein trafficking (12, 26). Login to comment

[hide] Correction of both NBD1 energetics and domain inte... Cell. 2012 Jan 20;148(1-2):150-63. Rabeh WM, Bossard F, Xu H, Okiyoneda T, Bagdany M, Mulvihill CM, Du K, di Bernardo S, Liu Y, Konermann L, Roldan A, Lukacs GL
Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function.
Cell. 2012 Jan 20;148(1-2):150-63., [PMID:22265408]

Abstract [show]
The folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that DeltaF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore DeltaF508 CFTR biogenesis. Instead, both DeltaF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of DeltaF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.

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69 These results in concert with the effect of F508E, F508R, F508G, F508S, F508D, and F508N mutations revealed that the CD4T-NBD1 PM density was proportional to the domain stability if the NBD1 Tm was >38 C (Figures 3D and S4D). Login to comment

[hide] Solubilizing mutations used to crystallize one CFT... Chem Biol. 2008 Jan;15(1):62-9. Pissarra LS, Farinha CM, Xu Z, Schmidt A, Thibodeau PH, Cai Z, Thomas PJ, Sheppard DN, Amaral MD
Solubilizing mutations used to crystallize one CFTR domain attenuate the trafficking and channel defects caused by the major cystic fibrosis mutation.
Chem Biol. 2008 Jan;15(1):62-9., [PMID:18215773]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) Cl(-) channel. F508del, the most frequent CF-causing mutation, disrupts both the processing and function of CFTR. Recently, the crystal structure of the first nucleotide-binding domain of CFTR bearing F508del (F508del-NBD1) was elucidated. Although F508del-NBD1 shows only minor conformational changes relative to that of wild-type NBD1, additional mutations (F494N/Q637R or F429S/F494N/Q637R) were required for domain solubility and crystallization. Here we show that these solubilizing mutations in cis with F508del partially rescue the trafficking defect of full-length F508del-CFTR and attenuate its gating defect. We interpret these data to suggest that the solubilizing mutations utilized to facilitate F508del-NBD1 production also assist folding of full-length F508del-CFTR protein. Thus, the available crystal structure of F508del-NBD1 might correspond to a partially corrected conformation of this domain.

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146 However, the effect produced by F494N is not completely unexpected, as structural crosstalk between the side chain of the F508 residue (e.g., F508R) and the Q loop or g-phosphate switch (e.g., residues W496 and M498) has been previously shown to occur (Massiah et al., 1999). Login to comment

[hide] Comparison of the bacterial HelA protein to the F5... J Bacteriol. 1997 Dec;179(24):7869-71. Goldman BS, Sherman DA, Kranz RG
Comparison of the bacterial HelA protein to the F508 region of the cystic fibrosis transmembrane regulator.
J Bacteriol. 1997 Dec;179(24):7869-71., [PMID:9401049]

Abstract [show]
The HelA protein of Rhodobacter capsulatus is the ATP-binding-cassette subunit of an exporter complex required for cytochrome c biogenesis. By primary sequence comparisons the F88 residue of HelA is similar to the F508 residue of the cystic fibrosis transmembrane regulator (CFTR) protein. Previous studies have established that CFTR F508delta or F508R proteins are defective but F508C is functional. Our results demonstrate that the HelA F88 mutants functionally mimic the phenotypes of known CFTR F508 mutants. The phenotypes of additional HelA mutants and the in vivo steady-state levels of these proteins are also reported.

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7 Previous studies have established that CFTR F508⌬ or F508R proteins are defective but F508C is functional. Login to comment
43 It has been demonstrated that the CFTR F508⌬ and a site-directed mutant, CFTR F508R, are defective in intracellular transport and processing, and thus folding (4). Login to comment
55 It is particularly intriguing that the HelA F88R and the CFTR F508R mutants are both defective, whereas with other substitutions (e.g., cysteine) function is retained. Login to comment
63 The CFTR mutation F508R (marked with a asterisk) was found to be defective in COS-7 cells (4) and was not associated with a CF patient. Login to comment

[hide] Defective intracellular transport and processing o... Cell. 1990 Nov 16;63(4):827-34. Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, Smith AE
Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.
Cell. 1990 Nov 16;63(4):827-34., [PMID:1699669]

Abstract [show]
The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

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130 Also included is F508R, in which we changed the residue at 508 rather than deleting it. Login to comment
132 The mutation of phenylalanine 508 to arginine also results in CFTR that does not mature. Login to comment
137 CFTR Mutants Mutant CF Exon CFTR Domain A B C Wild type R334W K464M Al507 AF508 F508R s5491 G551 D N894,900Q K1250M Tthllll Y 7 N 9 Y 10 Y 10 N 10 Y 11 Y 11 N 15 N 20 N 22 TM6 NBDl NBDl NBDl NBDl NBDl NBDl ECD4 NBD2 Term - + ++ - + ++ - + - - + - - + - - + - - + - - + ++ + - - - + ++ - + - The known association with CF (Y, yes; N, no), exon localization, domain location, and presence (+ ) or absence (- ) of bands A, B, and C of mutant CFTR species is shown. Login to comment
169 All the mutations around 508 studied here (AF508, Al507, F508R) fail to generate mature CFTR, whereas of mutations at the second site, S549l does not produce mature CFTR but G551D does. Login to comment

[hide] CFTR gene mutations and asthma in the Norwegian En... Respir Med. 2006 Dec;100(12):2121-8. Epub 2006 May 5. Munthe-Kaas MC, Lodrup Carlsen KC, Carlsen KH, Skinningsrud B, Haland G, Devulapalli CS, Pettersen M, Eiklid K
CFTR gene mutations and asthma in the Norwegian Environment and Childhood Asthma study.
Respir Med. 2006 Dec;100(12):2121-8. Epub 2006 May 5., [PMID:16678395]

Abstract [show]
BACKGROUND: Several candidate genes have been implicated in the etiology of asthma, including the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR gene result in derangements of mucociliary clearance. Homozygotes for CFTR mutations develop cystic fibrosis (CF), a disorder characterized mainly by lung and pancreas disease. OBJECTIVE: To investigate whether there was an increased frequency of CFTR mutations in asthma patients. METHODS: Seven hundred and three subjects aged 10-11 years from the environment and childhood asthma (ECA) study were included in the present study. Possible associations between asthma, reduced lung function, bronchial hyperresponsiveness (BHR), and increased or decreased nitrogen oxide (NO) levels (based on structural parental interview, spirometry, PD20 methacholine challenge test and exhaled NO measurements), and the five most common CFTR mutations in Norway (DeltaF508, R117H, R117C, 4005+2T-->C, 394delTT), the modulating polymorphisms IVS8(TG)mTn and the IVS8-5T were investigated. RESULTS: No association were found between asthma, reduced lung function, BHR or exhaled NO levels and CF heterozygosity. However, the IVS8(TG)11T7 haplotype was associated with normal lung function. CONCLUSIONS: Our results do not support the hypothesis that CFTR mutations or polymorphisms play a role in the pathogenesis of asthma in children. However, the distribution of Tn(TG)m haplotypes differed between individuals with reduced lung function and individuals with normal lung function.

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46 PCR and fragment analysis for the 394delTT and delF508 were performed in a multiplex reaction with the following primers: 394F (forward) 50 -FAM-GCAGAGAATGGGATAGA- GAGC-, 394R (reverse) 50 -ATTCACCAGATTTCGTA- GTC- and F508F (forward) 50 -HEX-GCCTGGCACCAT- TAAAGAA-and F508R (reverse) 50 -AGTTGGCATGC- TTTGATGAC-. Login to comment

[hide] Requirements for efficient correction of DeltaF508... Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023. Mendoza JL, Schmidt A, Li Q, Nuvaga E, Barrett T, Bridges RJ, Feranchak AP, Brautigam CA, Thomas PJ
Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences.
Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023., [PMID:22265409]

Abstract [show]
Misfolding of DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the DeltaF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores DeltaF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

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187 See also Table S2. (C) F508K, F508R, and F508K in combination with I539T, G550E, R553M, R555K, and 3M mutations increase folding yield of NBD1, but exhibit no corresponding increase in CFTR maturation yield (dark blue circles and line, m = 0.03, R = 0.40) (&#b1;SEM, n = 9 along x axis and n = 3 along y axis). Login to comment