ABCMdb

ABCC7 p.Arg553Met

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Publications

PMID: 12110684 [PubMed] DeCarvalho AC et al: "Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 20032308 [PubMed] Roy G et al: "Interplay between ER exit code and domain conformation in CFTR misprocessing and rescue."

No. Sentence Comment

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

PMID: 20233947 [PubMed] He L et al: "Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR."

No. Sentence Comment

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

PMID: 20590134 [PubMed] Loo TW et al: "The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface."

No. Sentence Comment

64 It was recently reported that rescue of ΔF508-CFTR byother suppressor mutations inNBD1(I539T,G550E,R553M, R555K) was drastically reduced in wild-type CFTR lacking NBD2 (ΔNBD2) (20). Login to comment
129 A similar effect was observed when the combination of four NBD1 suppressormutations(I539T,G550E,R553M,R555K) was introduced into ΔF508-CFTR (20). Login to comment

PMID: 20667826 [PubMed] Thibodeau PH et al: "The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis."

No. Sentence Comment

104 The introduction of the single mutations, G550E, R553M or R553Q, and R555K, has previously been shown to partially rescue the ⌬F508 trafficking defect in CFTR and restore channel activity at the plasma membrane (Fig. 1A) (19-21). Login to comment
142 The introduction of the -3M mutations (G550E, R553M, R555K) rescues the trafficking defects associated with the ⌬F508 mutation and restores near wild type function. Login to comment

PMID: 20706124 [PubMed] Lucarelli M et al: "A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation."

No. Sentence Comment

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

PMID: 20837481 [PubMed] Pagant S et al: "Intragenic suppressing mutations correct the folding and intracellular traffic of misfolded mutants of Yor1p, a eukaryotic drug transporter."

No. Sentence Comment

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

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

No. Sentence Comment

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

PMID: 21602569 [PubMed] Yu W et al: "Probing conformational rescue induced by a chemical corrector of F508del-cystic fibrosis transmembrane conductance regulator (CFTR) mutant."

No. Sentence Comment

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

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

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

PMID: 9379898 [PubMed] Wemmie JA et al: "Mutational analysis of the Saccharomyces cerevisiae ATP-binding cassette transporter protein Ycf1p."

No. Sentence Comment

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

PMID: 22722932 [PubMed] Hudson RP et al: "Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

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

PMID: 22680785 [PubMed] Liu X et al: "Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity."

No. Sentence Comment

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

PMID: 22406676 [PubMed] Aleksandrov AA et al: "Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR."

No. Sentence Comment

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

PMID: 22210114 [PubMed] Dong Q et al: "Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-DeltaF508 processing and alter its gating defect."

No. Sentence Comment

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

PMID: 21965669 [PubMed] Wang W et al: "Thermally unstable gating of the most common cystic fibrosis mutant channel (DeltaF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops."

No. Sentence Comment

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

PMID: 19477416 [PubMed] Kim Chiaw P et al: "Functional rescue of DeltaF508-CFTR by peptides designed to mimic sorting motifs."

No. Sentence Comment

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

PMID: 9511928 [PubMed] Seibert FS et al: "Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein."

No. Sentence Comment

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

PMID: 9188468 [PubMed] Qu BH et al: "Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding."

No. Sentence Comment

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

PMID: 7686820 [PubMed] Welsh MJ et al: "Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis."

No. Sentence Comment

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

PMID: 7682896 [PubMed] Teem JL et al: "Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast."

No. Sentence Comment

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

PMID: 14596935 [PubMed] Owsianik G et al: "Rescue of functional DeltaF508-CFTR channels by co-expression with truncated CFTR constructs in COS-1 cells."

No. Sentence Comment

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

PMID: 16712779 [PubMed] Tummler B et al: "Rescue of F508del CFTR: Commentary on "F508del CFTR with two altered RXR motifs escapes from ER quality control but its channel activity is thermally sensitive"."

No. Sentence Comment

23 doi:10.1016/j.bbamem.2006.03.033 Hegedus' finding also provides a clue why second-site mutations in the RXR motif in the dodecapetide of NBF1 such as R553Q, R553M and R555K partially corrected the F508del CFTR mutant phenotype in model systems [16] and in CF patients [17]. Login to comment

PMID: 22265409 [PubMed] Mendoza JL et al: "Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences."

No. Sentence Comment

18 Additional second-site revertant mutations I539T, G550E, R553M, and R555K, within the portion of CFTR NBD1 included in the chimera, were also identified (DeCarvalho et al., 2002; Teem et al., 1993, 1996). Login to comment
19 The R553M, I539T, and the combination of G550E-R553M-R555K (3M) mutations correct the folding and stability defects of the DF508 NBD1 domain in isolation (DeCarvalho et al., 2002; Hoelen et al., 2010; Pissarra et al., 2008; Qu et al., 1997; Thibodeau et al., 2010) but only partially restore maturation of the full-length mutant protein (Hoelen et al., 2010; Pissarra et al., 2008; Thibodeau et al., 2010). Login to comment
127 The surface view A B I539T G550E R553M R555K 3M WT F S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 3 Relative Yield NBD1 ( -gal.) 25 30 35 40 45 0.0 0.5 1.0 Temperature (C ) Relative Turbitity 0 1 2 3 4 -5 0 5 10 WT F I539T I539T F S573E R555K D529F Relative Yield NBD1 ( -gal.) Tm Figure 3. Login to comment
166 B C A B 0 1 2 3 0 1 2 Relative Yield NBD1 (b2;-gal.) Relative Yield CFTR (ELISA) WT ࢞F WT ƊF S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 Relative Yield CFTR (ELISA) I539T G550E R553M R555K 3M Figure 4. Login to comment
172 See also Figure S5. (B) The influence of the 508-coupled mutations (green circles), four second-site suppressor mutations (I539T, G550E, R553M, and R555K) and three suppressors in combination (G550E, R553M, and R555K) (orange circles) on F508 background on relative maturation of full-length CFTR and relative NBD1 folding yield is correlated (green line, m = 0.75, R = 0.85). Login to comment
185 Previously identified second-site suppressor (I539T, G550E, R553M, R555K, and 3M) but not the 508-coupled mutants (D529F and S573E) increase the yield of DF508 NBD1. Login to comment
187 See also Table S2. (C) F508K, F508R, and F508K in combination with I539T, G550E, R553M, R555K, and 3M mutations increase folding yield of NBD1, but exhibit no corresponding increase in CFTR maturation yield (dark blue circles and line, m = 0.03, R = 0.40) (&#b1;SEM, n = 9 along x axis and n = 3 along y axis). Login to comment
219 When R1070W is combined with mutations that improve DF508 NBD1 folding yield, I539T, G550E, R553M, R555K, and 3M (open triangles), the correlation between NBD1 folding and CFTR maturation in the wild-type protein is restored (m = 0.77, R = 0.47, black line) (&#b1;SEM). Login to comment

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

No. Sentence Comment

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

PMID: 23104983 [PubMed] He L et al: "Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein."

No. Sentence Comment

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

PMID: 24685677 [PubMed] Pranke IM et al: "Biosynthesis of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

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

PMID: 24949105 [PubMed] Cebotaru L et al: "Complement yourself: Transcomplementation rescues partially folded mutant proteins."

No. Sentence Comment

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

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

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

PMID: 26149808 [PubMed] Chong PA et al: "Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization."

No. Sentence Comment

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