ABCMdb

ABCC7 p.Gly550Glu

ClinVar:	c.1648G>T , p.Gly550* ? , not provided c.1648G>A , p.Gly550Arg ? , not provided
CF databases:	c.1648G>T , p.Gly550* D , CF-causing c.1648G>A , p.Gly550Arg (CFTR1) D , The above mutation was detected by DGGE and identified direct sequencing.
Predicted by SNAP2:	A: D (59%), C: D (80%), D: D (91%), E: D (85%), F: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (80%), T: D (85%), V: D (71%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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

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

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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

[hide] Impact of the deltaF508 mutation in first nucleoti... J Biol Chem. 2005 Jan 14;280(2):1346-53. Epub 2004 Nov 3. Lewis HA, Zhao X, Wang C, Sauder JM, Rooney I, Noland BW, Lorimer D, Kearins MC, Conners K, Condon B, Maloney PC, Guggino WB, Hunt JF, Emtage S
Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure.
J Biol Chem. 2005 Jan 14;280(2):1346-53. Epub 2004 Nov 3., 2005-01-14 [PMID:15528182]

Abstract [show]
Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

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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

[hide] Side chain and backbone contributions of Phe508 to... Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26. Thibodeau PH, Brautigam CA, Machius M, Thomas PJ
Side chain and backbone contributions of Phe508 to CFTR folding.
Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26., [PMID:15619636]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

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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

[hide] Processing of CFTR: traversing the cellular maze--... Pediatr Pulmonol. 2005 Jun;39(6):479-91. Amaral MD
Processing of CFTR: traversing the cellular maze--how much CFTR needs to go through to avoid cystic fibrosis?
Pediatr Pulmonol. 2005 Jun;39(6):479-91., [PMID:15765539]

Abstract [show]
Biosynthesis of the cystic fibrosis transmembrane conductance regulator (CFTR), like other proteins aimed at the cell surface, involves transport through a series of membranous compartments, the first of which is the endoplasmic reticulum (ER), where CFTR encounters the appropriate environment for folding, oligomerization, maturation, and export from the ER. After exiting the ER, CFTR has to traffic through complex pathways until it reaches the cell surface. Although not yet fully understood, the fine details of these pathways are starting to emerge, partially through identification of an increasing number of CFTR-interacting proteins (CIPs) and the clarification of their roles in CFTR trafficking and function. These aspects of CFTR biogenesis/degradation and by membrane traffic and CIPs are discussed in this review. Following this description of complex pathways and multiple checkpoints to which CFTR is subjected in the cell, the basic question remains of how much CFTR has to overcome these barriers and be functionally expressed at the plasma membrane to avoid CF. This question is also discussed here.

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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

[hide] Nucleotide binding domains of human CFTR: a struct... Cell Mol Life Sci. 2005 Sep;62(18):2112-23. Eudes R, Lehn P, Ferec C, Mornon JP, Callebaut I
Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.
Cell Mol Life Sci. 2005 Sep;62(18):2112-23., [PMID:16132229]

Abstract [show]
Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.

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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

[hide] Revertant mutants G550E and 4RK rescue cystic fibr... Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17891-6. Epub 2006 Nov 10. Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD
Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17891-6. Epub 2006 Nov 10., 2006-11-21 [PMID:17098864]

Abstract [show]
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. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wt- and V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control.

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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

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

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

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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

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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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

[hide] Molecular dynamics analysis of the wild type and d... Biochimie. 2010 Jan;92(1):51-7. Epub 2009 Sep 23. Bisignano P, Moran O
Molecular dynamics analysis of the wild type and dF508 mutant structures of the human CFTR-nucleotide binding domain 1.
Biochimie. 2010 Jan;92(1):51-7. Epub 2009 Sep 23., [PMID:19781595]

Abstract [show]
Mutations of CFTR (Cystic Fibrosis transmembrane Conductance Regulator), a membrane protein expressed in the epithelium that forms a chloride channel, cause a chronic, developmental and hereditary disease, known as Cystic Fibrosis. The most common mutation is the deletion of F508, a residue present in the first nucleotide binding domain (NBD1). We studied the thermodynamic properties of NBD1 wild type (WT) and mutant (dF508), starting from the crystallographic structures in the Protein Data Bank using the techniques of Molecular Dynamics. The two structures were similarly stable at room temperature, showed no change enthalpy or entropy, maintaining the same dimensions and the same order of magnitude of atomic fluctuations; the only difference was the energy of interaction with the solvent, in which the mutant appears slightly disadvantaged; these differences between the two models are at microscopic level and relate to local variations (in residues at 8 A from F508) of the surface exposed to the solvent. We also found a decrease in the mutant of about 30 times of affinity for ATP compared to WT.

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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

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

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

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

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

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

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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

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

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

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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

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

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

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27 These suppressor mutations (I539T, G550E, R553M/Q, and R555K) promote ⌬F508-CFTR maturation and trafficking to the cell surface, and also restore channel activity (16). Login to comment
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

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

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

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

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

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

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

[hide] The cystic fibrosis-causing mutation deltaF508 aff... J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28. Thibodeau PH, Richardson JM 3rd, Wang W, Millen L, Watson J, Mendoza JL, Du K, Fischman S, Senderowitz H, Lukacs GL, Kirk K, Thomas PJ
The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.
J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28., 2010-11-12 [PMID:20667826]

Abstract [show]
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the DeltaF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the DeltaF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that DeltaF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

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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

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

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

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

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

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

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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

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

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

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

[hide] The primary folding defect and rescue of DeltaF508... PLoS One. 2010 Nov 30;5(11):e15458. Hoelen H, Kleizen B, Schmidt A, Richardson J, Charitou P, Thomas PJ, Braakman I
The primary folding defect and rescue of DeltaF508 CFTR emerge during translation of the mutant domain.
PLoS One. 2010 Nov 30;5(11):e15458., [PMID:21152102]

Abstract [show]
In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues DeltaF508 CFTR to the cell surface, but only I539T repaired DeltaF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length DeltaF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of DeltaF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis.

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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

[hide] Biochemical and biophysical approaches to probe CF... Methods Mol Biol. 2011;741:365-76. Schmidt A, Mendoza JL, Thomas PJ
Biochemical and biophysical approaches to probe CFTR structure.
Methods Mol Biol. 2011;741:365-76., [PMID:21594797]

Abstract [show]
The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain integral membrane protein central to epithelial fluid secretion (see Chapter 21). Its activity is defective in the recessive genetic disease cystic fibrosis (CF). The most common CF-causing mutation is F508del in the first nucleotide binding domain (NBD1) of CFTR. This mutation is found on at least one allele of more than 90% of all CF patients. It is known to interfere with the trafficking/maturation of CFTR through the secretory pathway, leading to a loss-of-function at the plasma membrane. Notably, correction of the trafficking defect by addition of intragenic second-site suppressor mutations, or the alteration of bulk solvent conditions, such as by reducing the temperature or adding osmolytes, leads to appearance of functional channels at the membrane--thus, the rescued F508del-CFTR retains measurable function. High-resolution structural models of NBD1 from X-ray crystallographic data indicate that F508 is exposed on the surface of the domain in a position predicted by homologous ABC transporter structures to lie at the interface with the intracellular loops (ICLs) connecting the transmembrane spans. Determining the relative impact of the F508del mutation directly on NBD1 folding or on steps of domain assembly or both domain folding and assembly requires methods for evaluating the structure and stability of the isolated domain.

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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

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

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

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

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

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

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

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

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

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122 In a recent study of four of the CFTR suppressor mutations located in NBD1 (I539T, G550E, R553M, and R555K), it was found that they only restored maturation of mutants that had processing mutations in NBD1 but not those that had processing mutations in other domains such as NBD2 (N1303K) or TMD2 (L1065P or R1066C) (66). Login to comment
329 It appears that the ΔF508 mutation inhibits folding of NBD1 and its ability to stably associate with other domains resulting in altered CFTR-chaperone interactions, ER retention,andenhanceddegradation(65).Second-sitesuppressor mutations in NBD1 (such as I539T/G550E/R553M/R555K) can restore interdomain assembly (65, 66) to yield a more stable ΔF508-CFTR molecule (64, 66). Login to comment
333 Other suppressor mutations in NBD1 such as G550E appear to rescue ΔF508-CFTR by altering the conformation of NBD1 (62). Login to comment

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

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

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

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

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

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5 Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Login to comment
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

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

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

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

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

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

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

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

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

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120 (i) A genetic approach identified second-site suppressor mutations, including I539T, G550E, R553M/Q, and R555K (18-21, 25, 26). Login to comment
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

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

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

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

[hide] Twenty years after cystic fibrosis gene identifica... Pathol Biol (Paris). 2011 Jun;59(3):131-3. Epub 2009 Nov 5. Edelman A, Fritsch J, Ollero M
Twenty years after cystic fibrosis gene identification: Where are we and what are we up to?
Pathol Biol (Paris). 2011 Jun;59(3):131-3. Epub 2009 Nov 5., [PMID:19896304]

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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

[hide] Mild processing defect of porcine DeltaF508-CFTR s... Biochem Biophys Res Commun. 2008 Aug 15;373(1):113-8. Epub 2008 Jun 12. Liu Y, Wang Y, Jiang Y, Zhu N, Liang H, Xu L, Feng X, Yang H, Ma T
Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease.
Biochem Biophys Res Commun. 2008 Aug 15;373(1):113-8. Epub 2008 Jun 12., [PMID:18555011]

Abstract [show]
Recent efforts have made significant progress in generating transgenic pigs with the DeltaF508-CFTR mutation to model the lung and pancreatic disease of human cystic fibrosis. However, species differences in the processing and function of human, pig and mouse DeltaF508-CFTR reported recently raise concerns about the phenotypic consequence of the gene-targeted pig model. The purpose of the present study was to characterize the DeltaF508 mutant of porcine CFTR to evaluate the severity of its processing defect. Biochemical and immunofluorescence analysis in transfected COS7 and FRT cells indicated that pig DeltaF508-CFTR efficiently targets to the plasma membrane and is present mainly as the mature glycosylated protein. Functional characterization in stably transfected FRT cells by fluorometric and electrophysiological assays supported efficient plasma membrane targeting of pig DeltaF508-CFTR. The mild cellular processing defect of pig DeltaF508-CFTR suggests that its gene-targeted pig model may not develop the lung and pancreatic phenotypes seen in CF patients.

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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

[hide] Molecular basis for the ATPase activity of CFTR. Arch Biochem Biophys. 2008 Aug 1;476(1):95-100. Epub 2008 Apr 8. Cheung JC, Kim Chiaw P, Pasyk S, Bear CE
Molecular basis for the ATPase activity of CFTR.
Arch Biochem Biophys. 2008 Aug 1;476(1):95-100. Epub 2008 Apr 8., [PMID:18417076]

Abstract [show]
CFTR is a member of the ABC (ATP binding cassette) superfamily of transporters. It is a multidomain membrane protein, which utilizes ATP to regulate the flux of its substrate through the membrane. CFTR is distinct in that it functions as a channel and it possesses a unique regulatory R domain. There has been significant progress in understanding the molecular basis for CFTR activity as an ATPase. The dimeric complex of NBD structures seen in prokaryotic ABC transporters, together with the structure of an isolated CF-NBD1, provide a unifying molecular template to model the structural basis for the ATPase activity of CFTR. The dynamic nature of the interaction between the NBDs and the R domain has been revealed in NMR studies. On the other hand, understanding the mechanisms mediating the transmission of information from the cytosolic domains to the membrane and the channel gate of CFTR remains a central challenge.

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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

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

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

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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

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

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

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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

[hide] Gout-causing Q141K mutation in ABCG2 leads to inst... Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5223-8. doi: 10.1073/pnas.1214530110. Epub 2013 Mar 14. Woodward OM, Tukaye DN, Cui J, Greenwell P, Constantoulakis LM, Parker BS, Rao A, Kottgen M, Maloney PC, Guggino WB
Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules.
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5223-8. doi: 10.1073/pnas.1214530110. Epub 2013 Mar 14., [PMID:23493553]

Abstract [show]
The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (DeltaF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in DeltaF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein-protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.

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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

[hide] Effects of the gout-causing Q141K polymorphism and... Biochem Biophys Res Commun. 2013 Jul 19;437(1):140-5. doi: 10.1016/j.bbrc.2013.06.054. Epub 2013 Jun 22. Saranko H, Tordai H, Telbisz A, Ozvegy-Laczka C, Erdos G, Sarkadi B, Hegedus T
Effects of the gout-causing Q141K polymorphism and a CFTR DeltaF508 mimicking mutation on the processing and stability of the ABCG2 protein.
Biochem Biophys Res Commun. 2013 Jul 19;437(1):140-5. doi: 10.1016/j.bbrc.2013.06.054. Epub 2013 Jun 22., [PMID:23800412]

Abstract [show]
ABCG2 is an important multidrug transporter involved also in urate transport, thus its mutations can lead to the development of gout and may also alter general drug absorption, distribution and excretion. The frequent ABCG2 polymorphism, Q141K, is associated with an elevated risk of gout and has been controversially reported to reduce the plasma membrane expression and/or the transport function of the protein. In the present work we examined the stability and cellular processing of the Q141K ABCG2 variant, as well as that of the DeltaF142 ABCG2, corresponding to the DeltaF508 mutation in the CFTR (ABCC7) protein, causing cystic fibrosis. The processing and localization of full length ABCG2 variants were investigated in mammalian cells, followed by Western blotting and confocal microscopy, respectively. Folding and stability were examined by limited proteolysis of Sf9 insect cell membranes expressing these ABCG2 constructs. Stability of isolated nucleotide binding domains, expressed in and purified from bacteria, was studied by CD spectroscopy. We find that the Q141K variant has a mild processing defect which can be rescued by low temperature, a slightly reduced activity, and a mild folding defect, especially affecting the NBD. In contrast, the DeltaF142 mutant has major processing and folding defects, and no ATPase function. We suggest that although these mutations are both localized within the NBD, based on molecular modeling their contribution to the ABCG2 structure and function is different, thus rescue strategies may be devised accordingly.

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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

[hide] Requirements for efficient correction of DeltaF508... Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023. Mendoza JL, Schmidt A, Li Q, Nuvaga E, Barrett T, Bridges RJ, Feranchak AP, Brautigam CA, Thomas PJ
Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences.
Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023., [PMID:22265409]

Abstract [show]
Misfolding of DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the DeltaF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores DeltaF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

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

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

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

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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

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

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

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

[hide] Mechanisms of CFTR Folding at the Endoplasmic Reti... Front Pharmacol. 2012 Dec 13;3:201. doi: 10.3389/fphar.2012.00201. eCollection 2012. Kim SJ, Skach WR
Mechanisms of CFTR Folding at the Endoplasmic Reticulum.
Front Pharmacol. 2012 Dec 13;3:201. doi: 10.3389/fphar.2012.00201. eCollection 2012., [PMID:23248597]

Abstract [show]
In the past decade much has been learned about how Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) folds and misfolds as the etiologic cause of cystic fibrosis (CF). CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM) segments into the endoplasmic reticulum (ER) membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain-domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jig-saw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s) caused by DeltaF508 that provides a molecular target(s) for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients.

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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

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

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

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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

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

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

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

[hide] Dynamics intrinsic to cystic fibrosis transmembran... Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522. Chong PA, Kota P, Dokholyan NV, Forman-Kay JD
Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability.
Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522., [PMID:23457292]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTR's disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF-causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1-ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.

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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

[hide] Managing the underlying cause of cystic fibrosis: ... Paediatr Drugs. 2013 Oct;15(5):393-402. doi: 10.1007/s40272-013-0035-3. Galietta LJ
Managing the underlying cause of cystic fibrosis: a future role for potentiators and correctors.
Paediatr Drugs. 2013 Oct;15(5):393-402. doi: 10.1007/s40272-013-0035-3., [PMID:23757197]

Abstract [show]
Cystic fibrosis (CF), a severe genetic disease, is caused by mutations that alter the structure and function of CFTR, a plasma membrane channel permeable to chloride and bicarbonate. Defective anion transport in CF irreversibly damages the lungs, pancreas, liver, and other organs. CF mutations cause loss of CFTR function in multiple ways. In particular, class 3 mutations such as p.Gly551Asp strongly decrease the time spent by CFTR in the open state (gating defect). Instead, class 2 mutations impair the maturation of CFTR protein and its transport from the endoplasmic reticulum to the plasma membrane (trafficking defect). The deletion of phenylalanine 508 (p.Phe508del), the most frequent mutation among CF patients (70-90 %), destabilizes the CFTR protein, thus causing both a trafficking and a gating defect. These two defects can be overcome with drug-like molecules generically called correctors and potentiators, respectively. The potentiator Kalydeco (also known as Ivacaftor or VX-770), developed by Vertex Pharmaceuticals, has been recently approved by the US FDA and the European Medicines Agency (EMA) for the treatment of CF patients carrying at least one CFTR allele with the p.Gly551Asp mutation (2-5 % of all patients). In contrast, the corrector VX-809, which significantly improves p.Phe508del-CFTR trafficking in vitro, is still under study in clinical trials. Because of multiple defects caused by the p.Phe508del mutation, it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action. This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect.

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

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

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

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

[hide] Revertants, low temperature, and correctors reveal... Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004. Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, Da Paula AC, Williams J, Hirst S, Gomes CM, Amaral MD
Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.
Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004., [PMID:23890012]

Abstract [show]
Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

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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

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

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

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

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

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

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

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

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

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45 2PT, S492P/A534P/I539T; 4PT, 2PT + S422P/S434P; 3SS, G550E/R553M/R555K; 4SS, 3SS + I539T; ƊRI, deletion of RI amino acids 404-435; combo, ƊRI + 2PT + 3SS. Login to comment
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

[hide] Biophysical characterisation of calumenin as a cha... PLoS One. 2014 Aug 13;9(8):e104970. doi: 10.1371/journal.pone.0104970. eCollection 2014. Tripathi R, Benz N, Culleton B, Trouve P, Ferec C
Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.
PLoS One. 2014 Aug 13;9(8):e104970. doi: 10.1371/journal.pone.0104970. eCollection 2014., [PMID:25120007]

Abstract [show]
The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.

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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

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

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

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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