ABCMdb

PMID: 25083918

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., [PubMed]

Sentences

No. Mutations Sentence Comment

30 ABCC7 p.Arg1070Trp Furthermore, the combination of maximal NBD1 stabilization and interface modification by the paradoxical R1070W interface mutation disrupts the normal control of channel activity by phosphorylation. Login to comment 35 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp Rabeh et al. found that, while several of the solubilizing and suppressor mutations caused only a modest promotion of ƊF508 CFTR maturation, their effects were greater when they were combined with the NBD1/CL4 interface substitution R1070W or V510D [33], the latter also having been shown to have a direct effect on NBD1 thermostability [10]. Login to comment 45 ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Ala534Pro ABCC7 p.Ser434Pro ABCC7 p.Ser422Pro ABCC7 p.Ser492Pro 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 55 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp In contrast, each of the interface substitutions, V510D (lane 8) and R1070W (lane 9), had much smaller effects. Login to comment 61 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp The rates of appearance of the mature products and the levels reached were increased further when the R1070W mutation was added to the combined NBD1 changes but not when V510D was added. Login to comment 65 ABCC7 p.Ile539Thr ABCC7 p.Ser492Pro 1-WT; 2-S492P; 3-I539T; 4. Login to comment 66 ABCC7 p.Arg553Gln ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Ser492Pro S492P/I539T; 5-G550E/R553Q/R555K; 6-combo. Login to comment 72 ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro ABCC7 p.Ser495Pro 2PT, S492P/ A534P/I539T; 3PT, 2PT + S495P. Login to comment 74 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met (*) In full-length CFTR, R553M was introduced instead of R553Q in isolated NBD1. Login to comment 75 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys 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 84 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp ABCC7 p.Val510Asp The surface level of ƊF508 CFTR with the combined NBD1 stabilizing mutations (ƊF/combo) reached ~90% that of WT CFTR (Fig. 1c), and the level was somewhat further increased when V510D or R1070W was added to the combination (ƊF/combo/V510D and ƊF/combo/R1070W). Login to comment 87 ABCC7 p.Arg1070Trp Functional activity measured using this iodide efflux assay was similar for the ƊF508/combo and the ƊF508/4PT/R1070W, indicating the effectiveness of the combination of multiple NBD1 stabilizing mutations without interface modification in promoting ƊF508 CFTR channel activity. Login to comment 88 ABCC7 p.Val510Asp The addition of V510D to the ƊF508/combo did not further increase its peak of iodide efflux. Login to comment 89 ABCC7 p.Arg1070Trp It was initially surprising to observe that the addition of R1070W to the ƊF508/combo apparently greatly diminished iodide efflux (Fig. 1d, bottom panel). Login to comment 91 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp This apparent inability of baby hamster kidney (BHK) cells expressing ƊF508/combo/R1070W CFTR to be effectively stimulated by forskolin cocktail suggested that the ƊF508/combo/R1070W CFTR channel might be constitutively active, a possibility that was subsequently confirmed at the single-channel level (see Fig. 5 below). Login to comment 93 ABCC7 p.Ser492Pro ABCC7 p.Ser495Pro In addition to the S492P substitution found in three non-mammalian species that are relatively insensitive to the destabilizing influence of the ƊF508 mutation [13], we also included a second Q-loop proline substitution, S495P present in shark CFTR. Login to comment 95 ABCC7 p.Ile539Thr ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Ser492Pro S492P or I539T alone slightly increased the Tm of NBD1 (ƊTm = 2-3 &#b0;C) similar to the affect of the solubilization mutations F494N and Q637R combined. Login to comment 96 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro 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 97 ABCC7 p.Ser495Pro The S495P substitution alone in the isolated NBD1 of human CFTR caused the largest increase in Tm (5.99 &#b1; 0.33 &#b0;C) of any single change tested (Fig. 2a, lower panel). Login to comment 98 ABCC7 p.Ser495Pro Therefore, we compared the effect of S495P with that of other NBD1 stabilizing mutations on the maturation of ƊF508 CFTR. Login to comment 99 ABCC7 p.Ser495Pro As shown in Fig. 2b, the S495P substitution in ƊF508 CFTR resulted in substantial maturation. Login to comment 100 ABCC7 p.Ile539Thr ABCC7 p.Ser492Pro The effect of this single mutation was in contrast to that of S492P, which only increased maturation substantially when present together with I539T. Login to comment 101 ABCC7 p.Ile539Thr ABCC7 p.Ser495Pro Combination of I539T with S495P, however, did cause a further increase in maturation and the effects of the two proline substitutions also were additive. Login to comment 111 ABCC7 p.Arg1070Trp The R1070W mutation in CL4 has different effects on the gating of WT and ƊF508 CFTR channels. Login to comment 112 ABCC7 p.Arg1070Trp (a) Western blot indicating enhancement of maturation of mutant R1070W and ƊF508 CFTRs by combined NBD1 stabilizing mutants (combo). Login to comment 113 ABCC7 p.Arg1070Trp (b and c) Single-channel recordings of WT (b) and ƊF508 CFTR (c) containing the R1070W mutant at 25 &#b0;C and 35 &#b0;C. All point histograms used to calculate single-channel parameters are shown to the left of each tracing. Login to comment 116 ABCC7 p.Arg1070Trp (d) WT and ƊF508/R1070W CFTR single-channel recordings during continuous temperature ramp of 1 &#b0;C/min from 30 to 35 &#b0;C, maintained at 30 &#b0;C for 3 min before initiating the ramp and held at 35 &#b0;C for 2 min. Login to comment 117 ABCC7 p.Arg1070Trp The moment of ƊF508/R1070W functional inactivation is shown by an arrow above the tracing and appeared after about 1 min at 35 &#b0;C. X-scale bar below trace is 60 s and Y-scale bar is 1 pA. 1.5 mM, reflecting low-affinity binding. Login to comment 123 ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro Figure 3e shows that both BIA and BEIA further strongly increased maturation of the NBD1 stabilized variant ƊF508/2PT (S492P/A534P/I539T). Login to comment 125 ABCC7 p.Val510Asp Turning to the interface modified variants (Fig. 3f), BEIA also caused a large further increase in the maturation of the ƊF508/V510D protein. Login to comment 126 ABCC7 p.Arg1070Trp The BEIA influence on the other interface substituted form of the protein, R1070W, was less pronounced, particularly at the lower concentrations of the compound. Login to comment 129 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp The disease-associated CL4 mutation R1070W differentially influences WT and ƊF508 CFTR R1070W is a relatively rare disease-associated mutation in the CL4 region of CFTR. Login to comment 131 ABCC7 p.Arg1070Trp Homology models of CFTR three-dimensional structure indicated that the R1070-containing segment of CL4 was proximal to F508 on the NBD1 surface [29-32] and Thomas et al. reasoned that the introduction of the aromatic tryptophan side chain by R1070W substitution might compensate for the loss of the phenylalanine side chain due to the absence of F508 at the interface between domains [8]. Login to comment 132 ABCC7 p.Arg1070Trp They showed that the R1070W mutation substantially improved the maturation of ƊF508 CFTR. Login to comment 135 ABCC7 p.Arg1070Trp Combined NBD1 stabilizing mutations promote maturation and function of R1070W CFTR in both ƊF508 and WT backgrounds. Login to comment 136 ABCC7 p.Arg1070Trp (a) Single-channel current tracings of the WT, ƊF508/Combo and ƊF508/Combo/R1070W CFTRs at 25 &#b0;C. All point histograms, single-channel conductances and open probabilities are shown as in Fig. 4. Login to comment 137 ABCC7 p.Arg1070Trp (b) Single-channel currents at 37 &#b0;C showing the influence of combined NBD1 stabilizing changes on ƊF508 and WT CFTR without and with the R1070W mutation. Login to comment 139 ABCC7 p.Arg1070Trp Combined NBD1 stabilizing mutations and R1070W result in phosphorylation-independent opening of ƊF508 CFTR channels. Login to comment 140 ABCC7 p.Arg1070Trp (a) Single-channel recordings at 37 &#b0;C of alkaline phosphatase treated ƊF508 CFTR containing combined NBD1 stabilizing mutations without (upper tracing) and with R1070W (middle tracing) as indicated. Login to comment 141 ABCC7 p.Arg1070Trp Lower tracing: single-channel recording at 37 &#b0;C of ƊF508/combo/R1070W with all 15 PKA phosphorylation sites mutated (15SA). Login to comment 145 ABCC7 p.Arg1070Trp Contrasting effects of R1070W on WT and ƊF508 CFTR is very evident at the level of biosynthetic processing as shown in Fig. 4a, where maturation of the WT is reduced while that of ƊF508 is promoted. Login to comment 148 ABCC7 p.Arg1070Trp While the opposite effects of R1070W on WT and ƊF508 CFTR maturation have been well documented, the corresponding functional consequences have not. Login to comment 150 ABCC7 p.Arg1070Trp The R1070W mutation alone resulted in very low channel open probabilities at both 25 &#b0;C and 35 &#b0;C (Fig. 4b). Login to comment 153 ABCC7 p.Arg1070Trp In contrast, robust channel gating was observed with the combined ƊF508/ R1070W variant with the expected elevation of Po as temperature was increased from 25 &#b0;C to 35 &#b0;C (Fig. 4c). Login to comment 156 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp Therefore, as with the much less active R1070W/WT form, this more active double mutant (ƊF508/R1070W) has a similar short functional lifetime at 35 &#b0;C confirming that it remains thermally unstable [15]. Login to comment 158 ABCC7 p.Arg1070Trp This behavior was especially evident when the single-channel activities of ƊF508 CFTR modified by combined NBD1 stabilizing changes with and without the R1070W interface mutation were compared with the unmodified WT at both 25 &#b0;C (Fig. 5a) and 37 &#b0;C (Fig. 5b). Login to comment 160 ABCC7 p.Arg1070Trp Strikingly, addition of the R1070W mutation to the NBD1 stabilized combination resulted in a very high open probability (0.91) even at this lower temperature. Login to comment 162 ABCC7 p.Arg1070Trp With the addition of R1070W, a nearly, completely locked open state was achieved with a Po of 0.98 (Fig. 5b, third tracing). Login to comment 164 ABCC7 p.Arg1070Trp The lower two tracings in Fig. 5b indicate that the combined stabilizing modifications of NBD1 also elevate the open probability of the WT channel with and without the R1070W mutation. Login to comment 165 ABCC7 p.Arg1070Trp Combined NBD1 stabilization and NBD1/CL4 interface modification result in constitutive ion channel activity of ƊF508 CFTR In iodide efflux experiments from whole cells expressing ƊF508/combo/R1070W CFTR, we had observed that the cells could not be efficiently loaded with the anion prior to the stimulation of cAMP production to activate protein kinase A (PKA) (Fig. 1d), suggesting the possibility that the apparent locked-open state might exist in the absence of the normally obligatory phosphorylation by PKA. Login to comment 168 ABCC7 p.Arg1070Trp In contrast, the NBD1 combo stabilized versions of the ƊF508 CFTR channels (Fig. 6a, middle tracing) and WT (Fig. 6b, middle tracing) responded very differently to addition of the R1070W mutation, the former channel becoming essentially locked open while the open probability of the latter was increased only modestly. Login to comment 169 ABCC7 p.Arg1070Trp To further verify that the high open probability state of ƊF508/combo/R1070W CFTR was reached independently of phosphorylation by PKA, we employed a construct in which serines or threonines at all 15 PKA phosphorylation sites were mutated to Ala (15SA) [39]. Login to comment 170 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp While the activity of the combo/ R1070W/WT CFTR was little changed by the 15SA modifications (Fig. 6b, lower tracing), the 15SA version of the ƊF508/combo/R1070W CFTR channel remained fully active even in the absence of PKA phosphorylation sites (Fig. 6a, lower tracing), confirming that the maximally NBD1 stabilized and NBD1/CL4 interface modified form of ƊF508 CFTR has lost normal regulatory control by phosphorylation. Login to comment 176 ABCC7 p.Arg1070Trp To further pursue this hypothesis here, we combined several of the most effective second-site changes in NBD1 with or without the interface stabilizing mutant R1070W and compared their effects on maturation, traffic to the cell surface and channel function. Login to comment 179 ABCC7 p.Ser495Pro Strikingly, the introduction of a proline residue at position 495 (S495P) in the Q-loop was found to cause the largest increase in the domain Tm and have the strongest restorative effect on maturation of the full-length protein of any single NBD1 second-site change and its influence was additive with others (Fig. 2). Login to comment 191 ABCC7 p.Ser495Pro As demonstrated in the current work the S495P mutation, two residues C-terminal of the Q-loop glutamine have the greatest impact. Login to comment 200 ABCC7 p.Arg1070Trp Turning to the impact of modifying the NBD1/CL4 interface, which may be a site of action of the VX-809 corrector [23,25], the R1070W mutation, shown by others to improve ƊF508 CFTR maturation [8], was the main tool we employed. Login to comment 201 ABCC7 p.Val510Asp We observed that the mutation alone promoted only a low level of maturation as did the other known interface change, V510D [43]. Login to comment 202 ABCC7 p.Arg1070Trp Not only were their effects small compared to those of NBD1 stabilizing changes but R1070W also caused only a small increment above the correction caused by combined NBD1 stabilization (Fig. 1). Login to comment 203 ABCC7 p.Arg1070Trp From a functional perspective, most significant was the finding that, when modified by combined NBD1 mutations and R1070W, the efficiently assembled ƊF508 channel was constitutively active, not requiring phosphorylation by PKA (Fig. 6). Login to comment 204 ABCC7 p.Arg1070Trp Although the mechanism underlying this effect remains to be elucidated, it occurs in the stabilized mutant protein only when the NBD1/CL4 interface is altered by both the absence of F508 and the presence of R1070W (Figs. Login to comment 206 ABCC7 p.Arg1070Trp The disease-associated R1070W mutation alone is destabilizing of WT CFTR but does not cause it to be constitutively active without phosphorylation even when it contains all the NBD1 stabilizing changes (Fig. 6). Login to comment 208 ABCC7 p.Arg1070Trp These findings do not detract from the idea of therapeutically targeting the interface in different ways since it clearly plays a role in the propagation of stabilizing effects between domains as evidenced by the fact that impaired maturation caused by R1070W can be overcome by NBD1 stabilizing changes (Fig. 4a). Login to comment