ABCMdb

ABCC7 p.Gln637Arg

None has been submitted yet.

Publications

PMID: 19176754 [PubMed] Du K et al: "Cooperative assembly and misfolding of CFTR domains in vivo."

No. Sentence Comment

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elevated melting temperature of the recombinant NBD1*-3S relative to its wt counterpart (Rabeh, Mulvihille and Lukacs, unpublished observations).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

PMID: 19927121 [PubMed] Kanelis V et al: "NMR evidence for differential phosphorylation-dependent interactions in WT and DeltaF508 CFTR."

No. Sentence 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

PMID: 19944699 [PubMed] Lewis HA et al: "Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry."

No. Sentence Comment

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more soluble NBD1 variants from other vertebrate species [F494N in the γ- phosphate switch from several fish species (unpublished results) and Q637R in the RE from mouse].5,37 One newly reported ΔF508 structure has only these two mutations (PDB ID 2BBT, construct hNBD1-2f- ΔF508, Rwork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

PMID: 20150177 [PubMed] Atwell S et al: "Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant."

No. Sentence 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

PMID: 20551307 [PubMed] Da Paula AC et al: "Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins."

No. Sentence Comment

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

PMID: 20687133 [PubMed] Protasevich I et al: "Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1."

No. Sentence Comment

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

PMID: 20687163 [PubMed] Wang C et al: "Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis."

No. Sentence Comment

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
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
199 This inference is supported by the location in the ABCa subdomain of the F508del mutation that strongly facilitates the initial unfolding transition and all but Q637R among the second-site suppressor mutations demonstrated in Figures 4 and Supporting Information Figure S8 to inhibit this transition. Login to comment

PMID: 21486785 [PubMed] Jih KY et al: "The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR."

No. Sentence Comment

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

PMID: 21594798 [PubMed] Kanelis V et al: "NMR spectroscopy to study the dynamics and interactions of CFTR."

No. Sentence Comment

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

PMID: 21182301 [PubMed] Loo TW et al: "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."

No. Sentence Comment

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

PMID: 22038833 [PubMed] Colas J et al: "Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect."

No. Sentence Comment

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

PMID: 18215773 [PubMed] Pissarra LS et al: "Solubilizing mutations used to crystallize one CFTR domain attenuate the trafficking and channel defects caused by the major cystic fibrosis mutation."

No. Sentence Comment

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
148 Curiously, Q637R lies in this helix, known as the RD1 region (residues 590-672) of NBD1. 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

PMID: 18215767 [PubMed] Deber CM et al: "Defining the defect in F508 del CFTR: a soluble problem?"

No. Sentence Comment

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

PMID: 23055971 [PubMed] Molinski S et al: "Functional Rescue of F508del-CFTR Using Small Molecule Correctors."

No. Sentence Comment

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

PMID: 23378596 [PubMed] Hunt JF et al: "Cystic fibrosis transmembrane conductance regulator (ABCC7) structure."

No. Sentence Comment

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

PMID: 23380248 [PubMed] Hanrahan JW et al: "Novel pharmacological strategies to treat cystic fibrosis."

No. Sentence Comment

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

PMID: 23924900 [PubMed] Ren HY et al: "VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1."

No. Sentence Comment

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

PMID: 24058550 [PubMed] Dawson JE et al: "Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR."

No. Sentence Comment

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
6 Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. 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
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
46 A mutation between C-terminal helices H8 and H9, Q637R, alters 15 N-1 H NMR HSQC spectral peak intensities that correspond to residues in the CL4-binding and RI-deletion sites, consistent with a change in dynamics within the spatially remote regions. 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
100 Results Q637R Mutation Alters Protein Dynamics in the RI-deletion Site and CL4-binding Site In order to probe the molecular mechanism of allostery in CFTR NBD1, we monitored effects on NMR spectra of NBD1 DRI DRE (residues 387-646, D405-436) upon perturbation by mutagenesis and ligand binding. 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
116 Like WT NBD1, Q637R NBD1 has heterogeneous peak lineshapes (Figure 2A). Login to comment
119 At 1 mM, WT and Q637R NBD1 tumble in solution like objects that are larger than a monomer, with a rotational correlation time of tc = 2765 ns (A. Chong, unpublished data) and 2764 ns, respectively. 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
128 The site of the Q637R mutations is located between the C-terminal helices H8 and H9 (residues 630-646), which are adjacent to a sheet containing b-strands S3/S9/S10 (residues 453-458, 615-629). Login to comment
136 While the Q637R mutation only changes the chemical shifts of neighboring residues (Figure S2), it perturbs the intensities of resonances in the C-terminal site nearby the mutation position, as well as for remote peaks near the site of the RI deletion and for some residues within the predicted CL4-binding site [8-10] (Figure 1A). Login to comment
144 Change in peak intensity due to H8/H9 mutation Q637R. Login to comment
146 Overlay of F494N (black) and F494N/Q637R (orange) NBD1 HSQC spectra. B. Login to comment
147 Ratio of peak intensities for NBD1 containing the Q637R mutation to those of NBD1 without this mutation, reflective of changes in dynamics, as a function of residue. Login to comment
154 Dynamics changes (absolute value of the intensity ratio minus one) due to Q637R mapped onto the structure of NBD1, highlighting long-range effects in the RI-deletion site and CL4-binding site. Login to comment
155 Q637R effects were probed using F494N NBD1 for improved solubility and spectral quality. Login to comment
160 Q637R, located between C-terminal helices H8 and H9, is marked with a red star due to lack of electron density in the structure. 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
179 Other differences are due to the lack of assignment for some residues near the site of the Q637R mutation (Figures 4G and 4H) and due to low spectral signal-to-noise. 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
220 Purple triangles in chart G indicate residues that could not be assigned for Q637R NBD1. Login to comment
221 The CL4:NBD1 proportions are 10:1 for Q637R NBD1 and its CL4:WT NBD1 control, but is 12.5:1 for the rest of the data. 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
225 Q637R (red star) is between C-terminal helices H8 and H9. Login to comment
298 We found evidence for an allosteric network revealed by both the Q637R mutation and CL4 peptide binding to NBD1 that connects the CL4-binding site predicted in CFTR homology models to two regulatory sites in NBD1, near to the RI and to where the RE/R regions are located in the full-length channel. 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
309 The mutations that can partially suppress the folding defects of F508del NBD1 are scattered across NBD1- V510D near CL4 in the channel, I539T directly opposite the NBD1:NBD2 interface, and Q637R near the start of the R region [12], hinting at the complex allosteric nature of the NBD1 folding landscape. Login to comment
312 The deletion site is perturbed by both CL4 binding and the Q637R mutation between C-terminal helices H8 and H9. Login to comment
313 The heterogeneous lineshapes observed in WT and Q637R NBD1 spectra indicate that there is exchange between conformations at different timescales. Login to comment
314 The Q637R mutation has far-reaching effects on the dynamics within the ensemble, altering dynamics in several distal locations, including in the RI-deletion site and in the CL4-binding site. 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
326 The compound binding also caused small chemical shift changes in the CL4-binding site [35], as expected for sites that are allosterically linked, demonstrating the reciprocal nature of the linkage and supporting our observation that a mutation between H8 and H9, Q637R, changes the dynamics of NBD1 residues at the CL4-binding site, as well as the RI-deletion site. Login to comment
337 (TIF) Figure S2 The effects of Q637R on NBD1 chemical shifts. 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
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

PMID: 24513531 [PubMed] Moran O et al: "On the structural organization of the intracellular domains of CFTR."

No. Sentence Comment

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

PMID: 24737137 [PubMed] Phuan PW et al: "Synergy-based small-molecule screen using a human lung epithelial cell line yields DeltaF508-CFTR correctors that augment VX-809 maximal efficacy."

No. Sentence Comment

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
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

PMID: 25083918 [PubMed] He L et al: "Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly."

No. Sentence Comment

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

PMID: 26627831 [PubMed] Ehrhardt A et al: "Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop."

No. Sentence Comment

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