ABCMdb

ABCC7 p.Gly550Glu

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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

2 Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Login to comment
3 Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Login to comment
5 The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. Login to comment
25 Here we used the STE/CFTR⌬F508 chimera system to identify novel amino acid substitutions just upstream (I539T) and within (G550E) the ABC signature motif of CFTR * This work was supported by National Institutes of Health Grant HL61234 and a Program Enhancement Grant from Florida State University Research Foundation (to J. L. T.). Login to comment
35 The G550E mutation introduces a negatively charged amino acid in the highly conserved LSGGQ core signature motif of CFTR NBD1. Login to comment
37 We assessed the effect of the G550E mutation on the PKA-dependent activation of wild type and mutant CFTR chloride channel. Login to comment
101 Two novel point mutations were isolated in the CFTR sequence that substantially rescued the H5-⌬F508 mating defect (Table I), resulting in the change of Ile-539 of the CFTR sequence to a Thr residue (I539T) and of Gly to Glu at the 550 position (G550E). Login to comment
102 Mutations I539T and G550E Partially Rescue CFTR⌬F508- The two novel ⌬F508 revertant mutations isolated in yeast were located either just upstream (I539T) or within (G550E) the CFTR NBD1 signature motif (Fig. 1). Login to comment
104 To evaluate the effect of the novel revertant mutations on CFTR⌬F508 processing, I539T and G550E mutations were introduced into the full-length CFTR⌬F508 cDNA (⌬F/I539T and ⌬F/G550E) for expression in mammalian cells. Login to comment
105 To test whether the combination of the I539T and G550E mutations would result in an additive or synergistic effect in correcting the ⌬F508 phenotype, we also constructed a CFTR⌬F508 allele containing both revertant mutations (⌬F/DB). Login to comment
110 We observed that I539T and, to a lesser extent, G550E partially rescued the CFTR⌬F508-processing defect. Login to comment
122 STE6/CFTR (H5) variant Mating efficiency % of H5 ⌬F508 0.28 Ϯ 0.04 ⌬F508/I539T 42.70 Ϯ 0.40 ⌬F508/G550E 79.90 Ϯ 4.50 Mutations in the ABC Signature Motif Region Rescue CFTR⌬F50835898 fected with CFTR wt respond to cAMP agonists with a rapid increase in Isc, reflecting increased chloride permeability (48). Login to comment
124 The ⌬F508 revertants CFTR⌬F/I539T and CFTR⌬F/G550E exhibited 6- and 12-fold increases in chloride current relative to CFTR⌬F508, respectively (Fig. 2B). Login to comment
126 To assess the effect of the ⌬F508 revertant mutations on CFTR wt chloride channel function, CFTRG550E, CFTRI539T, and a CFTR allele containing both I539T and G550E mutations (CFTRDB) were transiently expressed in FRT cells, and chloride current was measured after activation with 10 ␮M forskolin and 100 ␮M IBMX. Login to comment
129 The G550E Mutation Increases the Sensitivity of CFTR and CFTR⌬F508 to Activation by cAMP Agonists in FRT Cells Transiently Expressing CFTR-As shown in Fig. 2B, the G550E mutation was more effective than I539T in restoring the chloride channel function of CFTR⌬F508 (12-fold versus 6-fold increase), yet CFTR⌬F/G550E cell lysates contained lower levels of mature protein relative to CFTR⌬F/I539T (Fig. 2A). Login to comment
130 Interestingly, the G550E mutation occurs in a conserved residue, FIG. 2. Login to comment
131 The I539T and G550E mutations partially rescue CFTR⌬F508-processing and functional defects. Login to comment
144 To better understand the mechanism by which the G550E mutation improves the function of CFTR⌬F508, we tested its effect on activation of CFTR⌬F508 and CFTR wt by suboptimal concentrations of forskolin. Login to comment
146 Accordingly, CFTR wt, CFTR⌬F508, CFTRG550E, and CFTR⌬F/G550E were transiently expressed in FRT cells, and the transfected cell monolayers were assayed for transepithelial chloride current in response to activation by sub-optimal concentrations of forskolin (0.5 ␮M) in the absence of IBMX. Login to comment
148 The G550E mutation substantially increases the sensitivity of CFTR⌬F508 to PKA activation, increasing the level of chloride current activated by the sub-optimal concentration of forskolin (0.5 ␮M) from 4.66% of maximum activation for CFTR⌬F508 to 29.25% for CFTR ⌬F508/G550E (Fig. 3A). Login to comment
149 Although the G550E mutation did not increase the chloride channel activity of CFTR wt when the channels were activated with the optimal concentration of cAMP agonists (Fig. 2C), we observed that G550E did increase the sensitivity of CFTR wt to activation by the sub-optimal concentration of forskolin (compare 49.54 versus 77.2% of maximum Isc for CFTR wt and CFTR G550E, respectively) (Fig. 3A). Login to comment
150 Characterization of FRT Cell Lines Stably Expressing the CFTR⌬F508 Revertants-We obtained FRT cell lines stably expressing CFTR⌬F/I539T, CFTR⌬F/G550E, and CFTR⌬F/DB to further characterize the effect of the revertant mutations on CFTR⌬F508 processing and function. Login to comment
152 Results from CFTR immunoblotting analysis (Fig. 4A) and functional studies (Fig. 4B) confirmed the suppression of the CFTR ⌬F508 processing and chloride impermeability defects by I539T and G550E mutations, as observed for the transient expression experiments (Fig. 2, A and B). Login to comment
156 The G550E mutation increases sensitivity of CFTR wt and ⌬F508 to activation by forskolin after transient expression in FRT cells. Login to comment
161 B, representative tracings for no CFTR control, CFTR⌬F508, and CFTR⌬F/G550E. Login to comment
164 C, representative tracings for CFTR wt and CFTR G550E, as described in B. TABLE II Effect on CFTR-mediated transepithelial chloride currents of CF-causing mutations and ⌬F508 revertant mutations within the LSGGQ motif FRT cells monolayers transiently expressing the CFTR variants were mounted in Ussing chambers, and chloride current values were measured as described in Fig. 2B. Login to comment
167 CFTR variant Isc n % of CFTR wt CFTR S549R 1.70 Ϯ 0.11 4 CFTR G551D 1.17 Ϯ 0.12 4 CFTR ⌬F508 0.76 Ϯ 0.07 17 CFTR ⌬F/G550E 9.30 Ϯ 0.55 14 (*) CFTR ⌬F/G550D 6.06 Ϯ 0.63 12 (*) CFTR ⌬F/G550H 4.17 Ϯ 0.40 8 (*) Mutations in the ABC Signature Motif Region Rescue CFTR⌬F50835900 CFTR⌬F/G550E relative to FRT-CFTR ⌬F508 (Fig. 4B), although the mature band C was barely detectable, and the steady-state levels of band B were decreased for the revertant (Fig. 4A). Login to comment
168 These results suggest that the G550E mutation increases the channel activity of CFTR variants containing the ⌬F508 mutation. Login to comment
170 We also investigated the effect of the revertant mutations I539T and G550E on the temperature sensitivity of CFTR ⌬F508. Login to comment
174 Effect of I539T and G550E mutations on CFTR ⌬F508 temperature sensitivity and dose response to forskolin activation in FRT stable cell lines. Login to comment
175 A, Western blot analysis of the steady-state level of CFTR⌬F508, CFTR⌬F/I539T, CFTR⌬F/G550E, and CFTR⌬F/DB stably expressed in FRT cells. Login to comment
186 The G550E mutation attenuated the temperature sensitivity of CFTR⌬F508, as the low temperature treatment resulted in a 2-fold increase in chloride current for CFTR⌬F/G550E. Login to comment
187 The I539T mutation rendered CFTR⌬F508 and CFTR⌬F/G550E insensitive to incubation at 30 °C (Fig. 4B). Login to comment
188 The Isc measured after low temperature treatment of FRT-CFTR⌬F508 (12.27 ␮A/cm2 ) was comparable with the Isc of FRT-CFTR⌬F/G550E incubated at physiological temperature (15.57 ␮A/cm2 ) (Fig. 4B). Login to comment
189 Sensitivity of the CFTR ⌬F508 Revertants to cAMP Activation-The increase in sensitivity to forskolin activation observed for the CFTR⌬F/G550E mutant in transient expression (Fig. 3) we hypothesize to be an intrinsic property of the mutant channel. Login to comment
191 To assess the effect of G550E and I539T on the modulation of sensitivity of CFTR⌬F508 to activation while minimizing the potential effect of different channel levels at the plasma membrane, we compared the sensitivity to forskolin activation of CFTR ⌬F508 rescued by incubation for 48 h at 30 °C, with CFTR⌬F/G550E, CFTR⌬F/I539T, and CFTR⌬F/DB incubated at 37 °C. FRT monolayers stably expressing each CFTR variant were mounted in Ussing chambers, and the cAMP-activated chloride current was measured in response to decreasing concentrations of forskolin. Login to comment
193 The results demonstrate that CFTR variants containing the G550E mutation have increased sensitivity to forskolin activation (Fig. 4C). Login to comment
194 When CFTR⌬F/G550E, incubated at physiological temperature, was compared with CFTR⌬F508 we observed a significant increase in sensitivity to activation by 0.5 ␮M forskolin, confirming the results from transient expression (Fig. 3). Login to comment
195 FRT-CFTR⌬F/DB (containing both I539T and G550E) also exhibited a significant increase in sensitivity to activation relative to FRT-CFTR⌬F508 over the entire range of suboptimal forskolin concentrations tested. Login to comment
196 However, in contrast to the variants containing G550E, increased sensitivity to activation by suboptimal concentrations of cAMP agonist was not observed for the variant containing I539T alone. Login to comment
197 The G550E Mutation Increases the Sensitivity of CFTR wt to Activation by cAMP Agonists in FRT Cells Stably Expressing CFTR-Next, we investigated the dose-response for forskolin activation of CFTR wt and CFTRG550E stably expressed in FRT cells. Login to comment
201 Additional ⌬F508 Revertant Mutations at Position G550- The significant rescue of the ⌬F508 defect by the G550E mutation prompted us to screen for other ⌬F508 revertant mutations at this codon using site-directed mutagenesis. Login to comment
203 Because the G550E mutation replaces a Gly residue with the negatively charged Glu, we also constructed a CFTR⌬F/G550D variant to test whether the negative charge resulting from an aspartate substitution would have a similar effect on CFTR channel activation. Login to comment
208 CFTR⌬F/G550H and CFTR⌬F/G550D displayed 50 and 68% of CFTR⌬F/G550E Isc, respectively, demonstrating that these additional revertants were not as effective as G550E in suppressing the CFTR ⌬F508 defect. Login to comment
210 However, unlike the results observed for CFTR⌬F/ G550E, suboptimal forskolin concentration failed to activate CFTR⌬F/G550D (not shown). Login to comment
211 The G550E Mutation Modulates the CFTR ⌬F508 Response to Activation by Genistein and Millimolar Concentrations of IBMX-Several compounds have been isolated that optimize channel activity of phosphorylated CFTR by mechanisms that are independent of increase in cAMP (55, 56). Login to comment
212 The effect of the G550E mutation to increased sensitivity to cAMP-mediated activation of mutant and wt CFTR led us to ask if this mutation would modulate the response of CFTR⌬F508 channels to optimization by two such compounds. Login to comment
214 Effect of G550E mutation on CFTR dose response to forskolin activation in FRT stable cell lines. Login to comment
215 A, Western blot analysis of the steady-state level of protein for CFTR wt and CFTR G550E stably expressed in FRT cells, as described in Fig 2A. Login to comment
221 Mutations in the ABC Signature Motif Region Rescue CFTR⌬F50835902 IBMX and 50 ␮M genistein on the PKA-dependent activity of CFTR⌬F508, CFTR⌬F/I539T, CFTR⌬F/G550E, CFTR⌬F/DB, and CFTR wt. Login to comment
227 Genistein significantly increased the PKA-activated Isc for all the cell lines containing the ⌬F508 mutation, although it produced a smaller increase for cell lines containing the G550E mutation; compare 58 and 70% increase for CFTR⌬F508 and CFTR⌬F/I539T, respectively, with 45 and 25% for CFTR⌬F/G550E and CFTR⌬F/DB (Fig. 6). Login to comment
232 Interestingly, 2 mM IBMX did not affect the PKA-activated Isc of the FRT-CFTR wt, FRT-CFTR⌬F/ G550E, or FRT-CFTR⌬F/DB under the experimental conditions employed. Login to comment
233 DISCUSSION Two novel ⌬F508 revertant mutations were identified just upstream (I539T) and within (G550E) the core consensus ABC signature motif LSGGQ in the NBD1 of CFTR. Login to comment
235 Increased cAMP-activated chloride permeability was also observed in FRT monolayers expressing CFTR⌬F/I539T and CFTR⌬F/ G550E to levels 6- and 12-fold higher than CFTR⌬F508, respectively. Login to comment
236 The larger fraction of processed CFTR⌬F/I539T and CFTR⌬F/G550E observed relative to CFTR⌬F508, thus, represents functional channels localized at the plasma membrane. Login to comment
237 Furthermore, functional studies using a double revertant allele (CFTR⌬F/DB) showed that I539T and G550E mutations act synergistically to increase CFTR⌬F508 chloride currents to ϳ29% of CFTR wt, representing a 38-fold increase over the CF mutant. Login to comment
240 The I539T and G550E mutations were identified as revertants of the CF-causing mutation ⌬F508. Login to comment
241 It might, thus, be expected that these revertant mutations, identified by virtue of their effects to reverse the ⌬F508 defect, would be specific for suppression of ⌬F508. However, results by others suggest that G550E can partially rescue another processing-defective CF mutant, A561E (61). Login to comment
242 Possibly, the ⌬F508 and A561E mutations cause misfolding in a similar manner, allowing each to be partially compensated by G550E. Login to comment
244 In support of the latter possibility, it was observed that the combination of I539T and G550E within wild type CFTR increased functional activity. Login to comment
245 Further experiments will be necessary to determine the extent to which ⌬F508 revertant mutations I539T and G550E suppress other CF mutations within the NBD1 that are associated with defective protein processing. Login to comment
246 To further assess the effects of the revertant mutations on CFTR⌬F508, we compared the functional activity of CFTR⌬F508, CFTR⌬F/G550E, and CFTR⌬F/I539T under various experimental conditions. Login to comment
248 The I539T mutation, when introduced in either CFTR⌬F508 or CFTR⌬F/G550E, rendered these variants insensitive to low temperature treatment. Login to comment
251 Effect of the I539T and G550E mutations on CFTR ⌬F508 activation by 2 mM IBMX and 50 ␮M genistein. Login to comment
256 In contrast to I539T, G550E had little effect on CFTR⌬F508 temperature sensitivity, suggesting that it affects the protein folding pathway differently. Login to comment
260 Our results show that the G550E mutation decreased the concentration of forskolin required for half-maximal stimulation of all the CFTR variants tested, CFTR wt, CFTR⌬F508, and CFTR⌬F/I539T. Login to comment
261 Furthermore, G550E mutation improved PKA-dependent activity of CFTR variants bearing the ⌬F508 mutation under optimal and suboptimal forskolin concentration, and improved the wild type channel activity only under suboptimal concentrations of forskolin, but not when maximal PKA activity was promoted, suggesting that this mutation could specifically increase function of underphosphorylated CFTR. Login to comment
262 Alternatively, G550E could facilitate CFTR phosphorylation under suboptimal PKA activity. Login to comment
263 Although the role of G550E in CFTR phosphorylation remains to be determined, the effect of G550E to increase the PKA-dependent activity of CFTR⌬F508 is consistent with the higher levels of function associated with CFTR⌬F/G550E relative to the low levels of processed protein observed. Login to comment
264 Because IBMX and genistein are known to enhance the functional activity of CFTR⌬F508 (23-25), we assessed the effect of these molecules on CFTR⌬F/G550E, CFTR⌬F/I539T, and CFTR⌬F/DB. Login to comment
268 The effect of 2 mM IBMX on the activation of CFTR⌬F/I539T was similar to the effect observed for CFTR⌬F508. However, 2 mM IBMX did not increase the PKA-dependent currents of FRT-CFTR⌬F/G550E or FRT-CFTR⌬F/DB. Login to comment
269 We speculate that G550E could directly alter the binding of IBMX to CFTR⌬F508, impairing the increase in Po or further contributing to the decrease of current amplitude (24). Login to comment
270 Genistein significantly enhanced PKA-activated chloride currents of CFTR⌬F508, CFTR⌬F/G550E, CFTR⌬F/I539T, and CFTR⌬F/DB, although the currents mediated by CFTR variants containing the G550E mutation were stimulated to a lesser extent. Login to comment
273 The revertant mutations I539T and G550E did not preclude genistein enhancement of the PKA-dependent activity of CFTR⌬F508 implying that, similarly to the CF mutant, the revertants could be underphosphorylated at maximal PKA activity. Login to comment
279 The G550E mutation represents a non-conservative introduction of a negatively charged Glu residue, changing the LSGGQ core consensus signature sequence of NBD1 to LSEGQ. Login to comment
294 We speculate that G550E and I539T mutations could restore the LSGGQ-mediated interactions disrupted by the ⌬F508 mutation. Login to comment

PMID: 15528182 [PubMed] Lewis HA et al: "Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure."

No. Sentence Comment

73 We also investigated the effect of three suppressor mutations (G550E, R553Q, R555K) that have been observed to improve in vivo trafficking efficiency of STE6-CFTR chimeras containing the ⌬F508 mutation expressed in yeast (23-25). Login to comment
100 Crystal Structure of ⌬F508 hNBD1 Shows Minimal Conformational Changes but Substantive Changes in Surface Topography at the Putative Site of MSD1 Interaction-Crystals diffracting to a resolution of 2.3 Å were obtained for hNBD1-7a- ⌬F508, which contains seven mutations (F409L, F429S, F433L, G550E, R553Q, R555K, H667R) in addition to the deletion of Phe-508 (see Table II). Login to comment
139 The three suppressor mutations (G550E, R553Q, R555K) occur either in or immediately following the LSGGQ signature sequence at the N terminus of ␣-helix 5. Login to comment

PMID: 15619636 [PubMed] Thibodeau PH et al: "Side chain and backbone contributions of Phe508 to CFTR folding."

No. Sentence Comment

148 Note added in proof: Crystal structures of the human F508A missense NBD1 (with solublizing mutations F429S and H667R) and the corrected ∆F508 NBD1 (with three known suppressor mutations G550E, R553Q and R555K, and the solublizing mutations F409L, F429S, F433L and H667R) have been reported51. Login to comment

PMID: 15765539 [PubMed] Amaral MD et al: "Processing of CFTR: traversing the cellular maze--how much CFTR needs to go through to avoid cystic fibrosis?"

No. Sentence Comment

49 Indeed, by replacing either glycineat position550 by aglutamic acid residue (G550E) or isoleucine 539 by threonine (I539T), in cis with F508del it is possible to rescue both its membrane localization and channel activity.28 Another study showed that removal from F508del-CFTR of sequence signals, called arginine-framed tripeptide (AFT)-sequences (responsible for the ER retention or retrieval of other ion channels),29 permits nascent F508del-CFTR to mature and generate functional ClÀ channels at the cell surface.30 There are four of these arginine (R) sequences in CFTR: one in the N-terminal cytoplasmic domain (R29), two in NBD1 (R516 and R555), and one in the R domain (R766). Login to comment
51 Whether these AFT motifs are just ER-retention signals or, like G550E and I539T, also act as intrinsic folding determinants remains to be fully clarified. Login to comment
52 Interestingly, and shedding some light on the mechanism involved, both G550E and 4RK also rescue another NBD1 trafficking mutant that is buried in NBD1.32 The C terminus of CFTR, containing a binding motif for a number of proteins (discussed below), also constitutes an intrinsic factor influencing CFTR proteinstability. Login to comment

PMID: 16132229 [PubMed] Eudes R et al: "Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations."

No. Sentence Comment

177 Thus, like the G550E mutation affecting the NBD1 ABC signature [54], a R555 mutation may restore (to some extent) the interactions disrupted by the ∆F508 mutation. Login to comment

PMID: 17098864 [PubMed] Roxo-Rosa M et al: "Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms."

No. Sentence Comment

0 Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms Mo´ nica Roxo-Rosa*† , Zhe Xu‡ , Andre´ Schmidt*† , Ma´rio Neto*, Zhiwei Cai‡ , Cla´udio M. Soares§ , David N. Sheppard‡ , and Margarida D. Amaral*†¶ *Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal; †Centre of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal; ‡Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom; and §Institute of Chemistry and Biological Technology, New University of Lisbon, 2781-901 Oeiras, Portugal Communicated by Michael J. Welsh, University of Iowa College of Medicine, Iowa City, IA, September 22, 2006 (received for review June 9, 2006) The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Login to comment
2 G550E rescued the trafficking defect of A561E but not that of R560T. Login to comment
4 Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wtand V562I-CFTR than does G550E. Login to comment
6 Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. Login to comment
7 G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention͞retrieval mediated by AFTs. Login to comment
21 DeCarvalho et al. (26) demonstrated that substitution of G550 by a negatively charged amino acid (G550E) promoted the maturation of F508del-CFTR and enhanced CFTR-mediated Cl- permeability (26). Login to comment
25 To better understand how revertant mutants rescue F508del-CFTR, we selected for study the revertants G550E and 4RK for several reasons. Login to comment
26 First, they represent two very distinct types of amino acid residues (G550E, acidic; 4RK, basic). Login to comment
27 Second, they reside in distinct functional motifs (G550E, the LSGGQ motif of NBD1; 4RK, the AFTs). Login to comment
28 Third, they are likely to have different effects on the structure of CFTR because only two AFTs lie in NBD1 (like G550E), whereas the other two are on the N terminus and regulatory domain, respectively. Login to comment
30 Together, these reasons led us to hypothesize that G550E Author contributions: M.R.-R. and Z.X. contributed equally to this work; D.N.S. and M.D.A. designed research; M.R.-R., Z.X., A.S., M.N., and C.M.S. performed research; Z.C. analyzed data; and M.R.-R., D.N.S., and M.D.A. wrote the paper. Login to comment
37 47 ͉ 17891-17896 and 4RK act by different mechanisms. To explore this possibility, we tested the effects of G550E and 4RK on three additional CF mutations within NBD1: R560T, A561E, and V562I.ʈ We selected for study these CF mutants because (i) these residues constitute a hot spot for disease-causing mutations (seven mutations are associated with these three residues࿣ ); (ii) A561E and R560T are the second and fourth most frequent mutations among Portuguese and Irish CF patients, respectively (28); (iii) like G550E and R555K (one of the 4RK mutants), these mutations affect residues located between the LSGGQ and Walker B motifs of NBD1, which are highly conserved across species; and (iv) they all lie within the same ␣-helix (H5; G550-Y563) within the ATP-binding cassette ␣-subdomain of NBD1 (29, 30). Login to comment
38 To test the hypothesis that G550E and 4RK act by different mechanisms, we used biochemical and functional assays to investigate how these revertants rescue F508del-, R560T-, A561E-, and V562I-CFTR. Login to comment
47 Next, we investigated whether the revertants G550E and 4RK rescue the defective biosynthesis of R560T- and A561E-CFTR. Login to comment
49 Fig. 1A shows that G550E and 4RK were much less effective at rescuing A561E-CFTR compared with their effects on F508del-CFTR. Login to comment
50 In fact, band C of both A561E-4RK- and A561E- G550E-CFTR was detected only after a 24 h incubation with 2 mM 4-phenylbutyrate (4-PB), an enhancer of CFTR expression through transcriptional activation (Fig. 1B) (32). Login to comment
55 The mature form of CFTR (band C) was clearly observed for F508del- and A561E-CFTR (Fig. 1D) in the presence of G550E and 4RK, but G550E failed to correct the defective biosynthesis of R560T-CFTR (Fig. 1 A and D). Login to comment
56 Thus, the revertants G550E and 4RK rescue some, but not all, CF mutations in NBD1. Login to comment
57 Effect of the G550E and 4RK Revertants on the Turnover and Processing of CFTR Variants. Login to comment
58 Fig. 1A demonstrates that steady-state levels of band C for both wtand V562I-CFTR were increased by 4RK but not by G550E, most strikingly for V562I-CFTR. Login to comment
59 In contrast, the revertants had the opposite effect on F508del-CFTR, with G550E generating higher steady-state levels of band C than 4RK (Fig. 1A). Login to comment
60 These data suggest that G550E and 4RK might alter the processing of CFTR in different ways. Login to comment
62 For wt-CFTR, the presence of the revertants, 4RK or G550E (dotted and dashed lines in Fig. 2 D and G, respectively), did not alter either the turnover rate of band B (solid line in Fig. 2D) or the efficiency of its conversion into band C (compare dotted and dashed lines with solid line in Fig. 2G). Login to comment
63 Similarly, the turnover rate of band B of either F508del-4RK- or F508del-G550E-CFTR (dotted and dashed lines in Fig. 2E) was the same as that of F508del-CFTR (solid line, Fig. 2E). Login to comment
64 Moreover, Fig. 2H demonstrates identical processing efficiencies for F508del-4RK- and F508del-G550E-CFTR (compare the dotted and dashed lines). Login to comment
65 For V562I-CFTR, the turnover rate of V562I-G550E was slightly, but not significantly (P Ͼ 0.05), reduced compared with those of V562I- and V562I-4RK-CFTR (Fig. 2F). Login to comment
66 Furthermore, the efficiency of processing of V562I-4RK (dotted line, Fig. 2I) was slightly increased relative to those of V562I and V562I-G550E (solid and dashed lines, Fig. 2I, respectively). Login to comment
70 (A) WB of total protein (30 ␮g) from BHK cells stably expressing wt-, F508del-, R560T-, A561E-, or V562I-CFTR, alone or in cis with 4RK and G550E. Login to comment
75 (D) BHK cells expressing F508del-, R560T-, or A561E-CFTR alone or in cis with the revertants 4RK and G550E were analyzed by CFTR IP after pulse-labeling for 3 h. Labeled arrows indicate the positions of bands A, B, and C. Thus, the higher steady-state levels of band C for 4RK variants of both wtand V562I-CFTR (Fig. 1A) are explained only in part by a slight (but not significant) increase in the efficiency of processing band B to band C. Surprised that the revertants did not exert stronger effects on the processing of CFTR, we wondered how they might influence CFTR Cl-channel function. Login to comment
82 In contrast, G550E, which produced lower steady-state levels of band C than wt-CFTR (Fig. 1A), generated Ϸ1.5-fold higher efflux of I- (Fig. 3 A and F). Login to comment
83 Consistent with previous work (24) and our biochemical data (Figs. 1 and 2), both 4RK and G550E restored CFTR function to F508del, although the time course of the F508del-4RK response was delayed by 1 min compared with that of wt-CFTR (Fig. 3 B and F). Login to comment
84 In contrast to F508del-CFTR, G550E did not restore CFTR function to R560T and both revertants rescued only modestly the CFTR function of A561E (Fig. 3 C, D, and F). Login to comment
85 Interestingly, V562I-G550E generated an efflux of I- equivalent in magnitude and time-course to that of V562I-CFTR (Fig. 3 E and F) despite its lower steady-state levels of band C. However, 4RK, which generated higher steady-state levels of band Fig. 2. Login to comment
86 Turnover and processing of wt-, F508del-, and V562I-CFTR alone or in cis with 4RK and G550E. Login to comment
87 (A-C) BHK cells expressing wt-, G550E- and 4RK-CFTR (A); F508del-, F508del-4RK-, and F508del-G550E-CFTR (B); and V562I-, V562I-4RK-, and V562I-G550E-CFTR (C) were pulse-labeled for 20 min with 100 ␮Ci͞ml [35S]methionine and then chased for 0, 0.5, 1, 3, and 5 h. Login to comment
96 (A-E) Time courses of I-efflux from BHK cells stably expressing wt- (A), F508del- (B), R560T- (C), A561E- (D), and V562I- (E) CFTR in the absence and presence of the 4RK and G550E mutations. Login to comment
104 In contrast, the data suggest that G550E compensates for a decreased steady-state level of band C by enhancing the channel activity of wtand V562I-CFTR. Login to comment
105 A similar mechanism might explain how G550E rescues the activity of CF mutants. Login to comment
106 4RK and G550E Rescue F508del-CFTR Channel Gating with Different Efficacy. Login to comment
107 To test the idea that the revertant G550E enhances the Cl-channel function of wt and CF mutants, we used the patch-clamp technique to study the single-channel behavior of wt-, V562I-, and F508del-CFTR in the absence and presence of the revertants. Login to comment
112 Before examining the rescue of F508del-CFTR channel function by the revertants, we quantified the deficit in CFTR function caused by F508del and the effects of 4RK and G550E on wt-CFTR. Login to comment
116 The revertants 4RK and G550E enhanced wt-CFTR channel gating but with strikingly different efficacies. 4RK caused a small but not significant increase in Po by lengthening MBD 2-fold, whereas G550E increased the Po of wt-CFTR 1.5-fold by prolonging MBD 4.5-fold without altering IBI (Fig. 4 B-D). Login to comment
117 Moreover, G550E dramatically enhanced the ATP-sensitivity of wt-CFTR (wt-CFTR, Km ϭ 210 ␮M; G550E, Km ϭ 19 ␮M; data not shown). Login to comment
118 Like their effects on wt-CFTR, 4RK and G550E enhanced V562I-CFTR channel gating and rescued that of F508del-CFTR with different efficacies (Fig. 4). Login to comment
119 For example, Fig. 4A demonstrates that F508del-4RK- and F508del-G550E-CFTR both have prolonged channel openings compared with those of wtand F508del-CFTR. Login to comment
120 But more strikingly, the IBI for F508del-G550E-CFTR is dramatically shorter than that of F508del-CFTR and approaches that of wt-CFTR (Fig. 4A). Login to comment
121 Fig. 4C demonstrates that both revertants prolonged strikingly the MBD of F508del-CFTR compared with that of either wtor F508del-CFTR (4RK, 2.5-fold; G550E, 4-fold; both vs. F508del-CFTR). Login to comment
122 However, only G550E rescued the IBI defect of F508del-CFTR, reducing its IBI from 8-fold longer than wt-CFTR to only 2-fold longer (Fig. 4D). Login to comment
123 As a result, the Po of F508del-G550E-CFTR exceeded that of wt-CFTR, whereas that of F508del-4RK is only half that of wt-CFTR (Fig. 4B). Login to comment
124 To summarize, our data demonstrate that the revertant G550E efficaciously rescues the gating defect of F508del-CFTR, whereas rescue by 4RK is modest. Login to comment
125 We interpret these data to suggest that 4RK and G550E exert their effects on F508del by different mechanisms. Login to comment
128 The revertants 4RK and G550E rescue the cell surface expression of A561E, albeit not as effectively as F508del, whereas G550E is without effect on R560T. Login to comment
129 Of note, G550E, but not 4RK, rescues the defective channel gating of F508del, suggesting that G550E and 4RK rescue the expression and function of F508del by distinct mechanisms. To understand the structural basis by which R560T and A561E disrupt the processing of CFTR (refs. Login to comment
136 Single-channel activity of wt-, V562I-, and F508del-CFTR alone and in cis with 4RK and G550E. Login to comment
140 Columns and error bars are means Ϯ SEM (wt: n ϭ 6 for all data; 4RK: Po, n ϭ 2; MBD and IBI, n ϭ 1; G550E: Po, n ϭ 3; MBD and IBI, n ϭ 1; V562I: Po, n ϭ 5; MBD and IBI, n ϭ 2; V562I-4RK: Po, n ϭ 4; MBD and IBI, n ϭ 3; V562I-G550E: Po, n ϭ 8; MBD and IBI, n ϭ 3; F508del: n ϭ 10 for all data; F508del-4RK: Po, n ϭ 13; MBD and IBI, n ϭ 10; F508del-G550E: Po, n ϭ 5; MBD and IBI, n ϭ 4). Login to comment
163 DeCarvalho et al. (26) identified the revertant G550E by using STE6͞CFTR chimeras to search for F508del suppressor mutations. Login to comment
164 The authors demonstrated that G550E (i) partially rescues the processing defect of F508del-CFTR, (ii) enhances F508del-CFTR Cl- currents in Fischer rat thyroid (FRT) epithelia, and (iii) increases the sensitivity of F508del-CFTR to stimulation by cAMP agonists (26). Login to comment
166 Any difference in the level of F508del rescue between previous studies of 4RK (25) and G550E (26) and our own are most likely due to the use of different cell lines. Login to comment
167 Of note, our single-channel data provide an explanation for the effects of G550E on F508del-CFTR activity. Login to comment
169 This suggests that G550E might promote the binding of ATP to site 1 of the NBD dimer while slowing the hydrolysis of ATP at site 2, plausibly by stabilizing the NBD dimer (4). Login to comment
170 Thus, we speculate that the gating behavior of F508del-G550E-CFTR might be a consequence of a (partial) correction the F508del-induced folding defect of NBD1. Login to comment
179 Consistent with this idea, our single-channel data demonstrate that 4RK incompletely rescues the gating defect of F508del-CFTR, unlike the G550E revertant. Login to comment
196 In summary, our studies indicate that the location of CF mutations within the H5 ␣-helix of NBD1 determines the efficacy with which the revertants G550E and 4RK restore their function: Mutations located proximally are rescued better than those located distally. Login to comment
198 Moreover, our data demonstrate that the revertants 4RK and G550E produce distinct effects on F508del-CFTR: 4RK affects mainly the efficiency of processing, enabling the mutant to escape AFT-mediated ER retention͞ retrieval, with only limited rescuing of folding. Login to comment
199 In contrast, G550E appears to exert its effect directly on the conformation of NBD1, as it rescues efficaciously the gating defect of F508del-CFTR. Login to comment

PMID: 18463704 [PubMed] Serohijos AW et al: "Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding."

No. Sentence Comment

46 However, even in the presence of correcting mutants (G550E, R553Q, and R555K) [10,15-17] in our model of NBD1-DF508, we still observe a significant change in dynamics at the folding transition. Login to comment
284 Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, et al. (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment

PMID: 18597042 [PubMed] Mornon JP et al: "Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces."

No. Sentence Comment

97 These mutations are actually located outside the NBD1 structure core, in the regulatory insertion (F409L, F429S, F433L) and extension (R667H), or in the signature sequence region (G550E, R553Q, R555K), whose local conformations in the crystal structure and in our model are perfectly superimposable. Login to comment

PMID: 19781595 [PubMed] Bisignano P et al: "Molecular dynamics analysis of the wild type and dF508 mutant structures of the human CFTR-nucleotide binding domain 1."

No. Sentence Comment

20 Structures were obtained by X-ray crystallography, at a resolution of 2.55 Å and 2.30 Å for WT and mutant, respectively. These structures are both characterized by the presence of seven mutations: F409L, F429S, F433L, G550E, R553Q, R555K and H667R which make them more soluble and therefore more easily crystallizable [6,7]. Login to comment
152 The chosen PDB entries,1XMJ and 2BBO, contain seven mutations, F409L, F429S, F433L, G550E, R553Q, R555K and H667R, that were introduced to the NBD1, wild type and dF508 used for this study, to facilitate the crystallization of the polypeptide [6]. Login to comment
154 It is interesting to notice that several of these mutations (F429S, G550E, R555K), have been identified as ''rescue`` mutations [18-20], that improve the expression of the defective dF508 CFTR. 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: 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

32 Comparing these hNBD1 structures to each other and to wild-type murine NBD1 (mNBD1) suggested that the overall fold of NBD1 was retained in the ΔF508 mutant and that structural changes were localized near the site of the deleted phenylalanine residue.5 However, three of these solubility-enhancing mutations (G550E/ R553Q/R555K) were known to be in vivo suppressors of the trafficking defect caused by the ΔF508 mutation.51-53 Compared to mNBD1, no significant structural perturbations were observed in the vicinity of these suppressor mutation sites in the crystal structures of hNBD1, suggesting that they act indirectly to suppress the effects of the ΔF508 mutation.5 Indeed, folding studies of a wider variety of hNBD1 variants, including several without trafficking-suppressor mutations, indicated that these mutations increase the thermodynamic stability of NBD1,5 which could account for improved folding and maturation in vivo. Login to comment
41 This structure was obtained from the same hNBD1-7a protein construct that yielded the previously reported ΔF508 structure, which has seven solubilizing mutations including the three trafficking-suppressor mutations G550E/R553Q/R555K. Login to comment
133 The structures harboring the G550E/R553Q/ R555K suppressor mutation set are shown in bright green (F508) and bright red (ΔF508). Login to comment
139 This region contains F508, the LSGGQ signature sequence (at residues 548-552), and the G550E/ R553Q/R555K suppressor mutation sites. 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

226 The 389-678(F409L, F429S, F433L, G550E, R553Q, R555K, H667R) (hNBD1-7a) structures are shown without (dark blue) and with the DF508 mutation (light blue) (H. Lewis, in preparation). 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
29 In addition to a similar effect, G550E also decreases the interburst interval of the ⌬F508-CFTR channel, such that its open probability is higher than that of WT-CFTR (17). 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
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
91 As shown in Fig. 1B, R560T-CFTR, although not rescued by G550E alone (17), was very effectively rescued by the 4S combination. 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
128 The rescue of ⌬F508 by suppressor mutations is diminished in the absence of NBD2 According to a structural model of CFTR (18), some of the suppressor mutations (I539T and G550E) are located at the NBD1/NBD2 interface (Fig. 1A), and ⌬F508 is known to destabilize NBD2 (6, 7). 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: 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
292 By contrast, the revertant mutation G550E, which enhances both CFTR trafficking and gating, was proposed to correct the defective folding of F508del-CFTR (24). 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

34 Printed in the U.S.A. NOVEMBER 12, 2010•VOLUME 285•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35825 G550E, R553Q, and R555K (19-21). Login to 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: 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

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

26 Moreover, three second-site mutations, selected by Teem and coworkers to reverse the in vivo trafficking defect caused by F508del,37 appeared to stabilize hNBD1 against chemical unfolding.15 Because this ''Teem suppressor triplet`` (G550E, R553Q, and R555K) does not significantly alter the structure of F508del-hNBD1,18 its effect increasing the thermodynamic stability of hNBD1 seems likely to be responsible for reversing the deleterious effects of the F508del mutation. Login to comment
155 Second-Site Solubilizing/Suppressor Mutations Delay the Initial Unfolding Transition Figure 4 shows the isothermal denaturation behavior of F508del-hNBD1-D(RI,RE) constructs harboring additional point mutations known to suppress the trafficking defect caused by the F508del mutation.32,37,39,40 The G550E, R553Q, and R555K mutations in the Teem suppressor triplet were isolated in vivo using a selection for mutations restoring the export of a yeast CFTR homolog bearing the equivalent of the F508del mutation. Login to comment
179 This research program led to the identification of several point mutations that improve the solubility and yield of purified hNBD1, including the Teem suppressor triplet (G550E, R553Q, and R555K) isolated as suppressors of the in vivo trafficking defect of F508del-CFTR.37 However, surprisingly, the other efficacious solubilizing mutations chosen exclusively on the basis of polarity and consistency with the CFTR sequence profile also generally suppress the defective in vivo trafficking of F508del-CFTR.32,39 This correlation is readily explained if the initial unfolding transition in hNBD1 (left side of Fig. 5) controls both aggregation of the purified domain in vitro and aberrant ER retention and degradation of intact CFTR in vivo. 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: 21152102 [PubMed] Hoelen H et al: "The primary folding defect and rescue of DeltaF508 CFTR emerge during translation of the mutant domain."

No. Sentence Comment

3 Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues DF508 CFTR to the cell surface, but only I539T repaired DF508 NBD1. Login to comment
22 Teem and coworkers [19] identified two mutations, G550E and I539T, that both significantly increased plasma membrane levels of DF508 CFTR and improved channel activity [19,20,21]. Login to comment
26 Introduction of I539T, but not the G550E suppressor mutation, counteracted all folding defects within NBD1, whereas both mutations rescue CFTR trafficking to the cell surface. Login to comment
101 Only I539T but not G550E suppresses the DF508 phenotype in NBD1 As the folding defect in DF508 CFTR arose in NBD1, we asked whether this defect could be rescued in NBD1 as well. Login to comment
102 Teem and colleagues identified two suppressor mutations (G550E, I539T) in NBD1 that were located in the same subdomain as F508 (Figure 3B), and that each partially rescued DF508 CFTR from the ER [19]. Login to comment
107 For comparison we included the G550E suppressor mutation. Login to comment
108 The two mutations exerted very different effects in that G550E did not affect stability of wild-type or DF508 NBD1, while I539T measurably stabilized the domain (Figure 3D). Login to comment
111 I539T but not G550E fully restores the conformational defect in DF508 NBD1 To establish whether the improved stability of the I539T mutant was due to rescued conformation, we in vitro translated wild-type and DF508 NBD1 with or without suppressor mutations and monitored changes in proteolytic digestion (Figure 4A). Login to comment
112 Again, I539T, but not G550E, caused a dramatic effect on the proteinase K digest, particularly detectable at 5 mg/ml. Login to comment
114 Comparing longer exposures of the 100 mg/ml ProtK treatment of the NBD1 molecules revealed that only the I539T mutation restored the ,17 kDa protease resistant band that had been lost in DF508 NBD1, whereas G550E had no measurable impact (Figure 4B, N). Login to comment
119 We concluded that I539T but not G550E rescues the DF508 phenotype by completely restoring NBD1 conformation and stability. Login to comment
129 Both I539T and G550E partially restore ''band C`` levels of DF508 CFTR. Login to comment
133 To analyze whether the I539T suppressor improved CFTR maturation like G550E [20,21] we used a pulse-chase approach to monitor both rate and efficiency of DF508 CFTR rescue. Login to comment
136 Introducing either G550E or I539T within DF508 CFTR partially countered misfolding and enhanced export from ER to Golgi (Figure 6). Login to comment
137 The I539T suppressor was much more effective than G550E in rescuing DF508 CFTR: the majority of CFTR molecules now reached the Golgi complex, whereas some loss of signal still occurred for G550E, implying some residual degradation. Login to comment
139 We conclude that, while both mutations rescue full-length CFTR to the plasma membrane, the I539T mutation rescues the DF508 phenotype within the isolated NBD1 domain already during its synthesis whereas G550E practically bypasses the folding defect in NBD1 and rescues via an alternative mechanism. Login to comment
143 The I539T suppressor mutation but not G550E in the same subdomain rescued the defect and restored NBD1 conformation to wild-type, Figure 3. Login to comment
163 (A) Wild-type and DF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were in vitro translated in the presence of 35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Login to comment
165 (B) Longer exposure of the 100 mg/ml proteinase K digest of in vitro translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Login to comment
203 Reverting the DF508 phenotype While the I539T mutation rescued NBD1, G550E hardly affected the isolated DF508 NBD1 domain. Login to comment
210 Where G550E does not measurably rescue NBD1 misfolding, it does rescue CFTR functioning and therefore is likely to rescue at least one of the downstream domain assembly steps. Login to comment
211 The G550E mutation is located in the highly conserved ABC transporter signature motif of NBD1 (LSG550 GQ) and, according to the Sav1866 homology model [8], is in close proximity to NBD2. Login to comment
213 While G550E only slightly increases steady state plasma membrane levels of DF508 CFTR, it does enhance CFTR mediated chloride currents [19] and restores CFTR channel activity by increasing the duration of channel opening and thereby open probability (Po) [20]. Login to comment
239 The deletion of F508 and the reverting mutations G550E and I539T were introduced both in full-length CFTR and NBD1 by side directed mutagenesis using primers (amino acid change underlined, I539T: 59-CCAAGTTTGCAGAGAAAGACAATACCG- TTCTTGGAGAAGGTGGAATC-39 G550E: 59-GGAGAAGG- TGGAATCACACTGAGTGAGGGTCAACGAGCAAGAATT- TCTTTAGC-39 DF508: 59-GGCACCATTAAAGAAAATATC- ATTGGTGTTTCCTATGATGAATATAG-39) and all constructs were sequence verified. Login to comment

PMID: 21594797 [PubMed] Schmidt A et al: "Biochemical and biophysical approaches to probe CFTR structure."

No. Sentence Comment

30 Subsequently, in a screen for suppressor mutations of the F508del defect, the original R553Q suppressor mutation was identified as were I539T, G550E, R553Q, and R555K (18, 19). 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
333 Other suppressor mutations in NBD1 such as G550E appear to rescue ΔF508-CFTR by altering the conformation of NBD1 (62). 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
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
126 More pronounced inactivation was seen in G550E/ Table 1. Login to comment
139 (C) G550E/ ΔF508 CFTR (n = 4). 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
153 After two exposures to the elevated temperature, recovery, although evident, was much slower than that seen with G550E/ΔF508 or I539T/ΔF508 CFTR. Login to comment
247 Similarly, G550E, R555K, and R1070W; when combined individually with ΔF508, improved protein maturation at 37 °C to at most 18% of wt,4,6 but nevertheless significantly improved the thermal stability of the double mutant channels. 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
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
259 Recovery from partial inactivation was also seen with R555K and G550E, but unlike I539T, both of these second-site mutations also resulted in persistent, steady-state conductance at 37 °C. 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

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
451 Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment
455 Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment

PMID: 22265408 [PubMed] Rabeh WM et al: "Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function."

No. Sentence Comment

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

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
167 Other examples are the G550E and R555K mutations, which also partially rescued CFTR-ΔF508 processing and increased the Po by lengthening the burst duration. Login to comment
168 Although ΔF508 lengthened the interburst interval for both mutations, G550E reduced the magnitude of that increase (21, 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
266 Roxo-Rosa M, et al. (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. 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: 19896304 [PubMed] Edelman A et al: "Twenty years after cystic fibrosis gene identification: Where are we and what are we up to?"

No. Sentence Comment

58 Moreover, it has been observed by mutagenesis followed by heterologous expression of CFTR, that replacing glycine at position 550 by a glutamic acid residue (G550E) or isoleucine 539 by threonine (I539T), in cis in F508del-NBD1 leads to the delivery of functional F508del-CFTR to the plasma membrane. Login to comment

PMID: 18555011 [PubMed] Liu Y et al: "Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease."

No. Sentence Comment

149 The identified revertant mutations I539T, G550E, and R555K each partially res- Fig. 4. Login to comment
148 The identified revertant mutations I539T, G550E, and R555K each partially res- Fig. 4. Login to comment

PMID: 18417076 [PubMed] Cheung JC et al: "Molecular basis for the ATPase activity of CFTR."

No. Sentence Comment

107 The construct generated had several mutations to increase solubility of the domain (F409L, F429S, F433L, G550E, R553Q, R555K, H667R) in addition to the deletion of F508. 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

23 Some of these mutations represented sequence changes between human CFTR and CFTRs from other species, whereas others (G550E, R553Q, and R555K) had been previously identified as F508del-CFTR revertant mutations (Teem et al., 1996; deCarvalho et al., 2002; Chang et al., 1999). Login to comment
26 In addition, another revertant mutation, G550E, likely acts by altering the structure of NBD1 (Roxo-Rosa et al., 2006). Login to comment
155 Comparison with Other Revertants Using the same cellular system employed to investigate the solubilizing mutations, we recently examined the mechanism of action of two other F508del-CFTR revertants, G550E and 4RK, the simultaneous mutation of four arginine-framed tripeptides (AFTs), R29K, R516K, R555K, and R766K (Roxo-Rosa et al., 2006). Login to comment
157 G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. Login to comment
158 We also showed that both G550E and 4RK affected wt-CFTR (Roxo-Rosa et al., 2006). Login to comment
160 Moreover, whereas the revertants 4RK and G550E restored the iodide efflux of F508del-CFTR to wt levels (Roxo-Rosa et al., 2006), the magnitude of iodide efflux elicited by F508delD- and F508delT-CFTR was less than that of wt-CFTR. This indicates that the solubilizing mutations are not as effective as 4RK and G550E at augmenting either the cell-surface expression or function of F508del-CFTR. Login to comment
161 However, at steady state, F508delD and F508delT produced amounts of band C similar to those of G550E- or 4RK-F508del (present study and A.S. and M.D.A., unpublished data). Login to comment
165 By contrast, both revertant mutations markedly prolonged the MBD of F508del-CFTR, with G550E exceeding that of wt-CFTR by 4-fold (Roxo-Rosa et al., 2006). Login to comment
166 Moreover, only G550E suppressed the prolonged IBI of F508del-CFTR (Roxo-Rosa et al., 2006), reducing it to a level similar to that achieved here with F508delT-CFTR. Login to comment
167 We interpret these data to suggest that the conformation state(s) that solubilizing mutations achieve is closer to that of wt-CFTR than the conformation produced by the revertant G550E (Roxo-Rosa et al., 2006). Login to comment
168 Consistent with this interpretation, the revertants, especially G550E, extended the MBD and elevated the Po of wt-CFTR (Roxo-Rosa et al., 2006), whereas wt-CFTR containing the solubilizing mutations had the same MBD, IBI, and Po as wt-CFTR. Login to comment
169 We thus suggest that the solubilizing mutations act specifically on F508del-CFTR (in contrast to G550E) to abrogate its gating defect, possibly through correction of NBD1 and/or CFTR folding. Login to comment

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

No. Sentence Comment

18 The first human F508 del-NBD1 structure obtained carried seven additional mutations, three of which (suppressor mutations G550E, R553Q, and R555K) were known to rescue the F508 del-CFTR defect (Chang et al., 1999; DeCarvalho et al., 2002; Teem et al., 1996). Login to comment

PMID: 23493553 [PubMed] Woodward OM et al: "Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules."

No. Sentence Comment

159 Interestingly, the R193K mutation, homologous to the suppressor mutation R555K in CFTR, didn`t increase Q141K ABCG2 expression; but did appear to decrease the insoluble fraction of Q141K protein (Fig. S6D). Login to comment
325 Roxo-Rosa M, et al. (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment

PMID: 23800412 [PubMed] Saranko H et al: "Effects of the gout-causing Q141K polymorphism and a CFTR DeltaF508 mimicking mutation on the processing and stability of the ABCG2 protein."

No. Sentence Comment

113 Therefore all these three mutations were introduced into the corresponding regions of the ABCG2 DF142 construct (3R: G188E, R191Q, and R193K). Login to comment
122 While this G188E mutation does not increase the maturation of WT ABCG2 [11], the analogous G550E mutation promotes the maturation of WT CFTR [21] that again suggest fundamental differences between the NBDs of the two proteins. Login to comment
123 While this G188E mutation does not increase the maturation of WT ABCG2 [11], the analogous G550E mutation promotes the maturation of WT CFTR [21] that again suggest fundamental differences between the NBDs of the two proteins. 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

59 Similarly, the second site mutations, previously discussed with regard to their efficacy in stabilizing the isolated F508del-NBD1, i.e., the second site mutations in the ABC conserved core ATP binding subdomains (G550E, R553Q, and R555K) also promote improved processing of the full-length F508del-CFTR. Login to 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: 23248597 [PubMed] Kim SJ et al: "Mechanisms of CFTR Folding at the Endoplasmic Reticulum."

No. Sentence Comment

122 Mutations that increase NBD1 solubility and/or thermodynamic stability (I539T, G550E, R553Q, and others; Teem et al., 1993; DeCarvalho et al., 2002; Roxo-Rosa et al., 2006; Pissarra et al., 2008; Hoelen et al., 2010) and/or decrease backbone flexibility (Aleksandrov et al., 2012) can enhance both NBD1 folding yield in cells and trafficking efficiency of full length WT as well as ࢞F508 CFTR (Figure 3B). Login to comment

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

No. Sentence Comment

163 One such set, called the Teem set, comprised three point mutations (G550E/ R553Q/R555K) that previously were shown to promote improved biogenesis of F508del CFTR in tissue cultures cells, presumably by improving the stability of the protein (Teem et al. 1993; DeCarvalho et al. 2002). Login to comment
236 Note that the structures shown here contain seven point mutations included in hNBD1 constructs because of their beneficial influence on yield during purification-F409L, F429S, F433L, G550E, R553Q, R555K, and H667R. 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

40 The suppressor mutations G550E and I539T restore the DF508-CFTR proteolytic pattern to that of wild type CFTR. Login to comment

PMID: 23457292 [PubMed] Chong PA et al: "Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability."

No. Sentence Comment

200 Many of the suppressor mutations, like G550E, R553Q, and R555K (Teem et al. 1993, 1996), which are relatively distant from the F508 position, increase NBD1 thermal stability and reverse some of the processing and functional defects, presumably without reverting the surface changes caused by deletion of F508. Login to comment

PMID: 23757197 [PubMed] Galietta LJ et al: "Managing the underlying cause of cystic fibrosis: a future role for potentiators and correctors."

No. Sentence Comment

122 Some mutations, such as p.Gly550Glu, improve NBD1 stability [58]. Login to comment

PMID: 23865422 [PubMed] Loo TW et al: "Bithiazole correctors rescue CFTR mutants by two different mechanisms."

No. Sentence Comment

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

PMID: 23890012 [PubMed] Farinha CM et al: "Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction."

No. Sentence Comment

16 The second F508del-associated defect impairs CFTR interdomain folding, namely, (1) the NBD1-NBD2 dimerization interface (critical for channel activation and accounting for the F508del-CFTR gating defect; Dalemans et al., 1991), which can be rescued by the G550E revertant, and (2) the interaction of NBD1 with the fourth intracellular loop (ICL4) of TMD2 (Serohijos et al., 2008), shown to be reverted by either V510D (Loo et al., 2010) or R1070W, which both fill the pocket left empty by F508del (Thibodeau et al., 2010). Login to comment
23 Herein, we explored the MoA of VX-809 by analyzing its synergistic/additive effect with those of previously characterized genetic revertants, which rescue F508del-CFTR by causing different effects: 4RK affecting traffic (Roxo-Rosa et al., 2006), G550E (Roxo-Rosa et al., 2006) and R555K increasing channel gating by strengthening the NBD1:NBD2 dimer interface, and R1070W (Serohijos et al., 2008) and V510D (Wang et al., 2007a; Loo et al., 2010) by filling the NBD1:ICL4 interface. Login to comment
36 VX-809 Adds to VRT-325 and Corr-4a to Rescue F508del-CFTR but Exhibits Variable Effects on Genetic Revertants In order to characterize the rescue mechanism of VX-809 on F508del-CFTR, we then tested the effect of incubating it together with VRT-325 and Corr-4a on BHK cells stably expressing this mutant alone or in cis with the following genetic revertants: (1) 4RK (where the four AFTs were simultaneously mutated to lysines), (2) G550E, (3) R1070W, (4) V510D, or (5) R555K (Figures 1A-1E). Login to comment
41 Interestingly, however, analysis of the effects of the three compounds upon the revertants showed that VX-809 (but neither VRT-325 nor Corr-4a) is additive to G550E or R555K in correcting F508del-CFTR (by 38% and 32%, respectively), strongly suggesting that VX-809 acts differently from these two revertants. Login to comment
46 These analyses were performed for three of the revertants with different F508del-CFTR effects (4RK, traffic; G550E, NBD1:NBD2 dimer interface; and R1070W, NBD1:ICL4 interface). Login to comment
48 For example, an increase in F508del-G550E-CFTR processing is detected at 0.3 mM VX-809, whereas a 10-fold higher concentration is needed for significant rescue of F508del-CFTR (Figure 2C). Login to comment
57 Effect of Small Molecule Correctors on F508del-CFTR and Genetic Revertants (A-F) BHK cell lines stably expressing CFTR bearing F508del alone (A) or in cis with 4RK (B), G550E (C), R1070W (D), V510D (E), and R555K (F) were incubated for 24 hr with 6.7 mM VRT-325, 10 mM Corr-4a, or 3 mM VX-809 alone or in combination. Login to comment
72 We then assessed the effect of revertants G550E and R1070W by modeling (Figure 3F; Figures S2B and S2C), whereby G550E allows a salt bridge to form across the ATP binding site with Lys1250 and other residues from NBD2 (Figure 3F). Login to comment
87 Rescue of F508del-CFTR by Low Temperature Is Additive to Genetic Revertants To learn more about how low temperature rescues F508del-CFTR, we assessed its combined effect with that of the above genetic revertants: G550E, R1070W, 4RK, V510D, and R555K (Figures 4A and 4B). Login to comment
88 Results show that low temperature further increases processing levels of F508del-CFTR by the five genetic revertants, namely, V510D, G550E, R1070W, 4RK, and R555K, by an additional 35%, 65%, 38%, 27%, and 22%, respectively (compare gray and black bars in Figure 4B). Login to comment
96 (F) Change in F508del NBD1-NBD2 heterodimer interface by the second-site mutation G550E. Login to comment
101 Interestingly, these additive effects were observed not only for revertants promoting protein-autonomous folding (G550E, V510D, and R1070W) but also for the 4RK revertant, which bypasses the AFT-mediated ER retention. Login to comment
102 Combination of Different Genetic Revertants Is Also Additive Next, to assess the full potential for F508del-CFTR rescue, we combined the effects of folding and traffic revertants by producing stable BHK cell lines expressing F508del-G550E-CFTR, where 4RK, V510D, or R1070W were also added in cis, and analyzed processing (Figures 4C and 4D). Login to comment
103 Results in Figure 4C show that 4RK, V510D, and R1070W further increased processing of G550E-F508del-CFTR by another 12%, 59%, and 70%, respectively. Login to comment
104 In fact, the combined effects of G550E with either V510D or R1070W bring F508del-CFTR processing to z80%, i.e., close to levels of WT-CFTR, which can be further increased at 26 C reaching 88%. Login to comment
105 The combination of G550E with 4RK, although additive, has a more modest effect (32% in total) and is still additive to low temperature (Figure 4D, far right bar). Login to comment
106 Low Temperature Kinetics of F508del-CFTR Alone or with 4RK/G550E The increased effect of combining correctors, revertants, and low temperature is strongly suggestive of different MoAs. Login to comment
107 However, to further characterize the MoA of low temperature rescue, we studied the turnover rate and processing efficiency of F508del-CFTR or in cis with 4RK/G550E at 26 C in comparison to these variants at 37 C. Login to comment
123 The dotted line corresponds to levels of band C in F508del-G550E- CFTR. Login to comment
124 We then similarly assessed how low temperature affects the turnover and processing of F508del-CFTR bearing the two genetic revertants 4RK and G550E (Figure 6). Login to comment
125 Results from pulse-chase experiments of F508del-G550E-CFTR show a dramatic reduction in the turnover of the immature form (Figure 6A, left panel), which also corresponds to an increase in its processing efficiency (band C levels) when the chase was performed at 26 C (Figure 6A). Login to comment
128 The differential behavior observed for F508del-G550E- and F508del-4RK-CFTR at 26 C becomes most evident when these data are plotted as the total amount of band B + band C against time (Figures 6B and 6C). Login to comment
129 Indeed, whereas the total amount of F508del-G550E-CFTR at 26 C (Figure 6B, gray/dotted bars) onlybarelydecreasesincomparisontoitstotalat37 C(Figure6B, Figure 5. Login to comment
139 Altogether they indicate that 4RK is not as additive to low temperature in stabilizing F508del-CFTR as G550E. Login to comment
158 Low Temperature Stabilizes the Immature Form of G550E and 4RK Variants of F508del-CFTR and Acts Synergistically with G550E, but not 4RK, to Rescue F508del-CFTR (A-C) Pulse-chase experiments followed by IP were performed in BHK cells expressing (A) F508del-G550E (left panel) or F508del-4RK-CFTR (right panel) incubated at 26 C for 48 hr. After labeling with [35 S] methionine for 3 hr, chase was performed at 26 C (last five lanes in each panel). Login to comment
160 Percentage of bands B and C present at each time point of chase in F508del-G550E- (B) or F508del4RK-CFTR (C) incubated for the duration of the experiment (pulse + chase) at either 26 C or 37 C is shown in graph bars to illustrate the additive effect of low temperature and the genetic revertant G550E, but not 4RK, on F508del-CFTR rescue. Login to comment
164 Indeed, VX-809 (in contrast to either VRT-325 or Corr-4a) adds to the rescue by G550E and R555K (both acting at the NBD1:NBD2 interface), indicating that the compound and these two revertants probably correct distinct conformational cues of F508del-CFTR. Login to comment
165 In contrast, the additive effect of VX-809 to R1070W and V510D is rather modest, thus suggesting that this corrector acts more similarly to R1070W/V510D than to G550E/R555K. Login to comment
168 For example, F508del-G550E-CFTR required a 10-fold lower dose of VX-809 than F508del-CFTR for significant rescue. Login to comment
193 Furthermore, modeling also predicts that revertants G550E and R1070W act at different CFTR interdomain contacts disrupted by F508del: whereas G550E seems to restore the strength of the NBD1:NBD2 interface, R1070W rather promotes the NBD1:ICL4 interaction, as suggested previously (He et al., 2010; Serohijos et al., 2008) and in Figure S2A. Login to comment
195 Indeed, G550E, besides being able to promote rescue of F508del-CFTR (DeCarvalho et al., 2002), shows the largest combined effect with R1070W (or V510D). Login to comment
197 Moreover, the observed synergy of G550E or R555K with VX-809, but not VRT-325, is consistent with this model in which VRT-325 acts on the NBD1:NBD2 dimerization interface (also supported by the synergy between R1070W and VRT-325). Login to comment
210 Our data also show that low temperature, similar to chemical correctors, further increases processing levels of F508del-CFTR by the five genetic revertants, although to variable levels: V510D (by an additional 35%), G550E (65%), R1070W (38%), 4RK (27%), and R555K (22%). Login to comment
211 The strong synergistic effect of low temperature and G550E on the rescue of F508del-CFTR-efficient processing, but only modestly for 4RK, supported by pulse-chase data, suggest that low temperature and 4RK effects are mechanistically closer to each other (and thus related to bypassing the ER quality control) than low temperature and G550E. Login to comment
212 This is further supported by the additive effects of G550E and 4RK effects (Figure 4). Login to comment
214 Although 4RK can also be claimed to impact on F508del-CFTR folding (namely, through its two NBD1 changes: R516K and especially R555K), this is somewhat disproven by the additive effects of 4RK with G550E (Figure 4), the latter truly correcting F508del-NBD1 folding, as assessed by channel gating (Farinha and Amaral, 2005; Rosser et al., 2008; Roxo-Rosa et al., 2006). Login to comment
225 Assessing the synergistic/additive effects of investigational drug VX-809, one of the most promising to rescue the F508del-CFTR-trafficking defect, with those of genetic revertants as well as other correctors (VRT-325 and Corr-4a) or low temperature pointed to major insights into its MoA: (1) VX-809 is additive to both VRT-325 and Corr-4a, suggesting that each compound operates by a different MoA; (2) VX-809 is additive to low temperature rescue of the mutant almost to WT-CFTR levels; (3) VX-809, VRT-325, and Corr-4a show variable additive effects with the genetic revertants tested (4RK, G550E, and R1070W), thus providing clues for their possible action being exerted at specific protein binding pockets: VX-809 at the NBD1:TMD2 interface (and VRT-325 at NBD1:NBD2) or acting unspecifically (Corr-4a); and (4) VX-809 does not rescue the diacidic code traffic mutant in contrast to low temperature, which seems to act at trafficking surveillance checkpoints. Login to comment
231 EXPERIMENTAL PROCEDURES Cells and Culture Conditions BHK cell lines expressing F508del-4RK (R29K/R516K/R555K/R716K)-, F508del-G550E-, F508del-R1070W-, F508del-V510D-, F508del-R555K-, F508del-V510D/G550E-, F508del-G550E/R1070W-, DAA (D567A)-, 4RK- DAA-, DD/AA (D565A, D567A)-, 4RK-DD/AA-, and R560T-CFTR were produced and cultured as previously described (Roxo-Rosa et al., 2006). 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: 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: 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
66 S492P/I539T; 5-G550E/R553Q/R555K; 6-combo. 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: 25120007 [PubMed] Tripathi R et al: "Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator."

No. Sentence Comment

307 Further insights into the design parameters for such peptides are gained by the observation that certain suppressor mutations such as G550E and I539T can partially rescue the F508del-CFTR to the cell surface [6]. 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

38 Evidence that NBD1 destabilization is problematic for proper processing was provided by NBD1-thermostabilizing mutations distant from the F508del site, including G550E, R553Q, R555K, and deletion of the RI. Login to 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
364 Structural models have led to the prediction that the suppressor mutation G550E enhances dimerization through an electrostatic interaction with basic surfaces on NBD2 (59). Login to comment