ABCMdb

PMID: 22406676

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr Introduction of these prolines experimentally into full-length human ΔF508 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 *Corresponding author. Login to comment 40 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr This low-level maturation of rabbit ΔF508 CFTR may reflect primarily the I539T substitution (see below), Fig. 2. Login to comment 104 ABCC7 p.Ile539Thr Among the differences, in addition to the I539T substitution present in many non-human species, we noted a pattern where proline residues replaced other residues in the maturing compared to the non- maturing species (Fig. 5a). Login to comment 108 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr Since chicken CFTR appeared least influenced by the F508 deletion among the species studied, we tested the effect of its four proline substitutions on human ΔF508 CFTR with and without the I539T mutation. Login to comment 110 ABCC7 p.Ile539Thr However, the impact of the I539T substitution was much more dramatic when combined with proline replacements at the four positions at which they normally occur in the chicken sequence. Login to comment 111 ABCC7 p.Ile539Thr Although introduction of all four prolines in the absence of I539T does not promote the maturation of ΔF508 human protein, they have a major effect in its presence, increasing maturation to near the WT level (Fig. 5c). Login to comment 112 ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ser434Pro ABCC7 p.Ser434Pro ABCC7 p.Ser422Pro ABCC7 p.Ser422Pro Combination of introduction of a proline into the Q-loop at position 492 together with I539T to generate ΔF/PT was sufficient to allow a high level of maturation even without further addition of the substitutions in the I539 loop (A534P) to generate ΔF/2PT or also in the RI (S422P and S434P) to generate ΔF/4PT. Login to comment 115 ABCC7 p.Ile539Thr Figure 5d shows that the immature forms of all these variants decayed at similar rates (upper graph), consistent with earlier studies of WT and mutant human CFTR.24 However, formation of the mature species was least with I539T alone (ΔF/T) and increased incrementally with inclusion of one, two, and four proline substitutions as judged by the amounts formed by 2 and 4 h (lower graph). Login to comment 120 ABCC7 p.Ile539Thr With just the I539T (ΔF/T) substitution, a T1/2 of ~6 h was observed. Login to comment 121 ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro With the inclusion of S492P± A534P (ΔF/PT or ΔF/ 2PT), the turnover rates were indistinguishable from WT with a T1/2 of ~14 h. When prolines also replaced residues 422 and 434 in the RI to form ΔF/ 4PT, the rate appeared even slower than WT with a T1/2 of ~16 h. Thus, the biological stability of the Fig. 5. Login to comment 122 ABCC7 p.Ile539Thr Mimicking chicken CFTR (prolines and I539T) restores maturation and lifetime of ΔF508 CFTR. Login to comment 125 ABCC7 p.Ile539Thr The common I539T replacement is also indicated. Login to comment 128 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ser434Pro ABCC7 p.Ser434Pro ABCC7 p.Ser434Pro ABCC7 p.Ser434Pro ABCC7 p.Ser422Pro ABCC7 p.Ser422Pro ABCC7 p.Ser422Pro ABCC7 p.Ser422Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro (c) Western blot of WT and ΔF508 CFTR expressed in HEK-293 cells and ΔF508 modified with I539T (ΔF/T), S422P/S434P/S492P/A534P (ΔF/4P), I539T/S492P (ΔF/PT), I539T/S492P/A534P (ΔF/2PT), and I539T/S422P/S434P/S492P/A534P (ΔF/4PT). Login to comment 136 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro Thermostable binding of the nucleotide by the WT, which is lost in ΔF508 CFTR,19 was not restored after rescue in cells grown at 27 °C [(r)ΔF panel], but was to some extent by I539T (ΔF/T panel), to a greater extent when S492P was added (ΔF/PT panel), still further with A534P also added (ΔF/2PT panel), and to near the WT level with proline replacements at residues S492 and A534 as well (ΔF/4PT panel). Login to comment 139 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr At a fixed sub-physiological temperature (35 °C), the I539T/ΔF508 protein exhibited predominantly a fast flickering mode (ffm) with only rare full-conductance channel openings (Fig. 6a). Login to comment 140 ABCC7 p.Ser492Pro The addition of the S492P substitution generated a dominant full-conductance behavior (ΔF/PT), with a Po approximately half of the WT human channel at that temperature. Login to comment 143 ABCC7 p.Ser492Pro Addition of S492P to form ΔF/PT resulted in WT-like full-conductance gating at temperatures up to approximately 37 °C, above which there was a transition to the ffm (middle tracing). Login to comment 144 ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro Thus, as observed in the 35 °C fixed temperature recording (Fig. 6a), introduction of the Q-loop proline (S492P) had a significant stabilizing effect. Login to comment 151 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Val510Asp Thus, both the constant and varying temperature experiments demonstrated a requirement for a balance between thermal stability and channel activity in CFTR, consistent with considerations of such a relationship between stability and catalytic function of proteins in general.28 NBD1 stabilization restores an NBD1-CL4 interface In addition to destabilizing NBD1, deletion of F508 also disrupts interdomain contacts including the interface between the NBD1 surface and the cytoplasmic loop (CL)4 in MSD2 in which the residue normally participates.29 Specific second-site mutations on either side of this interface (e.g., R1070W or V510D) have been shown to promote maturation of ΔF508 CFTR.27,30,31 The current observations that the ΔF508 protein with NBD1 strongly stabilized by the proline and I539T substitutions had channel activity similar to the WT at physiological temperature suggested either that the NBD1-CL4 interface is not important for function or that it is adequately restored by NBD1 stabilization. Login to comment 155 ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys Using an otherwise Cys-less construct, we had previously observed methanethiosulfonate-mediated cross-linking between V510C and G1069C in WT CFTR.29,33 Figure 7 shows similar cross-linking of this pair of residues in ΔF/4PT CFTR, providing evidence that the stabilization of ΔF508 NBD1 can Fig. 6. Login to comment 176 ABCC7 p.Ile539Thr Both the RI and the SDR have been hypothesized to influence quaternary assembly of NBD1 with the rest of CFTR.29,33 Strikingly, substitution of S492 to proline in the context of the I539T mutation Fig. 7. Login to comment 177 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr Restoration of the ΔF508 CFTR NBD1-CL4 interface by proline and I539T mutations. Login to comment 178 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Val510Cys ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys ABCC7 p.Gly1069Cys ABCC7 p.Ala534Pro ABCC7 p.Ala534Pro ABCC7 p.Ser434Pro ABCC7 p.Ser434Pro ABCC7 p.Ser422Pro ABCC7 p.Ser422Pro ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less ΔF508-CFTR in the presence or absence of the 4PT mutations (S422P/S434P/S492P/A534P/I539T), with the Cys pair V510C/G1069C introduced at the CL4/NBD1 interface. Login to comment 182 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr Avian ΔF509 CFTR is destabilized by reversal of proline and I539T substitutions. Login to comment 188 ABCC7 p.Ile539Thr Such stabilization of the SDR was not observed in simulations with the I539T substitution alone (Fig. 9a). Login to comment 195 ABCC7 p.Ile539Thr Thus, while I539T alone did not appear to significantly improve the stability of ΔF508 NBD1, stabilization of the SDR via incorporation of prolines at different positions increased the stability of ΔF508 NBD1. Login to comment 216 ABCC7 p.Ile539Thr ABCC7 p.Ser492Pro ABCC7 p.Ser492Pro Introduction of a proline at S492 in the context of I539T increases the folding transition temperature of ΔF508 NBD1 to ~327 K, consistent with the observed decrease in thermal fluctuations upon S492P substitution (Fig. 9b, orange broken line). Login to comment 230 ABCC7 p.Ser492Pro The apparent additive effects of several substituted prolines acting independently have been observed in other proteins as well.47 Molecular dynamics simulations reveal that the SDR of NBD1 is stabilized by the S492P substitution, and the stability further increases with each proline substitution. Login to comment 237 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys 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 238 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr I539T has an additive effect with the 550/ 553/555 set.33 The I539T substitution alone promotes a low level of human ΔF508 CFTR maturation, and its normal presence in murine CFTR probably contributes to the partial maturation of the mouse ΔF508 variant15,21 as well as that of the rabbit (Fig. 2a). Login to comment 279 ABCC7 p.Val510Cys ABCC7 p.Gly1069Cys Briefly, HEK cells transiently expressing Cys-less CFTR constructs with V510C and G1069C substitutions on the WT or ΔF/4PT CFTR basis grown on 35-mm tissue culture dishes were harvested, washed twice in PBS, and resuspended in 60 μl of PBS. Login to comment 433 ABCC7 p.Val510Asp The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface. Biochemistry, 49, 6352-6357. 32. Login to comment 436 ABCC7 p.Val510Asp The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface. Biochemistry, 49, 6352-6357. 32. Login to comment 451 ABCC7 p.Gly550Glu Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment 455 ABCC7 p.Gly550Glu Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Login to comment