ABCMdb

PMID: 24412276

Loo TW, Clarke DM
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Val232Asp The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds Tip W. Loo a,b , David M. Clarke *,a,b a Department of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8 b Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 1. Login to comment 15 ABCC7 p.Val232Asp It appears that processing Biochemical Pharmacology 88 (2014) 46-57 A R T I C L E I N F O Article history: Received 11 November 2013 Received in revised form 30 December 2013 Accepted 31 December 2013 Available online 9 January 2014 Keywords: CFTR V232D processing mutation Cystic fibrosis Processing mutations Packing of TM segments Correctors A B S T R A C T Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Login to comment 17 ABCC7 p.Val232Asp Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Login to comment 19 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Ala ABCC7 p.Val232Asn The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. Login to comment 21 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asn ABCC7 p.Val232Asn The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Login to comment 23 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu ABCC7 p.Gln207Leu ABCC7 p.Gln207Ala The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. Login to comment 24 ABCC7 p.Val232Asp These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Login to comment 25 ABCC7 p.Val232Asp Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Login to comment 26 ABCC7 p.Val232Asp Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones. Login to comment 37 ABCC7 p.Val232Asp Although P-gp studies have demonstrated that formation of non-native hydrogen bonds between TM segments promotes folding [22,23], a study of the V232D (TM4) CF processing mutation predicted that hydrogen bonds in CFTR TM segments would have the opposite effect and inhibit folding [24]. Login to comment 38 ABCC7 p.Val232Asp Therien et al. [24] used helix-loop-helix fragments of the CFTR TM3/TM4 region to study the V232D (TM4) mutation and concluded that the aspartate residue formed a non-native hydrogen bond with Gln207 in TM3 to disrupt TM3/TM4 interactions. Login to comment 39 ABCC7 p.Val232Asp Based on the conclusion that V232D inhibited maturation by forming a non-native hydrogen bond with Gln207, it was proposed that over 75 other CF mutations in the TMDs may also inhibit maturation through non-native hydrogen bond formation between helices [24]. Login to comment 41 ABCC7 p.Val232Asp In this study, we used a mutational approach to test if the mechanism of the V232D processing defect was due to formation of a non-native hydrogen bond with Gln207 in full-length CFTR. Login to comment 42 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asn The rationale was that the V232N mutation should be just as effective as V232D to inhibit CFTR maturation and mutation of Gln207 to nonpolar residues should rescue the V232D mutant if the processing defect was caused by Asp232/Gln207 hydrogen bonds. Login to comment 70 ABCC7 p.Val232Asp Disulfide cross-linking analysis To compare the effect of V232D on packing of the TM segments, double cysteine mutants were generated for disulfide cross-linking analysis. Login to comment 71 ABCC7 p.Val510Ala Cys-less CFTR was constructed in which all endogenous cysteines were changed to alanine except that Cys590 and Cys592 were changed to leucine and Val510 was changed to alanine. Login to comment 80 ABCC7 p.Val232Asp Results 3.1. Replacement of Gln207 in TM3 with nonpolar residues does not rescue V232D CFTR The 1480 amino acids of CFTR are organized into two TMDs, each containing six TM segments, two NBDs, and an R domain [3] (Fig. 1A). Login to comment 85 ABCC7 p.Val232Asp The V232D mutation is a useful model system to examine the effects of processing mutations and correctors on CFTR maturation because it severely inhibits maturation such that CFTR accumulates as immature core-glycosylated protein in the ER. Login to comment 86 ABCC7 p.Val232Asp The V232D mutant however, can be efficiently rescued with correctors to yield mature protein at the cell surface with activity similar to the wild-type protein [6]. Login to comment 87 ABCC7 p.Val232Asp A previous study [24] on TM3/TM4 helix-loop-helix fragments suggested that V232D (TM4) caused CFTR misfolding by forming a non-native hydrogen bond with Gln207 (TM3). Login to comment 88 ABCC7 p.Val232Asp It was reported that the V232D mutation caused a TM3/TM4 helix-loop-helix fragment to migrate with increased mobility on SDS-PAGE gels relative to a wild-type fragment unless Gln207 was replaced by an amino acid such as leucine that did not form hydrogen bonds. Login to comment 89 ABCC7 p.Val232Gln ABCC7 p.Gln207Asp Further evidence for hydrogen bond formation was that the V232Q/Q207D mutant (reversal of the Gln and Asp positions) also migrated faster on SDS-PAGE gels [24]. Login to comment 90 ABCC7 p.Val232Asp ABCC7 p.Val232Asp If V232D inhibited CFTR maturation by forming a non-native hydrogen bond with Gln207, then we predicted that V232D would no longer inhibit maturation if Gln207 were replaced with an amino acid that could not form a hydrogen bond. Login to comment 96 ABCC7 p.Val232Asp Accordingly, Gln207 in the V232D mutant was replaced with a small (alanine) or large (leucine) amino acid that does not form hydrogen bonds. Login to comment 97 ABCC7 p.Val232Asp ABCC7 p.Gln207Leu The Q207L mutation had previously been reported to abolish hydrogen bond interactions with V232D in TM3/4 helix-loop-helix fragments [24]. Login to comment 99 ABCC7 p.Gln207Cys Gln207 does not appear to be essential for activity since it was found that the Q207C CFTR mutant was active when expressed in frog oocytes [32]. Login to comment 100 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu ABCC7 p.Gln207Cys ABCC7 p.Gln207Ala The V232D, V232D/Q207A, V232D/Q207L, and V232D/Q207C mutants were then expressed in the presence or absence of corrector VX-809 to test for maturation. Login to comment 101 ABCC7 p.Val232Asp Corrector VX-809 was used because it efficiently rescues V232D CFTR [33]. Login to comment 102 ABCC7 p.Val232Asp Whole cell SDS extracts were then subjected to immunoblot analysis. Fig. 1B shows that V232D severely inhibits maturation as only the 170 kDa immature protein was detected. Login to comment 103 ABCC7 p.Val232Asp Mature CFTR however, was observed when V232D was expressed in the presence of VX-809. Login to comment 104 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu ABCC7 p.Gln207Cys ABCC7 p.Gln207Ala By contrast, none of the Gln207 mutations rescued V232D CFTR (Fig. 1B and C) as no mature CFTR was observed when mutants V232D/Q207A, V232D/Q207L, or V232D/Q207C were expressed in the presence or absence of VX-809 (Fig. 1B). Login to comment 105 ABCC7 p.Val232Asp ABCC7 p.Gln207* These results suggest that the Q207X mutations likely had deleterious effects on mutant V232D, as the mutants could no longer be rescued with VX-809. Login to comment 107 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu ABCC7 p.Gln207Cys ABCC7 p.Gln207Ala Mutations to Gln207 inhibit CFTR maturation Since V232D but not mutants V232D/Q207A, V232D/Q207L, or V232D/Q207C could be rescued with VX-809 (Fig. 1B), we tested if maturation of CFTR was also sensitive to changes to Gln207 in a wild-type background. Login to comment 109 ABCC7 p.Val232Asp ABCC7 p.Gln207Ala Mutants V232D/Q207A, L, or C do not mature. Login to comment 112 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Ala (B) Whole cell extracts of cells expressing V232D or V232D/Q207A, L, or C mutants in the absence () or presence (+) of VX-809 were subjected to immunoblot analysis. Login to comment 116 ABCC7 p.Val232Asp An asterisk indicates significant (P < 0.05) inhibition when compared to V232D CFTR expressed in the presence of VX-809. Login to comment 117 ABCC7 p.Gln207Leu ABCC7 p.Gln207Cys ABCC7 p.Gln207Ala the Q207A, Q207L or Q207C changes. Login to comment 121 ABCC7 p.Gln207Asn A low amount (about 25%) of mature CFTR was observed only in mutant Q207N in the absence of VX-809. Login to comment 122 ABCC7 p.Gln207Trp ABCC7 p.Gln207Phe Expression in the presence of VX-809 promoted maturation (30-60% as mature product) of all of the mutants except Q207F or Q207W (Fig. 2A and B). Login to comment 125 ABCC7 p.Val232Asp The results suggest that Gln207 mutations might have an additive or synergistic effect on the V232D mutation. Login to comment 127 ABCC7 p.Val232Asp ABCC7 p.Val232Asn ABCC7 p.Val232Gln Inhibition of CFTR maturation correlates with the polarity of a Val232 mutation If V232D inhibits CFTR maturation by forming a non-native hydrogen bond with Gln207, then it would be expected that V232N and V232Q mutations would also severely inhibit maturation since asparagine and glutamine are structurally similar to aspartate and their amide groups can accept or donate two hydrogen bonds. Login to comment 128 ABCC7 p.Val232Asn ABCC7 p.Val232Gln Accordingly, mutants V232N and V232Q were constructed. Login to comment 129 ABCC7 p.Val232Glu Mutant V232E was also made because aspartic acid and glutamic acid have similar side chains. Login to comment 130 ABCC7 p.Val232Asp ABCC7 p.Val232Glu The mutants were expressed in HEK 293 cells and whole cell extracts subjected to immunoblot analysis. Fig. 3A and B shows that the expression of mutant V232E resembled that of V232D as it significantly inhibited maturation by about 40-fold (about 2% of the CFTR protein was present as the mature protein when compared to 80% for wild-type CFTR (Fig. 3)). Login to comment 131 ABCC7 p.Val232Asp ABCC7 p.Val232Glu ABCC7 p.Val232Asn ABCC7 p.Val232Gln By contrast, the amount of mature protein was about 20-fold higher (about 40%) for mutants V232N and V232Q compared to V232D or V232E (Fig. 3). Login to comment 132 ABCC7 p.Val232Asp ABCC7 p.Val232Glu These results suggested that charge rather than non-native hydrogen bonds might be responsible for the severe reduction in CFTR maturation caused by the V232D or V232E mutations. Login to comment 134 ABCC7 p.Val232Lys ABCC7 p.Val232Arg To test if other charged amino acids inhibited maturation, we constructed mutants V232R and V232K. Login to comment 137 ABCC7 p.Val232Asn ABCC7 p.Val232Asn ABCC7 p.Val232Gln ABCC7 p.Val232Gln The V232N and V232Q mutations modestly reduced the yield of mature CFTR by about half (80% for wild-type CFTR compared to about 40% for mutants V232N or V232Q) (Fig. 3). Login to comment 138 ABCC7 p.Val232Asn ABCC7 p.Val232Gln The V232N and V232Q mutations may have reduced maturation because of a change in polarity or because introduction of a larger side chain caused steric effects that disrupted packing of the TM segments. Login to comment 157 ABCC7 p.Val232Asp The results suggest that the V232D mutation may severely inhibit maturation because it introduces charge into a relatively hydrophobic region in the TM segments. Login to comment 159 ABCC7 p.Val232Asp 3.4. Rescue of Val232 processing mutants with correctors The V232D CFTR could be efficiently rescued with either VX-809 [19] or bithiazoles [33]. Login to comment 160 ABCC7 p.Val232Lys ABCC7 p.Val232Glu ABCC7 p.Val232Arg We tested if mutations to Val232 that severely inhibited maturation (V232E, V232K, or V232R) could be rescued with other correctors predicted to act as pharmacological chaperones such as 4a [33], VX-809 [19], 2b [35], 5a [12], or VX-325 [36]. Login to comment 163 ABCC7 p.Val232Glu Mutant V232E was rescued with all the correctors (35-70% mature product) (Fig. 4A and D). Login to comment 164 ABCC7 p.Val232Lys ABCC7 p.Val232Arg Mutant V232K was rescued with correctors 4a, VX-809 and VX-325 (25-60% mature product) (Fig. 4B and D) while mutant V232R could only be rescued with VX-809 (about 25% mature product) (Fig. 4C and D). Login to comment 165 ABCC7 p.Val232Arg The V232R mutant may have been difficult to rescue because arginine is the largest and most highly charged amino acid replacement. Login to comment 167 ABCC7 p.Val510Asp ABCC7 p.Val510Asp 3.5. Rescue of Val232 and Gln207 processing mutants with the V510D suppressor mutation Another approach to rescue CFTR processing mutants is to introduce the V510D suppressor mutation [37]. Login to comment 168 ABCC7 p.Val510Asp The V510D appears to act as a universal suppressor because it can promote maturation of mutants with processing mutations throughout the molecule. Login to comment 169 ABCC7 p.His1085Arg ABCC7 p.His1085Arg ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Val510Asp For example, V510D promotes maturation of mutants with processing mutations in TMD1 (V232D), TMD2 (H1085R) and NBD1 (DF508) whereas other suppressors such as I539T and R1070W promote maturation of DF508 CFTR but not mutants V232D or H1085R [19]. Login to comment 171 ABCC7 p.Val510Asp ABCC7 p.Val232Lys ABCC7 p.Val232Glu ABCC7 p.Val232Arg Accordingly, we introduced the V510D suppressor mutation into mutants V232E, V232K, or V232R. Login to comment 173 ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val232Glu ABCC7 p.Val232Glu The V510D was particularly effective in promoting maturation of V232E in the absence of VX-809 as the level of mature protein increased from less than 5% (Fig. 4A) to about 35% in mutant V510D/V232E (Fig. 5A and B). Login to comment 174 ABCC7 p.Val510Asp ABCC7 p.Val232Glu Expression of V510D/V232E in the presence of VX-809 yielded about 70% mature protein. Login to comment 175 ABCC7 p.Val510Asp ABCC7 p.Val232Lys ABCC7 p.Val232Arg The V510D suppressor caused a small increase in the amount of V232K mature protein (10%) but no detectable increase was observed in mutant V232R when expressed in the absence of VX-809 (Fig. 5A and B). Login to comment 176 ABCC7 p.Val510Asp ABCC7 p.Val232Lys The V510D/V232K mutant however, could still be efficiently rescued with corrector VX-809 to yield mature CFTR as the major product (about 70%). Login to comment 177 ABCC7 p.Val510Asp ABCC7 p.Val232Lys ABCC7 p.Val232Glu ABCC7 p.Val232Arg The relative maturation levels achieved using correctors or the V510D suppressor show that the V232E mutation could be more efficiently rescued than the V232K or V232R mutations. Login to comment 178 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val510Asp ABCC7 p.Val232Glu The properties of the V232E mutant are likely to be very similar to the V232D mutant as the V510D suppressor mutation [19] could also rescue the V232D mutant. Login to comment 179 ABCC7 p.Val510Asp ABCC7 p.Gln207* If V510D is a universal suppressor, then we predict that it would also promote maturation of the Q207X mutants. Login to comment 180 ABCC7 p.Val510Asp ABCC7 p.Gln207* Accordingly, the V510D mutation was introduced into mutants Q207X (X = A, L, C, E, F, W, N, and S) that were defective in maturation (Fig. 2). Login to comment 182 ABCC7 p.Gln207Leu ABCC7 p.Val510Asp ABCC7 p.Gln207Cys ABCC7 p.Gln207Asn ABCC7 p.Gln207Ser ABCC7 p.Gln207Glu It was observed that in the absence of VX-809, the V510D mutation significantly improved the maturation of Q207L, Q207C, Q207E, Q207N and Q207S (Fig. 6A and B). Login to comment 183 ABCC7 p.Gln207Leu ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Gln207Cys ABCC7 p.Gln207Asn ABCC7 p.Gln207Ser ABCC7 p.Gln207Glu Mature CFTR was the major product in Q207N/V510D (90% mature product) while mutants Q207L/V510D, Q207C/V510D, Q207E/V510D, and Q207S/V510D showed modest levels of mature CFTR (about 20-40% mature). Login to comment 184 ABCC7 p.Gln207Leu ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Gln207Cys ABCC7 p.Gln207Ala ABCC7 p.Gln207Phe ABCC7 p.Gln207Ser ABCC7 p.Gln207Glu In the presence of corrector VX-809 however, the amount of mature CFTR in mutants V510D/Q207A V510D/Q207L, V510D/Q207C, V510D/Q207E, V510D/Q207F and V510D/Q207S were significantly increased (25-85% mature product). Login to comment 185 ABCC7 p.Val510Asp ABCC7 p.Gln207Trp Corrector VX-809 did not significantly increase the amount of mature CFTR in mutant V510D/Q207W (Fig. 6A and B). Login to comment 188 ABCC7 p.Val232Lys ABCC7 p.Val232Glu ABCC7 p.Val232Arg Fig. 4. Rescue of V232E, V232K, and V232R mutants with correctors. Login to comment 189 ABCC7 p.Val232Lys ABCC7 p.Val232Glu ABCC7 p.Val232Arg Whole cell extracts of cells expressing mutants V232E (A), V232K (B), or V232R (C) in the absence (none) or presence of various correctors were subjected to immunoblot analysis. Login to comment 195 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Lys ABCC7 p.Gln207* ABCC7 p.Val232Glu ABCC7 p.Val232* The L305R(TM5) P-gp mutant resembles CFTR mutants V232D, V232E, and V232K The characteristics of the V232D/Q207X (Fig. 1) and V232X (Fig. 3) mutants suggest that the mechanism of the V232D mutation involves disruption of a hydrophobic pocket between TM segments. Login to comment 204 ABCC7 p.Val232Asp ABCC7 p.Val232Lys ABCC7 p.Val232Glu It appears that the CFTR V232D, V232E, and V232K processing mutationsresemble P-gp mutantsthatcontain acharged residueata hydrophobic interface between adjacent TM segments because they can be efficiently rescued (with VX-809). Login to comment 215 ABCC7 p.Val510Asp ABCC7 p.Gln207* To test if the rescued L305R mutant was active, histidine-tagged versions of the mutant and wild-type P-gp were expressed in HEK Fig. 6. Rescue of Q207X mutants with the V510D suppressor mutation. Login to comment 216 ABCC7 p.Gln207* (A) Whole cell extracts of cells expressing CFTR Q207X mutants in the absence () or presence (+) of VX-809 or were subjected to immunoblot analysis. Login to comment 220 ABCC7 p.Val510Asp ABCC7 p.Gln207* An asterisk indicates significant (P < 0.05) difference when compared to the Q207X mutant (without the V510D mutation) that was expressed in the absence of corrector. Login to comment 221 ABCC7 p.Val510Asp Fig. 5. Rescue of Val232 mutants with the V510D suppressor mutation. Login to comment 222 ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Val232Lys ABCC7 p.Val232Glu ABCC7 p.Val232Arg (A) Whole cell extracts of cells expressing CFTR mutants V232E/V510D, V232K/V510D or V232R/ V510D in the absence () or presence (+) of VX-809 or were subjected to immunoblot analysis. Login to comment 226 ABCC7 p.Val510Asp An asterisk indicates significant (P < 0.05) difference when compared to the mutant lacking V510D and expressed without corrector. Login to comment 241 ABCC7 p.Val232Asp We predict that the V232D CFTR mutant might be similar to L305R P-gp because it involves insertion of a charged residue into a hydrophobic pocket. Login to comment 254 ABCC7 p.Leu346Arg ABCC7 p.Val350Arg ABCC7 p.Phe229Arg ABCC7 p.Phe312Arg ABCC7 p.Phe354Arg ABCC7 p.Phe236Arg ABCC7 p.Phe310Arg Accordingly, wild-type CFTR and mutants F229R, F236R, F310R, F312R, L346R, V350R and F354R were expressed in HEK 293 cells and whole cell SDS extracts were subjected to immunoblot analysis. Login to comment 258 ABCC7 p.His1085Arg ABCC7 p.Val232Asp ABCC7 p.Gln1071Pro Cross-linking analysis suggests that V232D causes incomplete packing of the TM segments Previous studies on processing mutations in NBD1 (DF508) or in the fourth intracellular loop connecting TM segments 10 and 11 (Q1071P or H1085R) showed that they trapped CFTR at an early folding step resulting in incomplete packing of the TM segments [17,50]. Login to comment 263 ABCC7 p.Val232Asp To test if V232D trapped CFTR in a conformation with incomplete packing of the TM segments, the mutation was introduced into a Cys-less CFTR containing W356C(TM6) and W1145C(TM12). Login to comment 264 ABCC7 p.Val232Asp ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys Cys-less CFTR, mutants W356C/W1145C and V232D/W356C/W1145C were expressed in HEK 293 cells in the absence or presence of 5 mM VX-809. Membranes were prepared Fig. 8. Login to comment 270 ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys Cross-linked product was detected by SDS-PAGE followed by immunoblot analysis. Fig. 10A shows that the parent W356C/ W1145C CFTR readily matured in the absence of corrector VX-809 and was efficiently cross-linked with M8M or BMH (about 70% cross-linked product; Fig. 10B). Login to comment 271 ABCC7 p.Val232Asp ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys No cross-linked product was detected in Cys-less CFTR or in mutant V232D/W356C/W1145C when expressed in the absence of corrector VX-809 (Fig. 10A and B). Login to comment 272 ABCC7 p.Val232Asp ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys Expression of mutant V232D/W356C/W1145C in the presence of corrector however, promoted maturation of the protein such it could be efficiently cross-linked with either M8M or BMH (about 80-85% cross-linked product; Fig. 10B). Login to comment 273 ABCC7 p.Val232Asp These results suggest that the V232D mutation traps CFTR in a partially folded conformation with incomplete packing of the TM segments that can be corrected with VX-809. Login to comment 274 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val510Asp ABCC7 p.Val232Asn In summary, the results suggest that: (1) the mechanism of how V232D causes protein misfolding is unlikely to involve non-native hydrogen bond interactions with Gln207 because V232N yielded about 20-fold more mature CFTR than V232D; (2) it appears that the mechanism of protein misfolding by V232D mutation involves disruption of a hydrophobic pocket since the hydrophobicity of the substituted amino acid at position 232 correlated with the amount of mature product and the ability to correct the defects with correctors or the V510D suppressor mutation; (3) the V232D mutation traps CFTR as a partially folded intermediate that can be rescued by corrector VX-809 to yield a native structure. Login to comment 276 ABCC7 p.Val232Asp Discussion The results in this study suggest that the mechanism for inhibition of packing of TM segments caused by the V232D mutation may be different in the full-length protein than predicted by the TM3/4 helix-loop-helix model system. Login to comment 277 ABCC7 p.Val232Asp ABCC7 p.Val232Asp Therien et al. [24] studied the V232D mutation in the TM3/4 helix-loop-helix fragment and suggested that the fragment was a useful model system for studying the mechanism of the V232D processing mutation because most consecutive TM segments in membrane proteins were in contact with one another [51]. Login to comment 278 ABCC7 p.Val232Asp In the TM3/4 helix-loop-helix model system, it was predicted that the two TM segments would be adjacent to each other along their entire lengths to bring Gln207(TM3) in close contact with TM4 to form a non-native hydrogen bond with the V232D processing mutation. Login to comment 279 ABCC7 p.Val232Asp ABCC7 p.Val232Asp It was postulated that the V232D mutation formed a hydrogen bond with Gln207 because replacement of Gln207 with nonpolar residues reversed the abnormal migration pattern of the V232D TM3/4 fragment in SDS-PAGE gels. Login to comment 280 ABCC7 p.Val232Asp ABCC7 p.Gln207Leu They showed that the migration pattern of mutant V232D/Q207L in SDS-PAGE gels resembled that of the wild-type fragment. Login to comment 281 ABCC7 p.Val232Asp The results of TM3/4 helix-loop-helix model system studies were used to predict that hydrogen bond formation between V232D and Gln207 could alter the normal assembly and alignment of CFTR TM segments in full-length CFTR. Login to comment 282 ABCC7 p.Val232Asp Our studies on the full-length CFTR protein however, suggest that V232D inhibited maturation by disrupting a hydrophobic pocket. Login to comment 283 ABCC7 p.Gln207Asn ABCC7 p.Gln207Glu Furthermore, Gln207 itself appeared to be important for maturation since all mutations to Gln207, including the conservative changes Q207N and Q207E (Fig. 2) inhibited maturation of the protein. Login to comment 284 ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Val232Asp ABCC7 p.Gln207Leu ABCC7 p.Gln207Leu ABCC7 p.Gln207Cys ABCC7 p.Gln207Cys ABCC7 p.Gln207Ala Although mutants such as A207A, Q207L and Q207C could be rescued with corrector VX-809 (Fig. 2), the V232D mutation appeared to have an effect that was independent of that of Gln207 since mutants Q207A/V232D, Q207L/V232D and Q207C/V232D could no longer be rescued by VX-809 (Fig. 1B). Login to comment 285 ABCC7 p.Val232Arg Fig. 9. Replacement of hydrophobic residues predicted to be close to Val232 with arginine inhibits maturation. Login to comment 286 ABCC7 p.Leu346Arg ABCC7 p.Val350Arg ABCC7 p.Phe229Arg ABCC7 p.Phe312Arg ABCC7 p.Phe354Arg ABCC7 p.Phe236Arg ABCC7 p.Phe310Arg (A) Whole cell SDS extracts of cells expressing wild-type (WT), F229R, F236R, F310R, F312R, L346R, V350R and F354R CFTR mutants were subjected to immunoblot analysis. Login to comment 292 ABCC7 p.Val232Asp The V232D mutation inhibits packing of the TM segments. Login to comment 293 ABCC7 p.Val232Asp ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys (A) Membranes prepared from cells expressing mutants W356C/W1145C, V232D/W356C/W1145C or Cys-less CFTR in the absence (none) or presence of corrector VX-809 were treated without (none) or with cross-linkers (X-linkers) M8M or BMH for 10 min at 20 8C. Login to comment 301 ABCC7 p.Val232Asp The P-gp L305R mutation in TM5 resembles the CFTR V232D mutation because it also inhibits maturation by disrupting a TM4/5 hydrophobic interface (Fig. 7A). Login to comment 313 ABCC7 p.His1085Arg ABCC7 p.Val232Asp ABCC7 p.Gln1071Pro We predict that V232D inhibits CFTR maturation by a mechanism that is similar to that proposed for the Q1071P, H1085R [50] and DF508 [17,26] processing mutations (see Fig. 11). Login to comment 317 ABCC7 p.Val232Asp The V232D mutation traps CFTR at an early folding step with incomplete packing of the TM segments and incomplete domain assembly (Fig. 11B). Login to comment 321 ABCC7 p.Val232Asp Corrector 4a may also efficiently rescue V232D CFTR because it also appears to interact with the transmembrane domains [18,65]. Login to comment 323 ABCC7 p.Val232Asp In summary, mutational analysis suggests that the mechanism of protein misfolding by the V232D mutation in TM4 does not involve non-native hydrogen bond formation with Gln207 in TM3. Login to comment 325 ABCC7 p.Val232Asp Understanding the mechanism of protein misfolding by V232D CFTR and its rescue by correctors is important because more than 50 other CF mutations have been identified in the TM segments of CFTR that involve a change from nonpolar to polar/charged residue [66]. Login to comment 327 ABCC7 p.Val232Asp Model of inhibition of CFTR by V232D and rescue by corrector VX-809. Login to comment 329 ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys (A) CFTR mutant W356C/W1145C that does not contain any processing mutation can fold into a native structure resulting in proper contacts between the various domains and packing of the TM segments such that W356C and W1145C can be cross-linked with cross-linkers M8M or BMH (orange line). Login to comment 330 ABCC7 p.Val232Asp (B) The presence of V232D (red) in TM4 disrupts the hydrophobic environment and inhibits proper folding to trap the mutant protein as a partially folded intermediate. Login to comment 331 ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys Packing of the TM segments is incomplete such that W356C and W1145C cannot be cross-linked. Login to comment 332 ABCC7 p.Val232Asp ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys (C) Studies [19,20] suggest that corrector VX-809 interacts with TMD1 to induce V232D to complete the folding process to yield a native structure in which W356C and W1145C can be cross-linked (orange line). Login to comment