ABCMdb

ABCC7 p.Arg553Met

ClinVar:	c.1657C>T , p.Arg553* D , Pathogenic c.1657C>G , p.Arg553Gly ? , not provided c.1658G>A , p.Arg553Gln D , Pathogenic
CF databases:	c.1657C>T , p.Arg553* D , CF-causing c.1657C>G , p.Arg553Gly (CFTR1) ? , This mutation was found identified by DGGE and direct sequencing. This nucleotide change was observe in a French CF chromosome. c.1658G>A , p.Arg553Gln (CFTR1) ? , The amino acid change was found in a German CF patient on the maternal [delta]F508 CF chromosome associated with the haplotype 1-2-1-1-1-2 in J3.11(Msp) - KM.19 (Pst) - XV-2c - metH(Msp) - metH (Taq) - metD(taq). The paternal CF chromosome carries the 553X Stop mutation. So far, the R553Q mutation was not found on a small number of normal and of CF [delta]F508 or non-[delta]F508 chromosomes. Since this mutation occurs in the region of sequence identity with other membrane-associated proteins or transport systems that may contain glutamine instead of a basic amino acid at this position, we assume that this mutation may be neither a polymorphism nor may cayse disease but rather modulates the function of the [delta]F508 CFTR gene product.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (85%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (59%), 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: N, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: N, S: D, T: D, V: D, W: D, Y: D,

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[hide] Mutations in the nucleotide binding domain 1 signa... J Biol Chem. 2002 Sep 27;277(39):35896-905. Epub 2002 Jul 10. DeCarvalho AC, Gansheroff LJ, Teem JL
Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508.
J Biol Chem. 2002 Sep 27;277(39):35896-905. Epub 2002 Jul 10., 2002-09-27 [PMID:12110684]

Abstract [show]
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.

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103 Interestingly, the three ⌬F508 revertant mutations previously isolated using the STE6/CFTR system, R553Q, R553M, and R555K, are also located within the NBD1 signature motif (28, 29). Login to comment

[hide] NMR evidence for differential phosphorylation-depe... EMBO J. 2010 Jan 6;29(1):263-77. Epub 2009 Nov 19. Kanelis V, Hudson RP, Thibodeau PH, Thomas PJ, Forman-Kay JD
NMR evidence for differential phosphorylation-dependent interactions in WT and DeltaF508 CFTR.
EMBO J. 2010 Jan 6;29(1):263-77. Epub 2009 Nov 19., 2010-01-06 [PMID:19927121]

Abstract [show]
The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DeltaF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and DeltaF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild-type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N-terminal intracellular domain (ICD). Phosphorylation of DeltaF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full-length CFTR and expand our understanding of the molecular basis of the DeltaF508 defect.

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50 Resonance assignment of G550E/R553M/R555K NBD1-RE The weak intensity of many of the resonances and the limited stability of the WT NBD1-RE NMR samples precluded resonance assignment. Login to comment
51 Therefore, we used a variant of NBD1-RE containing the revertant mutations, G550E (DeCarvalho et al, 2002), R553M (Teem et al, 1993), and R555K (Teem et al, 1996). Login to comment
53 The G550E/R553M/R555K mutant NBD1-RE could be concentrated to B600 mM and was stable for 420 days, allowing NMR data for backbone resonance assignment to be recorded. Login to comment
54 More resonances are present in the spectra of the G550E/R553M/R555K mutant compared with WT NBD1-RE (Supplementary Figure 3), pointing to less severe broadening than in the spectra of WT protein because of differences in motion on the ms-ms timescale. Login to comment
55 Although not as extensive as observed for the WT NBD1-RE, spectra of the G550E/R553M/R555K mutant also show broadening, with some of the weak resonances having elevated R2 rates from ms-ms timescale motion (Supplementary Table 1). Login to comment
56 Relaxation data recorded on 360 and 550 mM samples of the G550E/R553M/R555K mutant were very similar for most residues, indicating that the elevated R2 rates are not caused by sample aggregation at high concentrations (Supplementary Table 1). Login to comment
57 Many resonances are weak, especially in the spectra of the lower concentrated sample of the G550E/ R553M/R555K mutant (i.e., Val562, Asp572, and Ser573), precluding reliable R2 values from being obtained for these residues. Login to comment
58 Importantly, for resonances observed for both the WT and G550E/R553M/R555K mutant forms of the protein, backbone chemical shifts are very similar (Supplementary Figure 3), allowing the straightforward transfer of assignments for most resonances. Login to comment
59 Using triple resonance experiments and specific labelling on Leu, the combination of Gly, Ser, Asp, and Asn residues, or aromatic residues, we have assigned 70% of the 1 HN and 15 N resonances in the G550E/R553M/R555K mutant and 60% of the 1 HN and 15 N resonances in WT NBD1-RE (Supplementary Figure 4a). Login to comment
79 The ribbon is coloured blue for residues for which we have resonance assignments, light grey for those not assigned, and dark grey for those assigned in the G550E/R553M/R555K mutant but not transferable to WT NBD1-RE. Login to comment
86 The secondary structures of the G550E/R553M/R555K mutant, WT, and DF508 NBD1-RE were determined using 1 HN and 15 N chemical shifts, as well as 13 Ca, 13 Cb, and 13 CO chemical shifts where available (Supplementary Figure 5). Login to comment
87 As expected from the similarity of the NMR spectra, secondary structures of the G550E/R553M/R555K mutant, WT, and DF508 NBD1-RE proteins are very similar and largely agree with that of the crystal structures. Login to comment
92 Interestingly, the unassigned residues in the G550E/ R553M/R555K mutant map to distinct regions on NBD1-RE (Supplementary Figure 4b). Login to comment
227 The significant destabilization of all forms of phosphorylated NBD1-RE (WT, DF508, and the G550E/R553M/R555K) precludes NMR resonance assignment required to further test this hypothesis. Login to comment
259 The average positions of the dynamic equilibrium from G550E/R553M/R55K and WT NBD1 were determined from the percentage of elevated R2 rates measured for each protein (Supplementary Table 1), whereas that of DF508 NBD1 was determined from the number of broadened residues compared with WT. Login to comment
265 Although the exact positions of the species may change depending on the type of data used, these data reflect the relative positions of the G550E/R553M/R555K mutant, WT, and DF508 NBD1-RE proteins. Login to comment
291 Materials and methods Sample preparation NBD1 from murine CFTR (residues 389-673 or 389-653) with the WT sequence, lacking Phe508 (DF508), or containing the revertant mutations G550E, R553M, and R555K (G550E/R553M/R555K) (Teem et al, 1993, 1996; Roxo-Rosa et al, 2006), was expressed as a 6x-His-Smt (SUMO) (Mossessova and Lima, 2000) fusion at 16 1C in BL21(DE3) Codon Plus cells grown in minimal media with 15 N- NH4Cl, 13 C-glucose, and/or 70% 2 H2O as required for NMR studies, and purified using standard chromatographic techniques, as previously described (Lewis et al, 2004, 2005). Login to comment
294 The G550E/R553M/R555K NBD1-RE mutant was used only for backbone resonance assignment (see Results). Login to comment
295 Purified WT, DF508, and G550E/R553M/R555K mutant proteins were exchanged into NMR buffer containing 20 mM Na phos, pH 7.0, 150 mM NaCl, 5 mM MgCl2, ATP or AMP-PNP, with 2 or 4% (v/v) glycerol. Login to comment
319 Backbone H, N, C, and Ca, and side chain Cb assignments for G550E/R553M/R555K NBD1-RE were obtained from standard triple resonance TROSY-based experiments (Sattler et al, 1999; Kanelis et al, 2001) and a 15 N-edited NOESY-HSQC spectrum (200 ms) recorded on samples of 0.5-0.6 mM G550E/R553M/R555K NBD1-RE that were uniformly 15 N and 13 C labelled and fractionally 2 H labelled to B50%. Login to comment
326 NMR assignments NMR resonance assignments for G550E/R553M/R555K NBD1-RE, WT NBD1-RE, and DF508 NBD1-RE have been deposited in the BioMag Res Bank under the accession codes 16367, 16393, and 16394, respectively. Login to comment

[hide] Interplay between ER exit code and domain conforma... Mol Biol Cell. 2010 Feb 15;21(4):597-609. Epub 2009 Dec 23. Roy G, Chalfin EM, Saxena A, Wang X
Interplay between ER exit code and domain conformation in CFTR misprocessing and rescue.
Mol Biol Cell. 2010 Feb 15;21(4):597-609. Epub 2009 Dec 23., 2010-02-15 [PMID:20032308]

Abstract [show]
Multiple mutations in cystic fibrosis transmembrane conductance regulator (CFTR) impair its exit from the endoplasmic reticulum (ER). We compared two processing mutants: DeltaF508 and the ER exit code mutant DAA. Although both have severe kinetic processing defect, DAA but not DeltaF508 has substantial accumulation in its mature form, leading to higher level of processing at the steady state. DAA has much less profound conformational abnormalities. It has lower Hsp70 association and higher post-ER stability than DeltaF508. The ER exit code is necessary for DeltaF508 residual export and rescue. R555K, a mutation that rescues DeltaF508 misprocessing, improves Sec24 association and enhances its post-ER stability. Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in DeltaF508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. We observed a conversion of the proteolytic pattern of DAA from the one resembling DeltaF508 to the one similar to wild-type CFTR during its maturation. Low temperature and R555K are additive in improving DeltaF508 conformational maturation and processing. Our data reveal a dual contribution of ER exit code and domain conformation to CFTR misprocessing and underscore the importance of conformational repair in effective rescue of DeltaF508.

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340 Interestingly, R553M, the substitution of the other arginine in the same RXR motif, rescues ⌬F508 CFTR as well (Teem et al., 1993), but through enhancing the folding yield of ⌬F508 NBD1 based on an in vitro study (Qu et al., 1997). Login to comment
343 Instead, like other second site mutations in NBD1, R555K and R553M substitutions might alter the conformation of CFTR in a way that repairs ⌬F508 conformation defects. Login to comment
344 The ER exit code "DAD", F508, and many ⌬F508 rescuing mutations (including R555K and R553M) reside in NBD1. Login to comment

[hide] Restoration of domain folding and interdomain asse... FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PMID:20233947]

Abstract [show]
Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.

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27 These suppressor mutations (I539T, G550E, R553M/Q, and R555K) promote ⌬F508-CFTR maturation and trafficking to the cell surface, and also restore channel activity (16). Login to comment
72 RESULTS Suppressor mutations restore folding mutations in NBD1 but not elsewhere Four suppressor mutations (I539T, G550E, R553M, and R555K) were originally found to rescue ⌬F508-CFTR maturation in a yeast mating screen using STE6/CFTR chimeras (14-16). Login to comment
79 B, C) HEK293 cells were transiently transfected with CFTR variants with maturation-compromising mutations introduced in different domains, in the presence or absence of single or combined suppressor mutations (4S: I539T/G550E/R4553M/R555K; 3S: G550E/R553M/R555K). Login to comment
84 Among these four suppressor mutations, R555K had the largest effect in promoting ⌬F508-CFTR maturation, while R553M had the least effect. Login to comment
85 The addition of G550E and R553M to R555K (3S) further increased its maturation, but no additional effect was detected by the addition of the fourth mutation I539T (4S) (Fig. 1A). Login to comment
112 Stable BHK cells overexpressing WT-CFTR and ⌬F508-CFTR with and without 4 suppressor mutations (I539T/G550E/R553M/R555K, ⌬F/ 4S) were pulse labeled with 100 ␮Ci/ml [35 S] methionine for 20 min, followed by 0, 1, 2, and 4 h chase. Login to comment
124 To test whether these NBD/CL interfaces not formed in ⌬F508-CFTR could be restored by the suppressor mutations, the 4 combined suppressor mutations, I539T/G550E/R553M/R555K (4S) were introduced into the ⌬F508-CFTR constructs with the Cys pairs at the potential interfaces. Login to comment
139 HEK293 cells were transiently transfected with Cys-less ⌬F508-CFTR in the presence or absence of suppressor mutations I539T/G550E/R553M/R555K, with Cys pairs introduced at CL2/NBD2 (A) or CL4/NBD1 (B) interfaces. Login to comment
146 However, when suppressor mutations (3S: G550E/R553M/R555K) were introduced into the N-half ⌬F508-CFTR, they did not promote complex glycosylation of the C half (Fig. 5A, lane 4), as they did in full-length CFTR (Fig. 1). Login to comment
154 HEK293 cells were transiently transfected with 1172X-CFTR or ⌬F508-1172X-CFTR in the presence or absence of single or combined suppressor mutations (4S: I539T/G550E/R4553M/R555K; 3S: G550E/R553M/R555K). Login to comment
162 N halves were either WT or carried the ⌬F508 mutation, in the absence or presence of suppressor mutations (3S: G550E/R553M/R555K). Login to comment

[hide] The V510D suppressor mutation stabilizes DeltaF508... Biochemistry. 2010 Aug 3;49(30):6352-7. Loo TW, Bartlett MC, Clarke DM
The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface.
Biochemistry. 2010 Aug 3;49(30):6352-7., 2010-08-03 [PMID:20590134]

Abstract [show]
Deletion of Phe508 (DeltaF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. The mutation severely reduces the stability and folding of the protein by disrupting interactions between NBD1 and the second transmembrane domain (TMD2). We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of DeltaF508-CFTR with V510D more efficiently than V510E. Promotion of DeltaF508-CFTR maturation did not require NBD2 as introduction of V510D into a DeltaNBD2/DeltaF508-CFTR mutant restored maturation to levels similar to that of full-length protein. The V510D mutation increased the half-life of mature DeltaF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. It was also observed that introduction of the V510R/R1070D mutations into DeltaF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. We propose that the V510D mutation in NBD1 promotes maturation and stabilizes DeltaF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2.

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64 It was recently reported that rescue of ΔF508-CFTR byother suppressor mutations inNBD1(I539T,G550E,R553M, R555K) was drastically reduced in wild-type CFTR lacking NBD2 (ΔNBD2) (20). Login to comment
129 A similar effect was observed when the combination of four NBD1 suppressormutations(I539T,G550E,R553M,R555K) was introduced into ΔF508-CFTR (20). 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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104 The introduction of the single mutations, G550E, R553M or R553Q, and R555K, has previously been shown to partially rescue the ⌬F508 trafficking defect in CFTR and restore channel activity at the plasma membrane (Fig. 1A) (19-21). Login to comment
142 The introduction of the -3M mutations (G550E, R553M, R555K) rescues the trafficking defects associated with the ⌬F508 mutation and restores near wild type function. Login to comment

[hide] A new complex allele of the CFTR gene partially ex... Genet Med. 2010 Sep;12(9):548-55. Lucarelli M, Narzi L, Pierandrei S, Bruno SM, Stamato A, d'Avanzo M, Strom R, Quattrucci S
A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
Genet Med. 2010 Sep;12(9):548-55., [PMID:20706124]

Abstract [show]
PURPOSE: To evaluate the role of complex alleles, with two or more mutations in cis position, of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the definition of the genotype-phenotype relationship in cystic fibrosis (CF), and to evaluate the functional significance of the highly controversial L997F CFTR mutation. METHODS: We evaluated the diagnosis of CF or CFTR-related disorders in 12 unrelated subjects with highly variable phenotypes. According to a first CFTR mutational analysis, subjects appeared to be compound heterozygotes for a classic mutation and the L997F mutation. A further CFTR mutational analysis was conducted by means of a protocol of extended sequencing, particularly suited to the detection of complex alleles. RESULTS: We detected a new [R117L; L997F] CFTR complex allele in the four subjects with the highest sweat test values and CF. The eight subjects without the complex allele showed the most varied biochemical and clinical outcome and were diagnosed as having mild CF, CFTR-related disorders, or even no disease. CONCLUSIONS: The new complex allele partially explains the variable phenotype in CF subjects with the L997F mutation. CFTR complex alleles are likely to have a role in the definition of the genotype-phenotype relationship in CF. Whenever apparently identical CFTR-mutated genotypes are found in subjects with divergent phenotypes, an extensive mutational search is mandatory.

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105 Both in vivo and in vitro studies have also highlighted cases in which there is one main mutation with the phenotypical effect that is worsened by a second mutation, which may even be a neutral variant when isolated, as occurs for F508C,38 R74W,41 S912L,44 and M470V.42 However, different effects have also been described, as in the case of the two M470 and R1235 variants, which give rise to a hyperactive CFTR when present on different alleles but have a suppressive effect when combined on the same allele.42 In addition, the finding of complex alleles in CFTR-RD seems to suggest that a second CFTR mutation may even lead to a partial reversion of the phenotype.43 Indeed, in a reduced number of complex alleles, the effect of the second mutation may partially correct the functional defect, thereby lessening the phenotypical effect, as has been demonstrated for the R553Q mutation in the [F508del; R553Q] complex allele by in vivo52 and in vitro53 studies and for the R553M mutation in the [F508del; R553M] complex allele by an in vitro study.53 A milder phenotypical effect has also been demonstrated for the [R334W; R1158X]54 and [-102T; S549R(TϾG)]55 complex alleles if compared with alleles carrying, respectively, isolated R1158X or S549R(TϾG). Login to comment

[hide] Intragenic suppressing mutations correct the foldi... J Biol Chem. 2010 Nov 19;285(47):36304-14. Epub 2010 Sep 13. Pagant S, Halliday JJ, Kougentakis C, Miller EA
Intragenic suppressing mutations correct the folding and intracellular traffic of misfolded mutants of Yor1p, a eukaryotic drug transporter.
J Biol Chem. 2010 Nov 19;285(47):36304-14. Epub 2010 Sep 13., 2010-11-19 [PMID:20837481]

Abstract [show]
ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-DeltaF670, similar to the predominant disease-causing allele in humans, CFTR-DeltaF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-DeltaF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and DeltaF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces.

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249 Remarkably, all suppressing mutations identified (I539T, G550E, R553M, and R555K) by this study are located within the NBD1 domain itself. Login to comment

[hide] NMR spectroscopy to study the dynamics and interac... Methods Mol Biol. 2011;741:377-403. Kanelis V, Chong PA, Forman-Kay JD
NMR spectroscopy to study the dynamics and interactions of CFTR.
Methods Mol Biol. 2011;741:377-403., [PMID:21594798]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a multi-domain membrane chloride channel whose activity is regulated by ATP at two nucleotide-binding domains (NBD1 and NBD2) and by phosphorylation of the regulatory (R) region. The NBDs and the R region have functionally relevant motions that are critical for channel gating. Nuclear magnetic resonance (NMR) spectroscopy is a highly useful technique for obtaining information on the structure and interactions of CFTR and is extremely powerful for probing dynamics. NMR approaches for studying CFTR are reviewed, using our previous NBD1 and the R region results to provide examples. These NMR data are yielding insights into the dynamic properties and interactions that facilitate normal CFTR regulation as well as pathological effects of mutations, including the most common disease mutant, deletion of F508 in NBD1.

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78 (b) HSQC spectrum of the G550E/R553M/R555K mutant NBD1-RE (398-673). Login to comment
102 The interacting peptide is in red and the NBD1-RE structure is colored blue for residues for which we have resonance assignments, light grey for those not assigned, and dark grey for those assigned in the G550E/R553M/R555K mutant but not transferable to WT NBD1-RE (19). Login to comment
134 The solubility of mNBD1 is greatly improved by the inclusion of the RE (14), which transiently populates helical structures that interact with NBD1 (19, 20), and incorporation of the revertant mutations, G550E, R553M, and R555K (43-45), yielding an NBD1-RE construct that is sufficiently soluble for NMR assignment experiments. Login to comment
140 Higher concentrations of glycerol and lower temperatures further stabilize the protein, but increase the viscosity of the solution, leading to Table 25.1 List of preferred CFTR constructs for NMR studies Construct Boundaries "Solubilizing" mutations mNBD1-RE 389-673 G550E, R553M, R555K hNBD1a 387-404, 437-646 None hNBD1-REa 387-404, 437-678 None hNBD1-RE 389-678 F494N hNBD1-RE 389-678 F429S, F494N, Q637R aThe RI (residues 405-436) have been deleted in these constructs. Login to comment
187 HSQC spectra recorded on samples specifically 15N labeled on Leu residues, aromatic residues (Phe, Tyr, and Trp), or the combination of Gly, Ser, Asp, and Asn residues were used to assist in identification of residue type in order to achieve 70% assignment of the G550E, R553M, R555K mutant NBD1-RE, which were then transferred to the WT protein (19), as the level of uniformity of lineshapes was greater for the G550E, R553M, R555K mutant than either WT or F508del (compare Fig. 25.2b, c). Login to comment

[hide] Probing conformational rescue induced by a chemica... J Biol Chem. 2011 Jul 15;286(28):24714-25. Epub 2011 May 21. Yu W, Chiaw PK, Bear CE
Probing conformational rescue induced by a chemical corrector of F508del-cystic fibrosis transmembrane conductance regulator (CFTR) mutant.
J Biol Chem. 2011 Jul 15;286(28):24714-25. Epub 2011 May 21., 2011-07-15 [PMID:21602569]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause loss of function of the CFTR channel on the apical surface of epithelial cells. The major CF-causing mutation, F508del-CFTR, is misfolded, retained in the endoplasmic reticulum, and degraded. Small molecule corrector compounds have been identified using high throughput screens, which partially rescue the trafficking defect of F508del-CFTR, allowing a fraction of the mutant protein to escape endoplasmic reticulum retention and traffic to the plasma membrane, where it exhibits partial function as a cAMP-regulated chloride channel. A subset of such corrector compounds binds directly to the mutant protein, prompting the hypothesis that they rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis directly by evaluating the consequences of a corrector compound on the conformation of each nucleotide binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. Interestingly, we found that VRT-325 was capable of partially restoring compactness in NBD1. However, VRT-325 had no detectable effect on the conformation of the second half of the molecule. In comparison, ablation of the di-arginine sequence, R(553)XR(555) (F508del-KXK-CFTR), modified protease susceptibility of NBD1, NBD2, and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together, these interventions restored processing of F508del-CFTR to near wild type. Importantly, however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. Importantly, this defect in channel activation can be fully corrected by the addition of the potentiator, VX-770.

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264 In addition, Thibodeau et al. (28) showed that in the context of the full-length protein, F508del-NBD1 exhibited enhanced protease sensitivity (in limited proteolysis studies) relative to WT-CFTR-NBD1 and further that the solubilizing mutations (G550E, R553M, and R555K) conferred protease resistance to F508del-NBD1. Login to comment

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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122 In a recent study of four of the CFTR suppressor mutations located in NBD1 (I539T, G550E, R553M, and R555K), it was found that they only restored maturation of mutants that had processing mutations in NBD1 but not those that had processing mutations in other domains such as NBD2 (N1303K) or TMD2 (L1065P or R1066C) (66). Login to comment
329 It appears that the ΔF508 mutation inhibits folding of NBD1 and its ability to stably associate with other domains resulting in altered CFTR-chaperone interactions, ER retention,andenhanceddegradation(65).Second-sitesuppressor mutations in NBD1 (such as I539T/G550E/R553M/R555K) can restore interdomain assembly (65, 66) to yield a more stable ΔF508-CFTR molecule (64, 66). Login to comment

[hide] Mutational analysis of the Saccharomyces cerevisia... Mol Microbiol. 1997 Aug;25(4):683-94. Wemmie JA, Moye-Rowley WS
Mutational analysis of the Saccharomyces cerevisiae ATP-binding cassette transporter protein Ycf1p.
Mol Microbiol. 1997 Aug;25(4):683-94., [PMID:9379898]

Abstract [show]
Ycf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein. These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.

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133 These mutants corresponded to CFTR alterations known to be associated with cystic fibrosis (G551D and G551S in CFTR, G756D and G756S in Ycf1p) as well as lesions that either disturb normal function (K464M in CFTR, K669M in Ycf1p) or act to suppress the phenotype of ⌬F508 CFTR (R553Q and R553M in CFTR, K758Q and K758M in CFTR). Login to comment
161 The two ⌬F508 suppressor mutations isolated in this study were R553Q and R553M. Login to comment
193 (Paddon et al., 1996) has shown that the R553Q and R553M mutations do not act by correcting a mislocalization defect in the Ste6p-⌬F508 CFTR chimera. Login to comment

[hide] Conformational changes relevant to channel activit... J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21. Hudson RP, Chong PA, Protasevich II, Vernon R, Noy E, Bihler H, An JL, Kalid O, Sela-Culang I, Mense M, Senderowitz H, Brouillette CG, Forman-Kay JD
Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21., [PMID:22722932]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between beta-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with beta-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

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315 A similar argument can be made for an underlying allosteric mechanism of suppression by the "3M" (G550E/R553M/R555K) mutations (17) which can improve CFTR processing in the absence of F508; even though they do not directly address the structural changes at the Phe-508 site, these mutations apparently change the equilibrium distribution of conformations accessed by NBD1 to be more similar to that of wild-type. Login to comment
313 A similar argument can be made for an underlying allosteric mechanism of suppression by the "3M" (G550E/R553M/R555K) mutations (17) which can improve CFTR processing in the absence of F508; even though they do not directly address the structural changes at the Phe-508 site, these mutations apparently change the equilibrium distribution of conformations accessed by NBD1 to be more similar to that of wild-type. Login to comment

[hide] Thermal instability of DeltaF508 cystic fibrosis t... Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15. Liu X, O'Donnell N, Landstrom A, Skach WR, Dawson DC
Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity.
Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15., [PMID:22680785]

Abstract [show]
Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or DeltaF508 CFTR was stable at 22 degrees C and increased at 28 degrees C, a temperature permissive for DeltaF508 CFTR expression in mammalian cells. At 37 degrees C, however, CFTR-mediated conductance was further enhanced, whereas that due to DeltaF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of DeltaF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of DeltaF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of DeltaF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 degrees C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated DeltaF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the DeltaF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.

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5 Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Login to comment
14 In contrast, the suppressor Received: January 6, 2012 Revised: June 6, 2012 Published: June 8, 2012 Article pubs.acs.org/biochemistry (c) 2012 American Chemical Society 5113 dx.doi.org/10.1021/bi300018e | Biochemistry 2012, 51, 5113-5124 mutation R553M was reported to be only modestly effective at correcting either defect.4,23 Thus, while second-site mutations can confer different phenotypes on ΔF508 CFTR with respect to maturation and channel function, the precise mechanisms by which they impact intramolecular interactions within, and external to, NBD1 remain poorly understood. Login to comment
18 We identified unique functional signatures for five second-site mutations, four in NBD1 (I539T, G550E, R553M, and R555K) and one in the fourth intracellular loop (ICL4, R1070W), and also investigated the relation of thermal stability to variations in channel gating brought about by intracellular cAMP, CFTR potentiators, and CFTR inhibitors. Login to comment
19 Consistent with previous studies, ΔF508 CFTR-mediated conductance, rescued by incubating oocytes at room temperature, decreased rapidly at 37 °C.5,22 When ΔF508 CFTR was expressed in the context of single, second site mutations, however, results ranged from complete protection from thermal inactivation at 37 °C (R553M) to profound inactivation that was fully reversed upon returning the bath to room temperature (I539T). Login to comment
124 Of the four NBD1 suppressor mutations tested only one, R553M, fully restored wt thermostability to ΔF508 CFTR channels. Login to comment
125 In contrast, pairing ΔF508 with R555K, a mutation that has been reported to be somewhat more effective than R553M at improving NBD1 folding and protein maturation,4,6,24 resulted in a channel that, although unable to sustain the initial increase in conductance evoked at 37 °C, was inactivated only slightly and returned to its prewarming level relatively rapidly when superfusate temperature was returned to 22 °C. Login to comment
135 Representative experiments for (A) R553M/ΔF508 CFTR (n = 3). Login to comment
146 There was no apparent correlation of the functional phenotype of the double mutant channels at 37 °C with the improvements reported for NBD1 folding and protein maturation,4,6 but the partial protection from thermal inactivation by R555K and G550E suggested that the effects might be correlated with the induction of increased Po.8,24 R553M, however, had been reported by Teem et al.24 not to increase Po of ΔF508 channels (34-36 °C), so we investigated the behavior of the double mutant in inside-out patches. Login to comment
147 We found that R553M/ΔF508 CFTR, like wt, exhibited a stable increase in Po at 35 °C (Figure 6). Login to comment
148 Po of R553M/ΔF508 CFTR channels was comparable to that of ΔF508 channels at 22 °C and increased similarly during the first minute of exposure to 35 °C. Login to comment
159 R553M/ΔF508 CFTR single-channels recorded from an inside-out patch using symmetric solutions (146 mM [Cl]- ) at pH 7.4. Login to comment
165 (H) Summary of changes in NPo for R553M/ΔF508 CFTR with time before and during warming to 35 °C. Login to comment
246 From this perspective, therefore, it was somewhat surprising that a single, second-site mutation, R553M, reported to only modestly improve ΔF508 protein maturation at 37 °C,4,6,23 nevertheless fully restored wt-like thermal stability to ΔF508 CFTR channel function. Login to comment
251 The three NBD1, second-site mutations that fully or partially protected ΔF508 CFTR channels from thermal inactivation at 37 °C, R553M, R555K, and G550E, share a common effect on ΔF508 CFTR channel function. Login to comment
252 They either maintain (R553M, present work) or increase (G550E8 , R555K24 ) the open probability of ΔF508 CFTR channels at 37 °C. Login to comment
254 Mendoza et al.6 reported that these three NBD1 suppressor mutations increased the yield of folded ΔF508 NBD1 in a cell-based assay from 0% (R553M) to 60% (R555K), although even a 60% increase represented less than 20% of the yield of wt protein under the same conditions. Login to comment
256 A fourth NBD1 suppressor mutation, I539T, in contrast to G550E, R553M, and R555K, is predicted to lie within an unstructured linker connecting two α-helical portions of NBD1. Login to comment
266 Like G550E, R553M, and R555K, this second-site mutation has been associated with increased open probability of the double mutant,7 an effect attributed to a partial improvement in the interaction between NBD1 and ICL4.29,57 Combining the ICL4 mutation with an NBD1 suppressor mutation on the ΔF508 background (R555K/R1070W/ΔF508), however, fully restored wt-like thermal stability at 37 °C, an "additive" effect similar to that reported by Mendoza et al 6 in their study of the effect of these mutations on NBD1 folding and ΔF508 CFTR protein yield. Login to comment
267 The rescue of wt thermal stability of channel function by the R553M mutation, however, indicates that the structural modifications introduced by combining ΔF508 with R1070W are not required for the thermal stabilization of channel gating. Login to comment

[hide] Allosteric modulation balances thermodynamic stabi... J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8. Aleksandrov AA, Kota P, Cui L, Jensen T, Alekseev AE, Reyes S, He L, Gentzsch M, Aleksandrov LA, Dokholyan NV, Riordan JR
Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR.
J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8., [PMID:22406676]

Abstract [show]
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human DeltaF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the gamma-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of DeltaF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human DeltaF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave DeltaF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

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237 A striking feature of the strong stabilizing effect of the proline substitutions was the essentially absolute dependence on the I539T substitution. This dependence contrasts the positive effects on ΔF508 CFTR maturation of other second site changes that are not wholly dependent on I539 T, such as those near the NBD1 signature sequence (G550E/R553M/R555K) and the RI. Login to comment

[hide] Human-mouse cystic fibrosis transmembrane conducta... Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):917-22. Epub 2011 Dec 30. Dong Q, Ostedgaard LS, Rogers C, Vermeer DW, Zhang Y, Welsh MJ
Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-DeltaF508 processing and alter its gating defect.
Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):917-22. Epub 2011 Dec 30., [PMID:22210114]

Abstract [show]
The DeltaF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the most common cause of cystic fibrosis. The mutation disrupts biosynthetic processing, reduces channel opening rate, and decreases protein lifetime. In contrast to human CFTR (hCFTR)-DeltaF508, mouse CFTR-DeltaF508 is partially processed to the cell surface, although it exhibits a functional defect similar to hCFTR-DeltaF508. To explore DeltaF508 abnormalities, we generated human-mouse chimeric channels. Substituting mouse nucleotide-binding domain-1 (mNBD1) into hCFTR partially rescued the DeltaF508-induced maturation defect, and substituting mouse membrane-spanning domain-2 or its intracellular loops (ICLs) into hCFTR prevented further DeltaF508-induced gating defects. The protective effect of the mouse ICLs was reverted by inserting mouse NBDs. Our results indicate that the DeltaF508 mutation affects maturation and gating via distinct regions of the protein; maturation of CFTR-DeltaF508 depends on NBD1, and the DeltaF508-induced gating defect depends on the interaction between the membrane-spanning domain-2 ICLs and the NBDs. These appear to be distinct processes, because none of the chimeras repaired both defects. This distinction was exemplified by the I539T mutation, which improved CFTR-DeltaF508 processing but worsened the gating defect. Our results, together with previous studies, suggest that many different NBD1 modifications improve CFTR-DeltaF508 maturation and that the effect of modifications can be additive. Thus, it might be possible to enhance processing by targeting several different regions of the domain or by targeting a network of CFTR-associated proteins. Because no one modification corrected both maturation and gating, perhaps more than a single agent will be required to correct all CFTR-DeltaF508 defects.

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120 (i) A genetic approach identified second-site suppressor mutations, including I539T, G550E, R553M/Q, and R555K (18-21, 25, 26). Login to comment
169 In addition, a variant that combined G550E with R553M and R553K increased processing and current, although the effect on channel kinetics was not tested (33). Login to comment

[hide] Thermally unstable gating of the most common cysti... J Biol Chem. 2011 Dec 9;286(49):41937-48. Epub 2011 Sep 30. Wang W, Okeyo GO, Tao B, Hong JS, Kirk KL
Thermally unstable gating of the most common cystic fibrosis mutant channel (DeltaF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops.
J Biol Chem. 2011 Dec 9;286(49):41937-48. Epub 2011 Sep 30., [PMID:21965669]

Abstract [show]
Most cystic fibrosis (CF) cases are caused by the DeltaF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of DeltaF508-CFTR can be "rescued" by culturing cells at 26-28 degrees C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a DeltaF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on DeltaF508-CFTR function. Low temperature-rescued DeltaF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 degrees C to 36.5 degrees C. A panel of CFTR correctors and potentiators that increased DeltaF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued DeltaF508-CFTR maturation and stabilized channel activity at 36.5 degrees C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued DeltaF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these DeltaF508-CFTR constructs were stable at 36.5 degrees C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) DeltaF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from DeltaF508-induced thermal instability, a finding that may inform alternative treatment strategies.

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65 The ⌬F508-CFTR construct with NBD1 suppressor mutations (G550E, R553M, R555K (3M/⌬F508)) was provided by Dr. Phillip Thomas (University of Texas Southwestern Medical Center, Dallas). Login to comment
137 Suppressor Mutations in NBD1 Correct Misfolding and Stabilize ⌬F508-CFTR Channel Activity at 36.5 °C-We have shown recently that three suppressor mutations (G550E, R553M, R555K (3M/⌬F508)) in NBD1 correct ⌬F508-CFTR maturation and misfolding and markedly increase its channel activity in excised patches at room temperature (43). Login to comment
180 Cells expressing G550E/R553M/R555K/⌬F508 (3M/⌬F508) were grown at 37 °C. Login to comment

[hide] Functional rescue of DeltaF508-CFTR by peptides de... Chem Biol. 2009 May 29;16(5):520-30. Kim Chiaw P, Huan LJ, Gagnon S, Ly D, Sweezey N, Rotin D, Deber CM, Bear CE
Functional rescue of DeltaF508-CFTR by peptides designed to mimic sorting motifs.
Chem Biol. 2009 May 29;16(5):520-30., [PMID:19477416]

Abstract [show]
The cystic fibrosis (CF)-causing mutant, deltaF508-CFTR, is misfolded and fails to traffic out of the endoplasmic reticulum (ER) to the cell surface. Introduction of second site mutations that disrupt a diarginine (RXR)-based ER retention motif in the first nucleotide binding domain rescues the trafficking defect of deltaF508-CFTR, supporting a role for these motifs in mediating ER retention of the major mutant. To determine if these RXR motifs mediate retention of the native deltaF508-CFTR protein in situ, we generated peptides that mimic these motifs and should antagonize mistrafficking mediated via their aberrant exposure. Here we show robust rescue of deltaF508-CFTR in cell lines and in respiratory epithelial tissues by transduction of RXR motif-mimetics, showing that abnormal accessibility of this motif is a key determinant of mistrafficking of the major CF-causing mutant.

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18 Previously, Teem and Welsh determined that second site revertant mutations in an endogenous RXR motif, proximal to the ABC signature sequence in NBD1 (including R553M and R555K in the motif: R553 AR555 ), partially restored trafficking of deltaF508-CFTR, suggesting that the motif in this region has particular functional significance (Teem et al., 1993, 1996). Login to comment
51 The current studies focus on the RXR motif residing in NBD1, proximal to the ABC signature motif and starting at the arginine at position 553, as it has been implicated in ER retrieval of deltaF508-CFTR with mutations R553M/Q or R555K leading to the enhanced surface expression of the major mutant (Teem et al., 1993, 1996). Login to comment

[hide] Cystic fibrosis: channel, catalytic, and folding p... J Bioenerg Biomembr. 1997 Oct;29(5):429-42. Seibert FS, Loo TW, Clarke DM, Riordan JR
Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein.
J Bioenerg Biomembr. 1997 Oct;29(5):429-42., [PMID:9511928]

Abstract [show]
The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.

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178 (1993) were able to identify two mutations, NBF1-located R553M and R553Q, which when introduced into AF508-CFTR partially restored the function of these chimeras. Login to comment

[hide] Localization and suppression of a kinetic defect i... J Biol Chem. 1997 Jun 20;272(25):15739-44. Qu BH, Strickland EH, Thomas PJ
Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding.
J Biol Chem. 1997 Jun 20;272(25):15739-44., [PMID:9188468]

Abstract [show]
A growing body of evidence indicates that the most common cystic fibrosis-causing mutation, DeltaF508, alters the ability of the cystic fibrosis transmembrane conductance regulator (CFTR) protein to fold and transit to the plasma membrane. Here we present evidence that the DeltaF508 mutation affects a step on the folding pathway prior to formation of the ATP binding site in the nucleotide binding domain (NBD). Notably, stabilization of the native state with 4 mM ATP does not alter the temperature-dependent folding yield of the mutant DeltaF508 NBD1 in vitro. In contrast, glycerol, which promotes DeltaF508-CFTR maturation in vivo, increases the folding yield of NBD1DeltaF and reduces the off pathway rate in vitro, although it does not significantly alter the free energy of stability. Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of DeltaF508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain. These results demonstrate that DeltaF508 is a kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native state ligands to promote folding. The implications for protein folding in general are that the primary sequence may not necessarily simply define the most stable native structure, but rather a stable structure that is kinetically accessible.

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4 Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of ⌬F508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. Login to comment
17 Two mutations at this position, R553M and R553Q, revert the mating phenotype of a ⌬F508 STE6-CFTR chimera in yeast (16). Login to comment
23 In the present study we use this system to examine the effects of the R553M second site mutation, the G551D functional mutation, the S549R maturation-defective mutation, glycerol, and ATP binding on the folding pathway and thermodynamic stability of NBD1. Login to comment
25 EXPERIMENTAL PROCEDURES Expression, Purification, and Folding of CFTR NBD1s-Oligonucleotide-mediated mutagenesis (19) was used to generate R553M, G551D, and S549R mutations in plasmid pBQ2.4 containing CFTR cDNAs. Login to comment
26 The mutant primers are as follows: R553M primer, 5Ј-GAAATTCTTGC- * This work was supported by National Institutes of Health Grant DK49835 and Welch Foundation Grant I-1284. Login to comment
36 Printed in U.S.A. This paper is available on line at http://www.jbc.org CATTTGACCTCCAC-3Ј (25 bases); G551D primer, 5Ј-CTTGCTCGTT- GATCTCCACTCAGTG-3Ј (25 bases); S549R primer, 5Ј-CGTTGAC- CTCCTCTCAGTGTGATTCC-3Ј (26 bases). Login to comment
38 Expression cassette polymerase chain reaction was employed to synthesize the cDNA fragments of CFTR NBD1-R553M, NBD1-G551D, and NBD1-S549R containing a 5Ј NdeI site, a 3Ј XhoI site, and a stop codon as described previously for NBD1 and NBD1⌬F (5). Login to comment
40 To construct the NBD1⌬F-R553M expression vector, pET28a NBD1-R553M and pET28a NBD1⌬F were digested with SphI. Login to comment
41 The larger fragment (5140 base pairs) from the pET28a NBD1-R553M plasmid digestion and the small fragment (720 base pairs) from the pET28a NBD1⌬F plasmid digestion were purified and ligated with T4 ligase. Login to comment
58 The expression levels of NBD1-R553M and NBD1⌬F-R553M are similar to NBD1 and NBD1⌬F (5). Login to comment
82 The Kd for ATP binding to the other NBD1s determined in similar experiments (data not shown) are presented in Table I. NBD1 and only 38% of the NBD1⌬F fold into the soluble conformation, whereas 96% of NBD1-R553M assumes the folded conformation at this temperature (Fig. 3A). Login to comment
83 Thus, the R553M mutation significantly enhances the folding yield of NBD1. Login to comment
84 For the double mutant NBD1⌬F-R553M the folding yield is indistinguishable from that of the wild-type. Login to comment
85 Thus, the second site mutation R553M effectively suppresses the ⌬F508 mutation defective folding yield in vitro. Login to comment
88 Significantly the R553M mutation increases the length of the lag phase and decreases the rate of change in light scattering, indicating that the rate of formation of the off pathway conformer is dramatically reduced in this mutant. Login to comment
89 Once again the double mutant NBD1⌬F-R553M dramatically increases the lag time and decreases the rate change in light scattering in comparison with NBD1⌬F. Login to comment
102 These results indicate that the inability of the ⌬F508 and S549R CFTR to transit to the apical membrane and the effect of the R553M suppressor cannot be explained simply by a reduction or enhancement in the free energy of stability of the mutant proteins. Login to comment
111 Protein NBD1 NBD1#2c;F NBD1-R553M NBD1⌬F-R553M NBD1-S549R NBD1-G551D Kd (␮M) 91 88 89 87 81 61 FIG. 3. Login to comment
112 The effects of the R553M mutation on the temperature-sensitive folding of NBD1 and the rate of formation of off pathway conformers. Login to comment
113 A, the effects of the R553M mutation on the folding yield of wild-type and NBD1⌬F were determined as described under "Experimental Procedures." Login to comment
114 Temperature-dependent folding of NBD1 (q), NBD1⌬F (E), NBD1-R553M (ç), and NBD1⌬F-R553M (É). Login to comment
117 NBD1 (solid line), NBD1⌬F (dotted line), NBD1-R553M (dashed line), and NBD1⌬F-R553M (dotted and dashed line). Login to comment
137 The 1.8 ␮M folded NBD1 (q), NBD1⌬F (E), NBD1-R553M (ç), NBD1⌬F-R553M (É), NBD1-G551D (f), and NBD1-S549R (Ⅺ) in 30 mM Tris-HCl, pH 8.0, 40 mM arginine, 0.2 mM EDTA, and 0.1 mM dithiothreitol were incubated with GdnHCl at the indicated concentration for 2 h. The sample was excited at 282 nm, and fluorescence emission spectra were collected. Login to comment
149 Protein ⌬GD,0 ⌬⌬GD,0 Cm m kJ/mol kJ/mol M kJ/mol/M NBD1 15.5 1.5 10.3 NBD1⌬F 14.4 -1.1 1.3 11.2 NBD1-R553M 16.6 1.1 1.4 11.7 NBD1⌬F-R553M 14.1 -1.4 1.4 10.1 NBD1-S549R 16.7 1.2 1.2 13.4 NBD1-G551D 16.6 1.1 1.4 11.7 reduction in the folding yield in vitro (5) and of the efficiency of maturation and transit to the plasma membrane in vivo (10). Login to comment
152 First, the R553M suppressor mutation effectively corrects the ⌬F508 folding defect in vitro but does not significantly alter the free energy of stability. Login to comment
153 Like ⌬F508, R553M may exert its effect on an intermediate formed during the process of folding. Login to comment
154 In both cases, the results indicate that the mutations are kinetic in nature but with opposite characteristics; R553M is a superfolder, whereas ⌬F508 is an ineffective folder. Login to comment
155 Results in vivo indicate that the R553M/⌬F508 CFTR double mutant only partially corrects the ⌬F508 maturation defect, and the fully mature mutant protein is only partially functional (16). Login to comment
156 The current results indicate that the diminished function of R553M observed in vivo is not due to a loss of the ability of NBD1 to bind ATP. Login to comment
39 The larger fragment (5140 base pairs) from the pET28a NBD1-R553M plasmid digestion and the small fragment (720 base pairs) from the pET28a NBD1DF plasmid digestion were purified and ligated with T4 ligase. Login to comment
56 The expression levels of NBD1-R553M and NBD1DF-R553M are similar to NBD1 and NBD1DF (5). Login to comment
80 The Kd for ATP binding to the other NBD1s determined in similar experiments (data not shown) are presented in Table I. NBD1 and only 38% of the NBD1DF fold into the soluble conformation, whereas 96% of NBD1-R553M assumes the folded conformation at this temperature (Fig. 3A). Login to comment
81 Thus, the R553M mutation significantly enhances the folding yield of NBD1. Login to comment
86 Significantly the R553M mutation increases the length of the lag phase and decreases the rate of change in light scattering, indicating that the rate of formation of the off pathway conformer is dramatically reduced in this mutant. Login to comment
87 Once again the double mutant NBD1DF-R553M dramatically increases the lag time and decreases the rate change in light scattering in comparison with NBD1DF. Login to comment
100 These results indicate that the inability of the DF508 and S549R CFTR to transit to the apical membrane and the effect of the R553M suppressor cannot be explained simply by a reduction or enhancement in the free energy of stability of the mutant proteins. Login to comment
109 Protein NBD1 NBD1DF NBD1-R553M NBD1DF-R553M NBD1-S549R NBD1-G551D Kd (mM) 91 88 89 87 81 61 FIG. 3. Login to comment
110 The effects of the R553M mutation on the temperature-sensitive folding of NBD1 and the rate of formation of off pathway conformers. Login to comment
115 NBD1 (solid line), NBD1DF (dotted line), NBD1-R553M (dashed line), and NBD1DF-R553M (dotted and dashed line). Login to comment
135 The 1.8 mM folded NBD1 (cf;), NBD1DF (E), NBD1-R553M (&#e7;), NBD1DF-R553M (&#c9;), NBD1-G551D (f), and NBD1-S549R (M) in 30 mM Tris-HCl, pH 8.0, 40 mM arginine, 0.2 mM EDTA, and 0.1 mM dithiothreitol were incubated with GdnHCl at the indicated concentration for 2 h. The sample was excited at 282 nm, and fluorescence emission spectra were collected. Login to comment
147 Protein DGD,0 DDGD,0 Cm m kJ/mol kJ/mol M kJ/mol/M NBD1 15.5 1.5 10.3 NBD1DF 14.4 21.1 1.3 11.2 NBD1-R553M 16.6 1.1 1.4 11.7 NBD1DF-R553M 14.1 21.4 1.4 10.1 NBD1-S549R 16.7 1.2 1.2 13.4 NBD1-G551D 16.6 1.1 1.4 11.7 reduction in the folding yield in vitro (5) and of the efficiency of maturation and transit to the plasma membrane in vivo (10). Login to comment
150 First, the R553M suppressor mutation effectively corrects the DF508 folding defect in vitro but does not significantly alter the free energy of stability. Login to comment
151 Like DF508, R553M may exert its effect on an intermediate formed during the process of folding. Login to comment

[hide] Molecular mechanisms of CFTR chloride channel dysf... Cell. 1993 Jul 2;73(7):1251-4. Welsh MJ, Smith AE
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell. 1993 Jul 2;73(7):1251-4., [PMID:7686820]

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89 In a study identifying second-site revertants of the AF508 mutation using STEG-CFTR chimeras in yeast, R553M and, to a lesser extent, R553Q partially reversed the localization and functional effects of the AF508 mutation (Teem et al., 1993). Login to comment

[hide] Identification of revertants for the cystic fibros... Cell. 1993 Apr 23;73(2):335-46. Teem JL, Berger HA, Ostedgaard LS, Rich DP, Tsui LC, Welsh MJ
Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast.
Cell. 1993 Apr 23;73(2):335-46., [PMID:7682896]

Abstract [show]
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (delta F508). We constructed STE6-CFTR chimeras with portions of the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBD1. The chimeras were functional in yeast, but mating efficiency decreased when delta F508 was introduced into NBD1. We isolated two delta F508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBD1 sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and Cl- channel gating defects caused by the delta F508 mutation. These results suggest that the NBD1s of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by delta F508 can be partially corrected by additional alterations in the protein.

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4 We isolated two AF508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBDl sequence. Login to comment
91 These yeast transformants each contained an H5-AF508 plasmid with a mutation at amino acid R553 of CFTR; in one case, R553 was replaced by methionine (H5-AF508/R553M), and in the other plasmid, R553 was replaced by glutamine (H5-AF508IR553Q). Login to comment
92 It is possible that other mutations within the R553-L558 region of the H5-AF508 plasmid could also result in increased mating efficiency; however, we proceeded to analyze the R553Q and R553M mutants in greater detail without further mutagenesis of the R553-L558 region. Login to comment
94 Whereas the mating efficiency of the H5-AF508 yeast strain is approximately lo/a of the H5 strain, yeast containing the H5-AF508/R553Q and H5-AF508/R553M plasmids mated at 3% and 3204 respec- 330 tively. Login to comment
96 However, when the mutations R553Q and R553M press the AF508 mating defect. Login to comment
97 The R553M mutation were introduced into CFTRAF508 (CFTRAF508/R553Q alone had little effect on H5 (H5R553M); when this mutant and CFTRAF508/R553M, respectively), CAMP-dependent was transformed into yeast, no further increase in mating anion permeability was restored. Login to comment
101 The R553Q and R553M mutations partially correct the defect in the H5-AF508 chimera and should also correct the defect in CFTRAF508 if a similar structure exists for NBDl in both proteins. Login to comment
102 As a test of this hypothesis, we introduced the R553Q and R553M mutations into CFTRAF508 cDNA and transfected mammalian cells with these constructs to determine whether the revertant mutations would correct the defect in CAMP-regulated Cl- transport of CFTRAF508. Login to comment
109 R553Q and R553M Suppress the CFTRAF508 Anion Transport Defect We assessed the effect of the R553Q and R553M mutations on CFTR function by assaying for CAMP-stimulated halide efflux using the halide-sensitive fluorophore 8-methoxy-N-(3sulfopropyl)-quinolinium (SPQ) (Illsley and Verkman, 1987). Login to comment
110 Expression of CFTR cDNA containing either the R553Q or the R553M mutation alone (without the AF508 mutation) in HeLa cells generated CAMP-stimulated halide efflux like wild-type CFTR (Figure 3). Login to comment
112 Relative Mating Efficiency of AF5OS Revertants Genotype H5R553M H5-AF508/R553M H&AF508/R553Q HSAF508 Mating Efficiency Relative to H5 (%) 77.4 f 3.7 34.2 f 7.0 3.2 i 0.8 1.1 f 0.5 Mating efficiencies were determined by quantitative mating assays and are expressed as a percentage relative to H5 (100%). Login to comment
118 Correction of CFTRAF508 Processing and Localization Cl- transport by CFTRAF508 containing the R553Q and R553M mutations would be detected only if the processing defect of CFTRAF508 was suppressed. Login to comment
122 CFTRAF508 is only present as the unglycosylated band A and the core glycosyl- 6000 -CFTR - AF508/R553M -c- R553M + AF508lR553Q I R553Q ,+ AF508 0 0 2 4 6 6 10 12 TIME (MIN) ated band B protein, consistent with its failure to traverse the Golgi complex and reach the plasma membrane (Cheng et al., 1990) (Figure 4). Login to comment
130 (B) Expression of wild-type CFTR (lane 1) or CFTR mutants CFTRAF508/R553M (lane 2) CFTRAF508IR553Q (lane 3) and CFTRAF508 (lane 4) in HeLa cells. Login to comment
140 R553Q and R553M mutations alone (without the AF508 mutation). Login to comment
141 As shown in Figure 4A, band C is present in cells expressing wild-type CFTR (lane 1) and also mutant CFTR containing either the R553Q or R553M mutation (lanes 2 and 3). Login to comment
143 Thus, the R5530 and R553M mutationsalone do not affect the glycosylation of CFTR. Login to comment
144 In cells transfected with the CFTRAF508/R553M (Figure 48, lane 2) a small increase in band C is detectable as compared with CFTRAF508 (lane 4). Login to comment
147 Thus, a detectable increase in band C occurs only with CFTRAF508/R553M as compared with CFTRAF508. Login to comment
148 However, the total amount of fully glycosylated CFTRAF508/R553M is still small relative to wild-type CFfR. Login to comment
153 However, when cells expressed CFTRAF508/ R553M, CFTR was detected at the plasma membrane (Figure 5C). Login to comment
156 These results are consistent with the observation that more CFTRAF5081 R553M than CFTRAF508Kt553Q is found in the band C Figure 5. lmmunolocalization of Wild-Type and Mutant CFTR HeLa cells were transfected with wild-type CFTR (A) and CFTR mutants CFTRAF508 (B), CFTRAF508/R553M (C), and CFTRAF5081R553Q (D). Login to comment
161 Thus, these CFTRAF508/R553Q CFTR data indicate that only the CFTRAF508/R553M mutant is To determine whether the suppressor mutations altered detectable in the plasma membrane at levels greater than the single-channel properties of CFTRAF508, the re- that observed for CFTRAF508. Login to comment
215 The CFTRAF508 revertant mutations R553Q and R553M were initially identified as revertants of the mating defect in yeast containing the H5-AF508 chimera, suggesting that the structure of NBDl in the chimera (and the effect of CF mutations on NBDl structure) resembles that of CFTR. Login to comment
223 Because wild-type phenotypes were observed with R553Q and R553M mutations alone, these mutations may be compensatory mutations that cause no detectable phenotype themselves. Login to comment
240 Although the R553Q mutation corrected the defect in the P, of CFTRAF508, it was less effective than R553M in correcting the processing defect of CFTRAF508. Login to comment
243 In this regard, one might predict that the R553M mutation (or the AF508/R553Q on both chromosomes) might have a greater effect in suppressing the AF508 Cl- transport defect in the sweat gland and other organs. Login to comment
275 Two such colonies were identified (which contained the R553Q and R553M mutations), plasmid DNA was isolated from each, and the DNA sequence of the NED1 region was determined. Login to comment
284 To express CFTR in HeLa cells transiently, cells were infected with recombinant vaccinia virus (vTF7-3) to express the T7 bacteriophage RNA polymerase and then transfected with plasmid DNA containing either wild-type CFTR (pTM-CFTR-4) or CFTR mutants (pTMCFTRAF508, pTMCFTRAF508/R553Q, pTMCFTRAF508/R553M, pTMCFTR/R553Q, and pTMCFTWR553M) under the control of the T7 promoter, essentially as described in Rich et al. (1990). Login to comment

[hide] Rescue of functional DeltaF508-CFTR channels by co... FEBS Lett. 2003 Nov 6;554(1-2):173-8. Owsianik G, Cao L, Nilius B
Rescue of functional DeltaF508-CFTR channels by co-expression with truncated CFTR constructs in COS-1 cells.
FEBS Lett. 2003 Nov 6;554(1-2):173-8., [PMID:14596935]

Abstract [show]
The most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), DeltaF508-CFTR, is misprocessed and subsequently degraded in the endoplasmic reticulum. Using the patch-clamp technique, we showed that co-expressions of DeltaF508-CFTR with the N-terminal CFTR truncates containing bi-arginine (RXR) retention/retrieval motifs result in a functional rescue of the DeltaF508-CFTR mutant channel in COS-1 cells. This DeltaF508-CFTR rescue process was strongly impaired when truncated CFTR constructs possessed either the DeltaF508 mutation or arginine-to-lysine mutations in RXRs. In conclusions, our data demonstrated that expression of truncated CFTR constructs could be a novel promising approach to improve maturation of DeltaF508-CFTR channels.

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153 The fact that second-site mutations in NBD1 of vF508-CFTR partially correct processing and functional defects of this mutant channel strongly supports this latter hypothesis (note that R553Q and R553M mutations correspond to the arginine in the RAR motif) [16^18]. Login to comment

[hide] Rescue of F508del CFTR: Commentary on "F508del CFT... Biochim Biophys Acta. 2006 May;1758(5):563-4. Epub 2006 Apr 7. Tummler B
Rescue of F508del CFTR: Commentary on "F508del CFTR with two altered RXR motifs escapes from ER quality control but its channel activity is thermally sensitive".
Biochim Biophys Acta. 2006 May;1758(5):563-4. Epub 2006 Apr 7., [PMID:16712779]

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23 doi:10.1016/j.bbamem.2006.03.033 Hegedus' finding also provides a clue why second-site mutations in the RXR motif in the dodecapetide of NBF1 such as R553Q, R553M and R555K partially corrected the F508del CFTR mutant phenotype in model systems [16] and in CF patients [17]. 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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18 Additional second-site revertant mutations I539T, G550E, R553M, and R555K, within the portion of CFTR NBD1 included in the chimera, were also identified (DeCarvalho et al., 2002; Teem et al., 1993, 1996). Login to comment
19 The R553M, I539T, and the combination of G550E-R553M-R555K (3M) mutations correct the folding and stability defects of the DF508 NBD1 domain in isolation (DeCarvalho et al., 2002; Hoelen et al., 2010; Pissarra et al., 2008; Qu et al., 1997; Thibodeau et al., 2010) but only partially restore maturation of the full-length mutant protein (Hoelen et al., 2010; Pissarra et al., 2008; Thibodeau et al., 2010). Login to comment
127 The surface view A B I539T G550E R553M R555K 3M WT F S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 3 Relative Yield NBD1 ( -gal.) 25 30 35 40 45 0.0 0.5 1.0 Temperature (C ) Relative Turbitity 0 1 2 3 4 -5 0 5 10 WT F I539T I539T F S573E R555K D529F Relative Yield NBD1 ( -gal.) Tm Figure 3. Login to comment
166 B C A B 0 1 2 3 0 1 2 Relative Yield NBD1 (b2;-gal.) Relative Yield CFTR (ELISA) WT ࢞F WT ƊF S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 Relative Yield CFTR (ELISA) I539T G550E R553M R555K 3M Figure 4. Login to comment
172 See also Figure S5. (B) The influence of the 508-coupled mutations (green circles), four second-site suppressor mutations (I539T, G550E, R553M, and R555K) and three suppressors in combination (G550E, R553M, and R555K) (orange circles) on F508 background on relative maturation of full-length CFTR and relative NBD1 folding yield is correlated (green line, m = 0.75, R = 0.85). Login to comment
185 Previously identified second-site suppressor (I539T, G550E, R553M, R555K, and 3M) but not the 508-coupled mutants (D529F and S573E) increase the yield of DF508 NBD1. Login to comment
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
219 When R1070W is combined with mutations that improve DF508 NBD1 folding yield, I539T, G550E, R553M, R555K, and 3M (open triangles), the correlation between NBD1 folding and CFTR maturation in the wild-type protein is restored (m = 0.77, R = 0.47, black line) (&#b1;SEM). Login to comment

[hide] Functional Rescue of F508del-CFTR Using Small Mole... Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012. Molinski S, Eckford PD, Pasyk S, Ahmadi S, Chin S, Bear CE
Functional Rescue of F508del-CFTR Using Small Molecule Correctors.
Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012., [PMID:23055971]

Abstract [show]
High-throughput screens for small molecules that are effective in "correcting" the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

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62 Employing biophysical methods, including circular dichroism, dynamic light scattering,and fluorescence,both groups confirmed that the introduction of "stabilizing mutations" residing in the ABC b1;-helical subdomain (G550E, R553M, R555K) and the structural diverse region (I539T), fully corrects defects in kinetic and thermal stability of the isolated F508del-NBD1 domain. Login to comment

[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]

Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

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148 A) èc;F508 with NBD1-stabilizing mutations: 4S, I539T/G550E/R553M/R555K; èc;RI, deletion of amino acid residues 404-435; 4PT, S422P/S434P/S492P/A534P/I539T. Login to comment

[hide] Biosynthesis of cystic fibrosis transmembrane cond... Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28. Pranke IM, Sermet-Gaudelus I
Biosynthesis of cystic fibrosis transmembrane conductance regulator.
Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28., [PMID:24685677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride (Cl(-)) channel. Mutations of its gene lead to the disease of cystis fibrosis (CF) among which the most common is the deletion of phenylalanine at position 508 (Phe508del). CFTR is a multi-domain glycoprotein whose biosynthesis, maturation and functioning as an anion channel involve multi-level post-translational modifications of CFTR molecules and complex folding processes to reach its native, tertiary conformation. Only 20-40% of the nascent chains achieve folded conformation, while the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations causing CF impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding the cellular basis of CF and has brought to light various potential targets for novel, promising therapies.

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1442 Mutations located in NBD1, such as I539T, G550E, R553M/Q and R555K, as well as R1070W in CL4 of MSD2 promote Phe508del-CFTR maturation and trafficking to the cell surface and also restore channel activity (DeCarvalho et al., 2002; Teem et al., 1993, 1996; Thibodeau et al., 2010). Login to comment

[hide] Complement yourself: Transcomplementation rescues ... Biophys Rev. 2014 Mar 1;6(1):169-180. Cebotaru L, Guggino WB
Complement yourself: Transcomplementation rescues partially folded mutant proteins.
Biophys Rev. 2014 Mar 1;6(1):169-180., [PMID:24949105]

Abstract [show]
Cystic Fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing DeltaF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the DeltaF508 deletion mutant. Transcomplementation rescues the processing and channel activity of DeltaF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.

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77 Using a mutagenesis approach, they were able to isolate two mutants, R553M and R553Q, which in combination with ƊF508 restored mating. Login to comment

[hide] Restoration of NBD1 thermal stability is necessary... J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30. He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR
Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly.
J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30., [PMID:25083918]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (DeltaF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The DeltaF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the DeltaF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

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45 2PT, S492P/A534P/I539T; 4PT, 2PT + S422P/S434P; 3SS, G550E/R553M/R555K; 4SS, 3SS + I539T; ƊRI, deletion of RI amino acids 404-435; combo, ƊRI + 2PT + 3SS. Login to comment
74 (*) In full-length CFTR, R553M was introduced instead of R553Q in isolated NBD1. Login to comment
75 Based on our single mutation analysis, the Tm difference between G550E/R553Q/R555K and G550E/R553M/R555K is less than 1 &#b0;C. Fig. 3. Login to comment
96 The S492P and I539T substitutions had additive affects such that ƊTm increased to 4.4 &#b0;C, and ƊTm was further increased to 8.4 &#b0;C when the additional mutations A534P/G550E/R553M/R555K were introduced. Login to comment

[hide] Deletion of Phenylalanine 508 in the First Nucleot... J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6. Chong PA, Farber PJ, Vernon RM, Hudson RP, Mittermaier AK, Forman-Kay JD
Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.
J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6., [PMID:26149808]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

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363 Interestingly, the combined suppressor mutations I539T, G550E, R553M, and R555K have a bigger positive effect on F508del CFTR when NBD2 is present (58), suggesting the importance of the NBD interaction and hinting that these NBD1-stabilizing mutations may also improve the ability of F508del NBD1 to dimerize with NBD2. Login to comment