ABCMdb

PMID: 22265409

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., [PubMed]

Sentences

No. Mutations Sentence Comment

16 ABCC7 p.Arg553Gln A patient with a less severe form of CF had DF508-R553Q on one allele and a nonsense mutation on the other (Do &#a8; rk et al., 1991). Login to comment 17 ABCC7 p.Arg553Gln A mating screen in yeast utilizing a CFTR-Ste6 chimera identified the same R553Q mutation as a suppressor of the loss-of-mating DF508 phenotype (Teem et al., 1993). Login to comment 18 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys 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 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg555Lys 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 24 ABCC7 p.Gly551Asp There is reasonable concern that this degree of correction will not provide therapeutic benefit based on the promising results with the more potent and efficacious potentiator of the rarer CF-causing G551D gating mutant (Accurso et al., 2010; Van Goor et al., 2009). Login to comment 62 ABCC7 p.Phe490Leu Ten of the sixteen positions are surface exposed; F490L and W496, like F508, are predicted to be at the ICL4 interface (Table 1; Figure S3). Login to comment 69 ABCC7 p.Glu583Gly The significance of the decrease in folding was <6E6 for 13 of the mutants and 0.002 for the least significant, E583G. Login to comment 70 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe Two 508-coupled mutants, D529F and S573E, dramatically increased the relative folding yield of NBD1 by 3.07- &#b1; 0.13-fold and 1.85- &#b1; 0.06-fold, respectively, a level comparable to that of the second-site suppressors identified in the STE6 chimera screen (Figure 3A). Login to comment 71 ABCC7 p.Ser573Glu ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe Thermal denaturation of purified D529F and S573E NBD1 revealed that S573E raised the Tm by 2 C-5 C relative to wild-type depending on ATP concentration (Figure S4B), indicating the improved folding yield in the complementation assay could be accounted for by increased stability of the native state (Figure 3B). Login to comment 72 ABCC7 p.Asp529Phe In stark contrast, D529F had no measurable effect on Tm indicating the complementation assay reflects changes in yield and pathway as well as changes in stability or solubility. Login to comment 76 ABCC7 p.Arg555Lys The R555K missense mutation F508 ICL4 ICL1 F508 A B 0 0.2 0.4 0.6 0.8 1 508 Consensus Walker B 389 673 Walker A 0 0.2 0.4 0.6 0.8 1 NBD1 508 TMD NBD R TMD2 NBD 1 1480 TMD NBD R TMD2 NBD Figure 2. Login to comment 90 ABCC7 p.Arg555Lys R555K increased the folding yield of NBD1, 3.26- &#b1; 0.07-fold over wild-type (Figure 3A). Login to comment 91 ABCC7 p.Arg555Lys The Tm of R555K NBD1 was 6.4 C higher than wild-type in 2 mM ATP. Login to comment 108 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe The same two mutations that improved NBD1 folding yield, D529F and S573E, increased the maturation efficiency of CFTR (Figure 4A, yellow and magenta bars, respectively). Login to comment 115 ABCC7 p.Ser573Glu ABCC7 p.Phe490Leu ABCC7 p.Phe490Leu ABCC7 p.Glu583Gly ABCC7 p.Glu583Gly ABCC7 p.Asp529Phe ABCC7 p.Asp529Phe ABCC7 p.Asp529Phe ABCC7 p.Asp529Phe ABCC7 p.His609Thr ABCC7 p.Leu526Ala ABCC7 p.Leu526Ala ABCC7 p.Leu526Ala ABCC7 p.Leu526Ala ABCC7 p.Phe575Thr ABCC7 p.Phe575Thr ABCC7 p.Tyr517Ile ABCC7 p.Tyr517Ile ABCC7 p.Tyr517Ile ABCC7 p.Tyr517Ile ABCC7 p.Pro574Ala ABCC7 p.Pro574Ala ABCC7 p.Pro574Ala ABCC7 p.Ser466Thr ABCC7 p.Ser466Thr ABCC7 p.Leu475Tyr ABCC7 p.Leu475Tyr ABCC7 p.Leu475Tyr ABCC7 p.Cys524Ala ABCC7 p.Cys524Ala ABCC7 p.Cys524Ala ABCC7 p.Cys524Ala ABCC7 p.Trp496Val ABCC7 p.Trp496Val ABCC7 p.Trp496Val ABCC7 p.Trp496Val ABCC7 p.Tyr563Val ABCC7 p.Tyr563Val ABCC7 p.Tyr563Val ABCC7 p.Tyr563Val ABCC7 p.Ala566Pro ABCC7 p.Ala566Pro ABCC7 p.Asp537Phe ABCC7 p.Asp537Phe Top 20 508-Coupled Positions within NBD1 Using Four Independent Statistical Methods ELSC McBASC OMES SCA 435 453 460 S466T S466T 468 470 472 473 473 474 474 L475Y L475Y L475Y F490L F490L W496V W496V W496V W496V 503 505 507 509 512 513 Y517I Y517I Y517I Y517I 520 520 521 C524A C524A C524A C524A L526A L526A L526A L526A D529F D529F D529F D529F D537F D537F 543 Y563V Y563V Y563V Y563V A566P A566P 569 569 S573E P574A P574A P574A F575T F575T 578 582 E583G E583G 587 591 595 598 602 604 604 604 H609T 617 630 630 640 The alignment of 493 sequences was used to calculate pairwise coupling scores using each method (ELSC, SCA, McBASC, and OMES). Login to comment 120 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe Interestingly, the two suppressors identified by the coupling analysis, D529F and S573E, had much more modest effects on NBD1 yield in the presence of the DF508 mutant as might be expected. Login to comment 127 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Ser573Glu ABCC7 p.Ser573Glu ABCC7 p.Phe490Leu ABCC7 p.Glu583Gly ABCC7 p.Asp529Phe ABCC7 p.Asp529Phe ABCC7 p.His609Thr ABCC7 p.Leu526Ala ABCC7 p.Phe575Thr ABCC7 p.Tyr517Ile ABCC7 p.Pro574Ala ABCC7 p.Ser466Thr ABCC7 p.Leu475Tyr ABCC7 p.Cys524Ala ABCC7 p.Trp496Val ABCC7 p.Tyr563Val ABCC7 p.Ala566Pro ABCC7 p.Asp537Phe 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 129 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe All mutations altered the relative yield relative to wild-type NBD1 as shown in the bar chart (&#b1; standard error of the mean [SEM], n = 9 except for WT, DF508, D529F, and S573E where n = 18). Login to comment 130 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe Two of the 508-coupled positions, D529F and S573E, increase the yield of NBD1. Login to comment 132 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe (B) Thermal denaturation of 5 mM purified recombinant WT, D529F, and S573E NBD1 in the presence of 2 mM ATP (left panel). Login to comment 133 ABCC7 p.Asp529Phe See also Figure S4. By contrast to the increased NBD1 folding yield, the D529F mutation (yellow circles) has no observable effect on thermal stability relative to WT (black circles). Login to comment 134 ABCC7 p.Ser573Glu S573E (magenta circles) increased the melting temperature 2 C. Login to comment 136 ABCC7 p.Asp529Phe For mutants on either WT (circles) or DF (triangles) backgrounds, the correlation between stability and folding yield, R = 0.94 when D529F is excluded (yellow circle) (right panel, &#b1;SEM). Login to comment 146 ABCC7 p.Arg1070Trp The R1070W sterically clashes with the F508 position in the model (Figure 6A, top) a prediction consistent with fact that it is a CF-causing mutation (Krasnov et al., 2008) that inhibits CFTR maturation when F508 is present (open circle, Figure 6A, bottom). Login to comment 148 ABCC7 p.Arg1070Trp The ability of the R1070W mutation to counteract the ICL4-NBD1 interface defect caused by the DF508 mutation, allowed assessment of the quantitative effects of suppression of both defects concurrently. Login to comment 149 ABCC7 p.Arg1070Trp When NBD1 suppressor mutations were introduced on top of R1070W and DF508, the slope was restored (m = 0.77, R = 0.47) (Figure 6B). Login to comment 152 ABCC7 p.Arg1070Trp Similarly, the R1070W interface mutant produces an 7-fold increase in the slope, but only a modest improvement in CFTR maturation. Login to comment 158 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Arg555Lys Mirroring the maturation results, correction of either the NBD1 defect (I539T, R555K, red bars) or the ICL4-NBD1 defect (R1070W, white bar) alone provided only modest improvements of function. Login to comment 166 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Ser573Glu ABCC7 p.Phe490Leu ABCC7 p.Glu583Gly ABCC7 p.Asp529Phe ABCC7 p.His609Thr ABCC7 p.Leu526Ala ABCC7 p.Phe575Thr ABCC7 p.Tyr517Ile ABCC7 p.Pro574Ala ABCC7 p.Ser466Thr ABCC7 p.Leu475Tyr ABCC7 p.Cys524Ala ABCC7 p.Trp496Val ABCC7 p.Tyr563Val ABCC7 p.Ala566Pro ABCC7 p.Asp537Phe 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 168 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe (A) The efficiency of full-length CFTR maturation was determined by ELISA (&#b1;SEM, n = 6 for WT, DF508, D529F, and S573E, n = 3 for other mutants). Login to comment 171 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe The two 508-coupled positions that increased NBD1 folding yield, D529F and S573E, also increased the maturation yield of CFTR (yellow and magenta bars, respectively). Login to comment 172 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys 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 173 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe The two F508- coupled position mutations, D529F and S573E, are colored in yellow and magenta circles, respectively. Login to comment 185 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe 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 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Phe508Arg ABCC7 p.Phe508Lys ABCC7 p.Phe508Lys 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 190 ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe Identified in this study, D529F and S573E improve folding of NBD1 in isolation and maturation of full-length CFTR in the wild-type background. Login to comment 197 ABCC7 p.Phe508Lys The experiments with the second-site suppressor mutations on the F508K background demonstrate, regardless of how well NBD1 folds, CFTR is unable to mature if the NBD1-ICL4 is disrupted. Login to comment 215 ABCC7 p.Arg1070Trp 0.0 0.5 1.0 1.5 0 25 50 75 100 WT F WT F Relative Yield CFTR (ELISA) Current Density (pA/pF) F I 5 3 9 T R 5 5 5 K R 1 0 7 0 W I 5 3 9 T R 5 5 5 K W T R 1 0 7 0 W 0.0 0.5 0.0 0.5 1.0 1.5 2.0 2.5 Relative Conductance F F-R1070W D C 0.0 0.5 1.0 1.5 2.0 0 1 WT F Relative Yield NBD1 ( -gal.) 0.0 0.5 1.0 1.5 2.0 0 1 WT F Relative Yield NBD1 ( -gal.) Relative Yield CFTR (ELISA) A + Forskolin/IBMX + Inh 172 60 sec 500 pA + Forskolin/IBMX + Inh 172 B Relative Yield CFTR (ELISA) Figure 6. Login to comment 217 ABCC7 p.Arg1070Trp The R1070W (open triangle) mutation in ICL4 modestly improves the relative yield of DF508 CFTR maturation and decreases the relative yield for CFTR wild-type (open circle) consistent with the predicted steric clash with the F508 side chain (top) (&#b1;SEM). Login to comment 219 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys 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 224 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr A representative trace of the corrected mutant, DF508-I539T-R1070W CFTR (cyan triangles) is more like wild-type (filled circles) than DF508 (filled triangles) (inset). Login to comment 233 ABCC7 p.Arg555Lys ABCC7 p.Ser573Glu ABCC7 p.Asp529Phe D529F, S573E, and R555K mutations of human wild-type NBD1 were expressed and purified as previously described (Thibodeau et al., 2005). Login to comment 241 ABCC7 p.Arg1070Trp ABCC7 p.Phe508Lys On the WT or DF508 CFTR models, F508K, R1070, R1070W residues were modeled using the PyMOL (Schrodinger, 2010) mutagenesis function (Figures 1B, 2B, rightmost panel, 5, 6, and S1). Login to comment