ABCMdb

ABCC7 p.Phe494Asn

Predicted by SNAP2:	A: N (78%), C: N (72%), D: D (66%), E: N (53%), G: N (53%), H: N (66%), I: N (87%), K: N (57%), L: N (82%), M: N (72%), N: N (72%), P: D (66%), Q: N (61%), R: N (57%), S: N (66%), T: N (78%), V: N (93%), W: N (53%), Y: N (87%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Diminished self-chaperoning activity of the DeltaF... PLoS Comput Biol. 2008 Feb 29;4(2):e1000008. Serohijos AW, Hegedus T, Riordan JR, Dokholyan NV
Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.
PLoS Comput Biol. 2008 Feb 29;4(2):e1000008., [PMID:18463704]

Abstract [show]
The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-DeltaF508 are essential information in correcting this pathogenic mutant.

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168 The solubilizing F494N mutation, which is adjacent to Q493, has also been shown to partially correct the folding defect of CFTR-DF508 [23]. Login to comment

[hide] Cooperative assembly and misfolding of CFTR domain... Mol Biol Cell. 2009 Apr;20(7):1903-15. Epub 2009 Jan 28. Du K, Lukacs GL
Cooperative assembly and misfolding of CFTR domains in vivo.
Mol Biol Cell. 2009 Apr;20(7):1903-15. Epub 2009 Jan 28., [PMID:19176754]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) architecture consists of two membrane spanning domains (MSD1 and -2), two nucleotide binding domains (NBD1 and -2), and a regulatory (R) domain. Several point mutations lead to the channel misprocessing, with limited structural perturbation of the mutant domain. To gain more insight into the basis of CFTR folding defect, the contribution of domain-wise and cooperative domain folding was assessed by determining 1) the minimal domain combination that is recognized as native and can efficiently escape the endoplasmic reticulum (ER) retention and 2) the impact of mutation on the conformational coupling among domains. One-, two-, three-, and most of the four-domain assemblies were retained at the ER. Solubilization mutations, however, rescued the NBD1 processing defect conceivably by thermodynamic stabilization. The smallest folding unit that traversed the secretory pathway was composed of MSD1-NBD1-R-MSD2 as a linear or split polypeptide. Cystic fibrosis-causing missense mutations in the MSD1, NBD1, MSD2, and NBD2 caused conformational defect in multiple domains. We propose that cooperative posttranslational folding is required for domain stabilization and provides a plausible explanation for the global misfolding caused by point mutations dispersed along the full-length CFTR.

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155 The N1*⌬F and N1*4D contains the same mutations as defined in a. N1*3S incorporates the F429S, F494N, and Q637R solubilization mutations. Login to comment
174 Five, remarkably, introducing three solubilization mutations (F429S, F494N, and Q637R) that were required to produce soluble, recombinant NBD1 in bacteria (Lewis et al., 2005), significantly increased the steady-state cell surface expression of the CD4Tl-N1*-3S at 37°C and suppressed the expression defect at 26°C (Figure 3, b and c). Login to comment
291 In support of this hypothesis, severalfold increase in the cell surface expression of CD4T-NBD1* was documented in the presence of those solubilization mutations (F429N, F494N, and Q637R) that were required for the recombinant NBD1 expression (Figure 3, b and c) (Lewis et al., 2005). Login to comment
293 Indeed, the F429N, F494N, and Q637R mutations thermodynamically stabilized the isolated NBD1 in the absence of domain-domain interactions as indicated by the elevated melting temperature of the recombinant NBD1*-3S relative to its wt counterpart (Rabeh, Mulvihille and Lukacs, unpublished observations). 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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107 Significant conformational changes, apart from differences in the local surface properties at the mutation site, were not observed in the crystal structures of DF508 NBD1-RE (also containing F429S, F494N, and Q637A mutations required for protein solubility and crystallization) (Lewis et al, 2004, 2005) and of DF508 NBD1 lacking the RI and the RE (PDB code 2PZF). Login to comment
249 The additional mutations (F494N, Q637A or F429S, F494N, and Q637R) in the DF508 NBD1-RE construct required for protein solubility and crystallization (Lewis et al, 2005) also partially rescue the trafficking and gating defects of full-length DF508 CFTR, suggesting that the crystal structure of DF508 NBD1-RE may correspond to a partially corrected conformation (Pissarra et al, 2008). Login to comment
250 It is noteworthy that the F429S mutation is in the RI and further promotes the revertant effect produced by the F494N/Q637 mutant (Pissarra et al, 2008). Login to comment

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

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

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48 These constructs have two solubilizing mutations selected because they are sequence variations naturally present in more soluble NBD1 variants from other vertebrate species [F494N in the γ- phosphate switch from several fish species (unpublished results) and Q637R in the RE from mouse].5,37 One newly reported ΔF508 structure has only these two mutations (PDB ID 2BBT, construct hNBD1-2f- ΔF508, Rwork =23.2 and Rfree =29.5 at 2.30 Å), while the other has an additional F429S mutation in a disordered region of the RI (PDB ID 2BBT, construct hNBD1-3-ΔF508, Rwork =22.6 and Rfree =29.1 at 2.05 Å). Login to comment
305 Equivalent structural analyses conducted on the F494N, Q637R, and H667R solubilizing mutations are described in detail in sections ST13-14 in the Supplementary Information. Login to comment

[hide] Structures of a minimal human CFTR first nucleotid... Protein Eng Des Sel. 2010 May;23(5):375-84. Epub 2010 Feb 11. Atwell S, Brouillette CG, Conners K, Emtage S, Gheyi T, Guggino WB, Hendle J, Hunt JF, Lewis HA, Lu F, Protasevich II, Rodgers LA, Romero R, Wasserman SR, Weber PC, Wetmore D, Zhang FF, Zhao X
Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant.
Protein Eng Des Sel. 2010 May;23(5):375-84. Epub 2010 Feb 11., [PMID:20150177]

Abstract [show]
Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

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65 Human NBD1 proteins utilized Clone name Sequencea 2935c469 Ser-NBD1[387-646(D405-436)] 2935c472 Ser-NBD1[387-646(D405-436,DF508)] 2935c492 Ser-NBD1[375-646(D405-436)] 2935c382b Ser-NBD1[389-678(F429S,F494N,Q637R)] 2935c371c Ser-NBD1[389-678(F429S,F494N,Q637R, DF508)] a All of the NBD1 proteins start with a non-native serine preceding the NBD1 sequence. Login to comment
114 Human NBD1 387-646(D405-436) is more stable and binds ATP tighter than non-truncated constructs Truncated and non-truncated NBD1 proteins were analyzed for their thermal unfolding properties: NBD1 387-646(D405-436) and NBD1 389-678[F429S,F494N,Q637R]. Login to comment
138 (B) The same analysis was conducted with NBD1[389-678(F429S,F494N,Q637R)] proteins (2935c382 and 2935c371). Login to comment
249 Many of the solubilizing mutations developed for non-truncated NBD1 are in the RI or Q-loop cleft (F409L, F429S, F433L and F494N) and might function by reducing these interactions. Login to comment

[hide] Folding and rescue of a cystic fibrosis transmembr... J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15. Da Paula AC, Sousa M, Xu Z, Dawson ES, Boyd AC, Sheppard DN, Amaral MD
Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.
J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15., 2010-08-27 [PMID:20551307]

Abstract [show]
Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys(584) interacts with residue Leu(581) in human CFTR Glu(584) interacts with Phe(581) in mouse CFTR. Introduction of the murine residue (Phe(581)) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.

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218 Single Channel Behavior of Processing Mutant K584E-CFTR-In previous research, we demonstrated that revertant (e.g. G550E-CFTR (24)) and solubilizing mutations (e.g. F429S/F494N/Q637R (13)) rescue defects in CFTR channel gating in addition to promoting the cell surface expression of F508del-CFTR. Login to comment

[hide] Thermal unfolding studies show the disease causing... Protein Sci. 2010 Oct;19(10):1917-31. Protasevich I, Yang Z, Wang C, Atwell S, Zhao X, Emtage S, Wetmore D, Hunt JF, Brouillette CG
Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1.
Protein Sci. 2010 Oct;19(10):1917-31., [PMID:20687133]

Abstract [show]
Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs +/-F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T(m)) by 6-7 degrees C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 +/- F508del: N(+/-MgATP) <==> I(T)(+/-MgATP) --> A(T) --> (A(T))(n). The equilibrium unfolding to intermediate, I(T), is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A(T), for which aggregation to (A(T))(n) and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A(T).

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44 hNBD1 Nameb Termini / Mutationsc Tm d DTm ¼ Tm D508 - Tm wt ( C) PDB ID 1 hNBD1-D(RI,RE) 2935c46917 387-646[D405-436] 57.7 þ 0.2 2PZE 1 (F508del)hNBD1D (RI,RE) 2935c47217 387-646[D405-436, F508del] 51.5 þ 0.3 À6.2 þ 0.3 2PZF 2 387-646[D405-436, V510D] 60.2 þ 0.4 2 387-646[D405-436, V510D, F508del] 53.0 þ 0.1 À7.2 þ 0.4 3 387-646[D405-436, F494N, Q637R] 59.2 3 387-646[D405-436, F494N, Q637R, F508del] 52.8 À6.4 4 387-646[D405-436, G550E, R553Q, R555K] 61.7 4 387-646[D405-436, G550E, R553Q, R555K,F508del] 55.7 À6.0 5 387-678[D405-436] 58.1 5 387-678[D405-436, F508del] 51.7 À6.2 6 hNBDI-315 2935c38217 389-678[F429S, F494N, Q637R] 49.8 þ 0.3 6 hNBDI-3F508del15 2935c37117 389-678[F429S, F494N, Q637R, F508del] 43.6 þ 0.1 À6.3 þ 0.3 2BBS 7 389-678[F429S, F494N, L636E5, Q637R] 50.5 þ 0.2 7 389-678[F429S, F494N, L636E, Q637R, F508del] 44.9 À6.2 þ 0.2 a DSC conducted at 1 mg/mL protein. Login to comment
61 The Teem suppressor triplet (pair 4)29 increases Tm by 4 , the V510D mutation (pair 2)30 by 2.5 , and F494N/ Q637R (pair 3) by 1.5 . Login to comment

[hide] Integrated biophysical studies implicate partial u... Protein Sci. 2010 Oct;19(10):1932-47. Wang C, Protasevich I, Yang Z, Seehausen D, Skalak T, Zhao X, Atwell S, Spencer Emtage J, Wetmore DR, Brouillette CG, Hunt JF
Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis.
Protein Sci. 2010 Oct;19(10):1932-47., [PMID:20687163]

Abstract [show]
The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.

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28 Surprisingly, several of these solubilizing surface mutations in hNBD1, identified in a screen focused exclusively on the in vitro solubility of hNBD1, were shown to suppress the in vivo trafficking defect of F508del-CFTR more strongly than the best existing pharmacological agents.32,38 Notably, the mutated residues (e.g., F429S, F494N, and Q637R) are not in direct contact with F508 and do not appear to be allosterically coupled.18 A similar hydrophobic-to-hydrophilic substitution in the immediate vicinity of F508, the V510D mutation, also strongly suppresses the in vivo trafficking defect of F508del-CFTR.39,40 It was proposed that these substitutions could block adventitious chaperone interactions that prevent proper ER export.18 However, there is as yet no concrete evidence explaining the tight correlation between the effects of mutations on the in vitro solubility properties of hNBD1 and the in vivo trafficking properties of human CFTR. Login to comment
118 Solubilizing surface mutations need to be introduced into full-length hNBD1 to obtain sufficient material for biophysical studies.15 Supporting Information Figure S5 compares the behavior of matched full-length and D(RI,RE) constructs containing F429S, F494N, and Q637R mutations15 in the absence or presence of the F508del mutation. Login to comment
159 The denaturation of hNBD1-D(RI,RE)-F508del is compared to that of the same construct containing in addition the Teem suppressor triplet37 (left), V510D39 (center), or F494N/Q637R15 (right). Login to comment
160 F494N/Q637R mutations were identified in a screen for surface substitutions that improve the solubility of purified hNBD1 in vitro.15 This screen focused on replacement of hydrophobic residues with more hydrophilic residues present at equivalent sites in other vertebrate CFTR sequences. Login to comment
161 These mutations also show significant efficacy in suppressing the trafficking defect caused by the F508del mutation in vivo in tissue culture cells,32 although they are less effective that the Teem suppressors,37 the V510D mutation,39,40 or deletion of the RI.41 The Teem suppressor triplet (Fig. 4A-C), the V510D mutation (Fig. 4D-F), and the F494N/Q637R mutations (Fig. 4G-I) all stabilize hNBD1 against the initial unfolding transition, shifting its midpoint 0.25-0.50 M higher in urea concentration. Login to comment
162 The magnitude of the SLS increase following the initial unfolding transition is greatly reduced by the Teem suppressor triplet and the V510D mutation and significantly reduced by the F494N/Q637R mutations. Login to comment
168 First, the SLS data in Figure 4I demonstrate that the F494N/Q637R mutation pair in the F508del domain is unique in suppressing the self-association that occurs without an increase in trp fluorescence before the onset of the initial unfolding transition. Login to comment
170 Second, the F494N/Q637R mutation pair appears to decrease the secondary structure content in the partially unfolded intermediate formed by both the F508del (Fig. 4G) and F508 (Supporting Information Fig. S8G) domains, while the Teem suppressor triplet may increase the secondary structure content in this state just in the F508del domain (Fig. 4A). Login to comment
189 See Lewis et al.39 for a detailed description of the subdomain organization of hNBD1 and the stereochemical effects of the F508del mutation, the Teem suppressor mutation triplet,37 and the F494N/Q637R solubilizing mutations.15 reveals that hNBD1 unfolds via two sequential transitions which have similar spectroscopic and aggregation properties whether unfolding is driven by urea or by heat. Login to comment
200 The Teem suppressor triplet and the F494N mutation are located on the opposite face of the ABCa subdomain from the V510D mutation, which is adjacent to the F508del site, and crystallographic studies indicate that the direct stereochemical effects of these different mutations are likely to be unrelated.18 The influence of all of these mutations on the initial unfolding transition suggests that it involves a global change in ABCa subdomain conformation. Login to comment

[hide] The most common cystic fibrosis-associated mutatio... J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11. Jih KY, Li M, Hwang TC, Bompadre SG
The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.
J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11., 2011-06-01 [PMID:21486785]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (DeltaF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), can increase the open probability (Po) to approximately 0.7, implying that the gating defect of DeltaF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of DeltaF508-CFTR channels are dramatically shortened, suggesting that the DeltaF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of DeltaF508-CFTR, namely the solubilizing mutation F494N/Q637R and DeltaRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with DeltaF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of DeltaF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for DeltaF508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.

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12 We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of F508-CFTR channels are dramatically shortened, suggesting that the F508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of F508-CFTR, namely the solubilizing mutation F494N/Q637R and RI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with F508-CFTR. Login to comment
143 We introduced into F508-CFTR the 'solubilizing mutations` F494N/Q637R (Pissarra et al. 2008) and the regulatory insertion deletion ( RI, deletion of residues 404-435) (Aleksandrov et al. 2010) to test whether they have any effects on the F508-CFTR gating defects described above. Login to comment
145 As seen in Figs 5A and 6A, in either case, the current relaxation upon removal of ATP and PPi was significantly slower compared with that for WT-CFTR (F494N/Q637R-CFTR: τ = 86.14 ± 12.61s, n = 6; RI-CFTR: τ = 75.33 ± 14.36 s, n = 7). Login to comment
147 For ligand exchange experiments, these mutations also significantly prolong the second phase of current changes upon switching the ligand from ATP to P-ATP (F494N/Q637R-CFTR: τ = 76.41 ± 12.31 s, n = 6; RI-CFTR: τ = 81.78 ± 6.66 s, n = 7) (Figs 5A, 6A and 7). Login to comment
149 We found that although the locked-open time (F494N/Q637R/ F508-CFTR: τ = 5.95 ± 0.36 s, n = 8; RI/ F508-CFTR: τ =5.52 ± 0.45 s, n = 11) and ligand exchange time (F494N/Q637R/ F508-CFTR: τ=8.44 ± 1.3s, n=6; RI/ Figure 5. Login to comment
150 Effects of 'solubilizing mutations`, F494N/Q637R, on WTand F508-CFTR channels A, representative current traces of F494N/Q637R-CFTR channels locked opened by 1 mM ATP and 2 mM PPi (left) and ATP/P-ATP ligand exchange (right). Login to comment
151 B, representative current traces of F494N/Q637R/ F508-CFTR channels locked opened by 1 mM ATP and 2 mM PPi (left) and ATP/P-ATP ligand exchange (right). Login to comment
157 F508-CFTR: τ = 8.95 ± 1.75 s, n = 4) of F494N/ Q637R/ F508 and F508/ RI channels are prolonged (Figs 5B, 6B and 7), they are still much shorter than those of WT channels. Login to comment
159 Besides prolonging the PPi locked-open time and the ligand exchange time, F494N/Q637R/ F508 and F508/ RI have been previously shown to improve the function of F508-CFTR (Pissarra et al. 2008; Aleksandrov et al. 2010). Login to comment
161 To our surprise, we found that P-dATP still increases the current dramatically for both compound mutants: 6.96 ± 0.17-fold increase for RI/ F508-CFTR (n = 5), and 12.36 ± 1.21-fold increase for F494N/Q637R-CFTR (n = 8) (Fig. 8). Login to comment
165 sol: solubilizing mutation, F494N/Q637R. Login to comment
170 Since P-dATP does not alter single channel conductance (Miki et al. 2010), the increase in the macroscopic current induced by P-dATP in F494N/Q637R/ F508 and F508/ RI (12-and 7-fold, respectively) suggests that the Po of these mutant channels is still lower than that of WT channels when ATP is used as the ligand. Login to comment
191 Effect of P-dATP on F494N/Q637R/ F508-CFTR and RI/ F508-CFTR Representative current traces of F494N/Q637R/ F508-CFTR (A) and RI/ F508-CFTR (B) in the presence of 50 μM P-dATP. Login to comment
192 C, current increase induced by P-dATP for F494N/Q637R/ F508-CFTR (n = 8) and RI/ F508-CFTR (n = 5). 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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136 Alternatively, the protein can be modified to increase solubility by specific point mutations (F494N and to a lesser degree F429S and Q637R) without deleting the RI (46). 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

[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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121 Suppressor mutations can rescueΔF508-CFTRbya variety ofmechanisms.Examplesinclude removal of the ER retention signals (arginine-framed trafficking motif mutations; R29K, R516K, R555K, and R766K) (61, 62), introduction of a combination of CFTR suppressor mutations (F949/Q637R or F29S/F494N/Q637R) that increase solubility of NBD1(63),orintroductionofsuppressormutationssuchasV510D (TMD1) (64) and R1070W(TMD2) (65) that restore NBD1-TMD2 interactions. Login to comment

[hide] Disruption of cytokeratin-8 interaction with F508d... Hum Mol Genet. 2012 Feb 1;21(3):623-34. Epub 2011 Oct 28. Colas J, Faure G, Saussereau E, Trudel S, Rabeh WM, Bitam S, Guerrera IC, Fritsch J, Sermet-Gaudelus I, Davezac N, Brouillard F, Lukacs GL, Herrmann H, Ollero M, Edelman A
Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect.
Hum Mol Genet. 2012 Feb 1;21(3):623-34. Epub 2011 Oct 28., [PMID:22038833]

Abstract [show]
We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.

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29 The first one shows experimentally that NBD1 destabilization occurs as a consequence of three solubilizing mutations, namely V510D, F494N and Q637R (21). Login to comment

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

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

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26 Both the R mutations (G550E, R553Q, and R555K) and S mutations (F409L, F429S, F433L, F494N, and H667R) could partially rescue the DF508 CFTR folding and functional defect (Lewis et al., 2005; Pissarra et al., 2008; Teem et al., 1993, 1996) and were assumed to stabilize the domain either alone or in combinations (1S, 3S, R, R1S, and R4S; see Figure 1B). Login to comment

[hide] A chaperone trap contributes to the onset of cysti... PLoS One. 2012;7(5):e37682. doi: 10.1371/journal.pone.0037682. Epub 2012 May 31. Coppinger JA, Hutt DM, Razvi A, Koulov AV, Pankow S, Yates JR 3rd, Balch WE
A chaperone trap contributes to the onset of cystic fibrosis.
PLoS One. 2012;7(5):e37682. doi: 10.1371/journal.pone.0037682. Epub 2012 May 31., [PMID:22701530]

Abstract [show]
Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of DeltaF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a 'chaperone trap'. Culturing cells at 30 C resulted in correction of DeltaF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that DeltaF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.

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229 NBD1-WT(F494N)-TTEVVMENVTAFWEEGFGELFE- KAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINF- KIERGQLLAVAGSTGAGKTSLLMMIMGELEPSEGKIKHS- GRISFCSQNSWIMPGTIKENIIFGVSYDEYRYRSVIKACQ- LEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYK- DADLYLLDSPFGYLDVLTEKEIFESCVCKLMANK- TRILVTSKMEHLKKADKILILHEGSSYFYGTF- SELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRF- SLEGDAPVS. Login to comment
230 NBD1-DF508 (F494N)-TTEVVMENVTAFWEEGFGELFE- KAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINF- KIERGQLLAVAGSTGAGKTSLLMMIMGELEPSEGKIKHS- GRISFCSQNSWIMPGTIKENIIGVSYDEYRYRSVIKACQ- LEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYK- DADLYLLDSPFGYLDVLTEKEIFESCVCKLMANK- TRILVTSKMEHLKKADKILILHEGSSYFYGTF- SELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRF- SLEGDAPVS. Login to comment
233 NBD1-WT(F494N)-TTEVVMENVTAFWEEGFGELFE- KAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINF- KIERGQLLAVAGSTGAGKTSLLMMIMGELEPSEGKIKHS- GRISFCSQNSWIMPGTIKENIIFGVSYDEYRYRSVIKACQ- LEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYK- DADLYLLDSPFGYLDVLTEKEIFESCVCKLMANK- TRILVTSKMEHLKKADKILILHEGSSYFYGTF- SELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRF- SLEGDAPVS. Login to comment
234 NBD1-DF508 (F494N)-TTEVVMENVTAFWEEGFGELFE- KAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINF- KIERGQLLAVAGSTGAGKTSLLMMIMGELEPSEGKIKHS- GRISFCSQNSWIMPGTIKENIIGVSYDEYRYRSVIKACQ- LEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYK- DADLYLLDSPFGYLDVLTEKEIFESCVCKLMANK- TRILVTSKMEHLKKADKILILHEGSSYFYGTF- SELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRF- SLEGDAPVS. Login to comment

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

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

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5 Although F508del-NBD1 shows only minor conformational changes relative to that of wild-type NBD1, additional mutations (F494N/Q637R or F429S/F494N/Q637R) were required for domain solubility and crystallization. Login to comment
33 However, these new F508del-NBD1 crystal structures still required either two (F494N/Q637R; Protein Data Bank [PDB] ID code: 2BBT) or three (F429S/F494N/ Q637R; PDB ID code: 2BBS) additional mutations for domain solubility and, hence, crystal formation (Lewis, 2005). Login to comment
35 To test these ideas, we investigated the effects of the mutations F494N/Q637R and F429S/F494N/Q637R on wt- and F508del-CFTR by studying: (1) the in vivo folding yield of NBD1, (2) the processing and trafficking of the full-length CFTR protein, and (3) the ClÀ channel function of CFTR. Login to comment
37 RESULTS While studying the effects of F508del on the structure of NBD1 from CFTR, Lewis (2005) introduced the mutations F494N/ Q637R (double; D) and F429S/F494N/Q637R (triple; T) into NBD1 to improve domain solubility and crystallization. Login to comment
42 Solubilizing Mutations Improve wt- and F508del-NBD1 Yield To explore whether F429S, F494N, and Q637R improve the yield of soluble NBD1, wt-NBD1 and F508del-NBD1 were expressed in bacterial cells in the absence and presence of the solubilizing mutations. Login to comment
144 Structural Implications An interesting aspect of the action of the solubilizing mutations (double, F494N/Q637R; triple, F429S/F494N/Q637R) is their remote location from that of F508. Login to comment
146 However, the effect produced by F494N is not completely unexpected, as structural crosstalk between the side chain of the F508 residue (e.g., F508R) and the Q loop or g-phosphate switch (e.g., residues W496 and M498) has been previously shown to occur (Massiah et al., 1999). Login to comment
151 In support of this notion, our data indicate that Q637R, at least with one additional solubilizing mutation (F494N), might contribute to the increased solubility of F508del-NBD1 and to the partial rescue of F508del-CFTR trafficking and function. Login to comment
152 The third solubilizing mutation, F429S, further promotes the revertant effect produced by the double mutant (F494N/Q637R) on F508del-CFTR, as the triple mutant (F429S/F494N/Q637R) visibly increased maturation of F508del-CFTR as measured by the higher maturation yield at steady state of F508delT-CFTR compared with that of F508delD-CFTR (Figure 1C). Login to comment
175 The available crystal structure of F508del-NBD1 was determined after the introduction of additional mutations (F494N/Q637R or F429S/ F494N/Q637R) to help domain solubilization and crystal formation. Login to comment
182 Site-Directed Mutagenesis, Cells, and CFTR Expression To introduce the solubilizing mutations F494N/Q637R and F429S/F494N/ Q637R into wt- and F508del-CFTR cDNAs in the pNUT expression vector, we used the primers F429S, 50 -GGTGATGACAGCCTCTCCTTCAGTAATTTC TCA-30 ; F494N, 50 -CATTCTGTTCTCAGAATTCCTGGATTATGCCTGG-30 ; Q637R, 50 -GAACTCCAAAATCTAAGGCCAGACTTTAGCTC-30 and the QuikChange site-directed mutagenesis kit (Stratagene). Login to comment
187 Cell lines expressing different solubilizing mutations are referred to as follows: wtD-CFTR, F494N-Q637R-CFTR; F508delD-CFTR, F494N- F508del-Q637R-CFTR; wtT-CFTR, F429S-F494N-Q637R-CFTR; and F508delT-CFTR, F429S-F494N-F508del-Q637R-CFTR. Login to comment

[hide] Defining the defect in F508 del CFTR: a soluble pr... Chem Biol. 2008 Jan;15(1):3-4. Deber CM, Cheung JC, Rath A
Defining the defect in F508 del CFTR: a soluble problem?
Chem Biol. 2008 Jan;15(1):3-4., [PMID:18215767]

Abstract [show]
Previously reported crystal structures of CFTR F508 del-NBD1 were determined in the presence of solubilizing mutations. In this issue of Chemistry & Biology, Pissarra et al. (2008) show that partial rescue of the trafficking and gating defects of full-length CFTR occurs in vivo upon recapitulation of the solubilizing F494N/Q637R or F428S/F494N/Q637R substitutions in cis with F508 del.

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2 In this issue of Chemistry & Biology, Pissarra et al. (2008) show that partial rescue of the trafficking and gating defects of full-length CFTR occurs in vivo upon recapitulation of the solubilizing F494N/Q637R or F428S/F494N/Q637R substitutions in cis with F508 del. Login to comment
19 Two F508 del-NBD1 structures lacking the suppressor mutations but retaining certain other alterations (F494N/Q637R or F428S/F494N/ Q637R) necessary for protein solubility subsequently became available (Lewis et al., 2005, http://www.pdb.org). Login to comment
23 To address this question, Pissarra et al. (2008) report in this issue on the trafficking in mammalian cell lines of full-length CFTR proteins carrying wt, F508 del, or F508 del with the F494N/Q637R or F429S/F494N/ Q637R replacements. Login to comment
25 Strikingly, the solubilizing mutations, notably the triple mutant F429S/ F494N/Q637R, appeared to promote some N-glycan processing in F508 del CFTR, to produce Band C, along with bands of MW intermediate between those of Band B and Band C, suggesting that these replacements partially rescue the trafficking defect. 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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34 The first stabilizing mutations were identified in the ABC conserved, canonical subdomains, and cluster in the b1;-helical subdomain (G550R, R553Q, R555K), in the b3; switch (F494N), and ATP binding core subdomain (Q637R). Login to comment

[hide] Small heat shock proteins target mutant cystic fib... Mol Biol Cell. 2013 Jan;24(2):74-84. doi: 10.1091/mbc.E12-09-0678. Epub 2012 Nov 14. Ahner A, Gong X, Schmidt BZ, Peters KW, Rabeh WM, Thibodeau PH, Lukacs GL, Frizzell RA
Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier-dependent pathway.
Mol Biol Cell. 2013 Jan;24(2):74-84. doi: 10.1091/mbc.E12-09-0678. Epub 2012 Nov 14., [PMID:23155000]

Abstract [show]
Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27's ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4's impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin-proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.

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169 Purified NBD1 proteins containing a single suppressor mutation (1S, F494N) were incubated with or without purified components for 1 h at 27&#b0;C as illustrated and described in Materials and Methods. Login to comment
286 For cycloheximide chases, the culture medium was changed to one containing mutation, F494N, as previously described (Rabeh et al., 2012). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514. Hunt JF, Wang C, Ford RC
Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.
Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514., [PMID:23378596]

Abstract [show]
Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

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165 The other mutation sets that improved the yield of soluble hNBD1 involved substitution of surface-exposed residues in hNBD1 with more polar residues occurring at the same position in CFTR orthologs from other species (F429S/F494N/ Q637R found in PDB ID 2BBS, F494N/ Q637R found in PDB ID 2BBT, and F429S/ H667R found in PDB ID 1XMI). Login to comment
275 A series of second-site mutations in NBD1 have parallel effects in rescuing the trafficking defect in CFTR in vivo (DeCarvalho et al. 2002; Pissarra et al. 2008; Aleksandrov et al. 2010) and inhibiting molten globule formation by isolated NBD1 in vitro (G550E/R553Q/R555K, F494N/Q637R, or V510D) (Protasevich et al. 2010; Wang et al. 2010). Login to comment

[hide] Novel pharmacological strategies to treat cystic f... Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006. Hanrahan JW, Sampson HM, Thomas DY
Novel pharmacological strategies to treat cystic fibrosis.
Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006., [PMID:23380248]

Abstract [show]
Cystic fibrosis (CF) is a lethal disease caused by mutations in the CFTR gene. The most frequent mutation is deletion of a phenylalanine residue (DeltaF508) that results in retention of the mutant, but otherwise functional, protein in the endoplasmic reticulum (ER). There have been recent advances in the identification of chemically diverse corrector compounds that allow DeltaF508-CFTR protein to traffic from the ER to the plasma membrane. The most studied correctors fall into two categories, pharmacological chaperones that bind to the mutant protein and circumvent its recognition by the cellular protein quality control systems and proteostasis regulators that modify the cellular pathways responsible for protein quality control and trafficking. This review focuses on recent advances in the field, strategies for the development of drugs from corrector compounds for the treatment of CF, and identification of their targets and mechanism(s) of action.

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146 Conversely, pharmacological chaperones that restore the interface between NBD1 and MSD2 should be additive with the three solubilizing (3S) mutant in NBD1 (F494N, Q637R, F429S) [11]. Login to comment

[hide] Bithiazole correctors rescue CFTR mutants by two d... Biochemistry. 2013 Aug 6;52(31):5161-3. doi: 10.1021/bi4008758. Epub 2013 Jul 22. Loo TW, Bartlett MC, Clarke DM
Bithiazole correctors rescue CFTR mutants by two different mechanisms.
Biochemistry. 2013 Aug 6;52(31):5161-3. doi: 10.1021/bi4008758. Epub 2013 Jul 22., [PMID:23865422]

Abstract [show]
Better correctors are needed to repair cystic fibrosis transmembrane conductance regulator (CFTR) processing mutants that cause cystic fibrosis. Determining where the correctors bind to CFTR would aid in the development of new correctors. A recent study reported that the second nucleotide-binding domain (NBD2) was involved in binding of bithiazole correctors. Here, we show that bithiazole correctors could also rescue CFTR mutants that lacked NBD2. These results suggest that bithiazoles rescue CFTR mutants by two different mechanisms.

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24 It should be noted, however, that the ƊNBD2 CFTR used in this study was different from that used by Okiyoneda et al.18 The ƊNBD2 CFTR constructs in our study did not contain the G550E, R553Q, R555K, and F494N mutations or the three hemagglutinin tags in the fourth extracellular loop that could influence CFTR corrector interactions. Login to comment

[hide] VX-809 corrects folding defects in cystic fibrosis... Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7. Ren HY, Grove DE, De La Rosa O, Houck SA, Sopha P, Van Goor F, Hoffman BJ, Cyr DM
VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7., [PMID:23924900]

Abstract [show]
Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

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93 These mutations are termed solubilizing (S) mutations and were introduced into NBD1 in different combinations (Figure 5A, S2 [F429S, Q637R] and S3 [F429S, F494N, and Q637R]). Login to comment
178 S2 (F429S, Q637R) and S3 (F429S, F494N, and Q637R) are mutations introduced into NBD1 to increase the thermodynamic stability of NBD1 and thereby increase CFTR and F508del-CFTR (B and C) folding efficiency (Pissarra et al., 2008; Teem et al., 1993). Login to comment
184 %-Wt C-Band %-Wt C-Band C. F508 V510 R1070 ICL4 ICL2 F429 Q637 F494 NBD1 S2=F429S, Q637R S3=F429S, F494N Q637R FIGURE 4:ߒ Functional defects in CFTR caused by disease-related mutations in MSD1 are suppressed by VX-809. Login to comment

[hide] Allosteric coupling between the intracellular coup... PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013. Dawson JE, Farber PJ, Forman-Kay JD
Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR.
PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013., [PMID:24058550]

Abstract [show]
Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function.

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5 Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Login to comment
25 The maturation defects can be partially suppressed by mutations in NBD1 or the CL4 coupling helix, such as V510D and F494N/Q637R [11,16-21], and by the drug VX-809 [22,23], now in clinical trials. Login to comment
27 Many of the F508del-suppressor mutations, such as F494N, as well as the RI deletion, increase the solubility of the NBD1 domain [20,24]. Login to comment
29 Both the solubilizing double mutant F494N/Q637R and the deletion of the RI increase the locked open time of WT and F508del CFTR binding pyrophosphate [27]. Login to comment
50 Similar titration patterns were observed in NBD1 constructs containing Q637R and mutations near the CL4-binding site (F508del, F494N, and V510D), as well as in a CL4-NBD1 fusion. Login to comment
56 Mutant NBD1 constructs with F494N, F508del, V510D, Q637R or F494N/ Q637R were constructed with a Stratagene QuikChange site-directed mutagenesis kit. Login to comment
74 (The 1.0 mM F494N and F494N/Q637R NBD1 samples used 50 mM sodium phosphate to maintain consistent pH conditions.) Login to comment
77 1.0 mM F494N and F494N/Q637R NBD1 samples were used for peak intensity comparison. Login to comment
86 TROSY 15 N-1 H HSQC [40], T1 [41], and T1r [41] data were collected on 1 mM F494N, Q637R, and F494N/Q637R NBD1 samples using a cryoprobe-equipped 600 MHz Varian spectrometer. Login to comment
115 Q637R is a solubilizing mutant that, in combination with F494N, partially corrects the folding defects induced by F508del in CFTR. Login to comment
121 The solubilizing mutation, F494N, reduces this association tendency. Login to comment
122 Both F494N NDB1 and F494N/Q637R NBD1 have a rotational correlation time of 2262 ns. Login to comment
124 The experimental equivalence of F494N and F494N/Q637R NBD1 tc values reduces the probability that any difference in lineshape may be caused by a difference in association states. Login to comment
125 Both decreases and increases in peak intensity, quantified by ratios of the intensities of the F494N/Q637R NBD1 and F494N NBD1 resonances (Figure 2B), are indicative of changes in dynamics. Login to comment
127 y~ I(F494N Q637R) I(F494N) {1 &#f0;2&#de; These changes in intensity point to the Q637R mutation affecting the dynamics of F494N NBD1 in multiple locations. Login to comment
146 Overlay of F494N (black) and F494N/Q637R (orange) NBD1 HSQC spectra. B. Login to comment
155 Q637R effects were probed using F494N NBD1 for improved solubility and spectral quality. Login to comment
175 CL4 also binds to NBD1 proteins containing single F508del-suppressor mutations, V510D and F494N, located near the predicted CL4-binding site, and Q637R, which lies between H8 and H9 (Figures 4 and S4). Login to comment
218 A, C, E, G. Dvobs between spectra of apo and CL4 peptide:NBD1 are shown in green for F508del, F494N, V510D, and Q637R NBD1, respectively. Login to comment
223 Dvobs values are mapped onto NBD1 structure as in Figure 2: B. F508del, D. F494N, F. V510D, and H. Q637R. Login to comment
224 F508del (position of F508 indicated as black sticks), F494N (orange space-filling representation), and V510D (purple space-filling representation) are near the predicted CL4-binding site. Login to comment
299 Chemical shift changes upon CL4 binding provide evidence for this allosteric network observed with minor variations in five variants of isolated NBD1-WT, F508del, F494N, V510D, and Q637R, in a CL4-NBD1 fusion protein with slightly different CL4 boundaries and in different buffer conditions. Login to comment
325 F508del suppressor modifications are found in this region as well, including the substitution of mouse RE into human CFTR [77] and Q637R, which in combination with F494N helps suppress folding defects in CFTR [20]. Login to comment
338 Chemical shift changes in F494N NBD1 due to H8/H9 mutation Q637R limited to neighboring residues in S3/S9/S10 and H8/H9. Login to comment
339 The F494N mutation was used to improve solubility. Login to comment
357 Overlay of apo (black) and 12.5:1 CL4:NBD1 (red) spectra for A. F508del NBD1, B. F494N NBD1, C. V510D NBD1, and D. Q637R NBD1 mutants. Login to comment

[hide] On the structural organization of the intracellula... Int J Biochem Cell Biol. 2014 Jul;52:7-14. doi: 10.1016/j.biocel.2014.01.024. Epub 2014 Feb 7. Moran O
On the structural organization of the intracellular domains of CFTR.
Int J Biochem Cell Biol. 2014 Jul;52:7-14. doi: 10.1016/j.biocel.2014.01.024. Epub 2014 Feb 7., [PMID:24513531]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein forming an anion selective channel. Mutations in the gene encoding CFTR cause cystic fibrosis (CF). The intracellular side of CFTR constitutes about 80% of the total mass of the protein. This region includes domains involved in ATP-dependent gating and regulatory protein kinase-A phosphorylation sites. The high-resolution molecular structure of CFTR has not yet been solved. However, a range of lower resolution structural data, as well as functional biochemical and electrophysiological data, are now available. This information has enabled the proposition of a working model for the structural architecture of the intracellular domains of the CFTR protein.

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1241 However, as the native preparation of NBD1 and NBD2 tend to precipitate at relatively low concentration (>2.5 mg/ml; Galeno et al., 2011; Galfr&#e8; et al., 2012), to obtain protein concentrations compatible with the crystallization conditions, three to seven revertant mutations (F409L, F429S, F433L, G550E, R553Q, R555K, H667R, Roxo-Rosa et al., 2006; F429S, F494N, Q637R, Pissarra et al., 2008) have been introduced into the NBD1. Login to comment

[hide] Synergy-based small-molecule screen using a human ... Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15. Phuan PW, Veit G, Tan J, Roldan A, Finkbeiner WE, Lukacs GL, Verkman AS
Synergy-based small-molecule screen using a human lung epithelial cell line yields DeltaF508-CFTR correctors that augment VX-809 maximal efficacy.
Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15., [PMID:24737137]

Abstract [show]
The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, DeltaF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methy l-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation DeltaF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for DeltaF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 microM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a DeltaF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.

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43 The cloning and characterization of 3HA-tagged variants of ƊF508-CFTR, R1070W- ƊF508-CFTR, and 3S-ƊF508-CFTR (containing the F494N, Q637R, and F429S NBD1 suppressor mutations) were described (Okiyoneda et al., 2013). Login to comment
81 Isolation of recombinant human NBD1 containing a single suppressor mutation (1S; F494N) and melting temperature measurement were performed as described (Rabeh et al., 2012). Login to comment
101 Preferential correction of DF508-CFTR-3HA with the NBD1 stabilizing 3S mutations (F494N, Q637R, and F429S) compared with CFTR carrying the R1070W interface-stabilizing mutation has been taken as evidence that VX-809 preferentially stabilizes the interface between NBD1 and MSDs but not the NBD1 folding defect CFTR (Okiyoneda et al., 2013). 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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95 S492P or I539T alone slightly increased the Tm of NBD1 (ƊTm = 2-3 &#b0;C) similar to the affect of the solubilization mutations F494N and Q637R combined. Login to comment

[hide] Channel Gating Regulation by the Cystic Fibrosis T... J Biol Chem. 2015 Dec 1. pii: jbc.M115.704809. Ehrhardt A, Chung WJ, Pyle LC, Wang W, Nowotarski K, Mulvihill CM, Ramjeesingh M, Hong J, Velu SE, Lewis HA, Atwell S, Aller S, Bear CE, Lukacs GL, Kirk KL, Sorscher EJ
Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.
J Biol Chem. 2015 Dec 1. pii: jbc.M115.704809., [PMID:26627831]

Abstract [show]
In this study, we present data indicating a robust and specific domain interaction between the CFTR first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed binding kinetics were not altered by the common clinical mutation, F508del. Both intrinsic ATPase activity and CFTR channel gating were severely inhibited by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR disregulation has been implicated in pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease (COPD), acquired rhinosinusitis, pancreatitis and lethal secretory diarrhea (e.g. cholera). Based on clinical relevance of CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

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48 This protein contained solubilizing mutations F494N/Q637R. Login to comment

[hide] Non-native conformers of CFTR NBD1 are recognized ... J Biol Chem. 2015 Dec 1. pii: jbc.M115.685628. Gong X, Ahner A, Roldan A, Lukacs GL, Thibodeau PH, Frizzell RA
Non-native conformers of CFTR NBD1 are recognized by Hsp27 and conjugated to SUMO-2 for degradation.
J Biol Chem. 2015 Dec 1. pii: jbc.M115.685628., [PMID:26627832]

Abstract [show]
A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein (sHsp), Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27, it interacted preferentially with the mutant, and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, K447, obviated Hsp27 mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 vs. WT, and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: a) its modification decreased as [ATP] increased, reflecting stabilization of the NBD by ligand binding, b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification, and c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.

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61 Variants that contain a single solubilizing mutation, F494N, were used in most experiments; this mutation enhances the yield of both NBDs from bacterial expression. Login to comment
98 In vitro SUMOylation of NBD1 - WT or F508del NBD1 SUMOylation was performed in vitro using purified human 1S-NBD1, a variant containing a single solubilizing mutation (F494N) that was introduced into the NBD during crystallization trials (32). Login to comment