ABCMdb

PMID: 20687163

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., [PubMed]

Sentences

No. Mutations Sentence Comment

26 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys 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 28 ABCC7 p.Phe429Ser ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Surprisingly, several of these solubilizing surface mutations in hNBD1, identified in a screen focused exclusively on the in vitro solubility of hNBD1, were shown to suppress the in vivo trafficking defect of F508del-CFTR more strongly than the best existing pharmacological agents.32,38 Notably, the mutated residues (e.g., F429S, F494N, and Q637R) are not in direct contact with F508 and do not appear to be allosterically coupled.18 A similar hydrophobic-to-hydrophilic substitution in the immediate vicinity of F508, the V510D mutation, also strongly suppresses the in vivo trafficking defect of F508del-CFTR.39,40 It was proposed that these substitutions could block adventitious chaperone interactions that prevent proper ER export.18 However, there is as yet no concrete evidence explaining the tight correlation between the effects of mutations on the in vitro solubility properties of hNBD1 and the in vivo trafficking properties of human CFTR. Login to comment 118 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Solubilizing surface mutations need to be introduced into full-length hNBD1 to obtain sufficient material for biophysical studies.15 Supporting Information Figure S5 compares the behavior of matched full-length and D(RI,RE) constructs containing F429S, F494N, and Q637R mutations15 in the absence or presence of the F508del mutation. Login to comment 155 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys 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 156 ABCC7 p.Val510Asp ABCC7 p.Val510Asp They were subsequently introduced into intact F508del-CFTR and demonstrated to suppress its aberrant trafficking in vivo in tissue culture cells.37 Introducing all three mutations simultaneously into full-length hNBD1 led to improved yield and stability of the purified soluble domain after overexpression in E. coli.15 The V510D mutation was made to explore the molecular pathology caused by the F508del mutation after crystallographic studies demonstrated that F508del produces a large change in the conformation of val-510.15 The hydrophobic sidechain of val-510 is mostly buried on the surface of hNBD1 in the absence of the F508del mutation,18 at a site where it is likely to make direct packing interactions to the transmembrane domains of CFTR.35 However, its backbone conformation is altered so that it projects from the surface of hNBD1 and is completely solvent-exposed in the presence of the F508del mutation.18 Surprisingly, the V510D mutation strongly suppresses the trafficking defect caused by the F508del mutation in vivo in tissue culture cells.39,40 Finally, the Figure 4. Login to comment 159 ABCC7 p.Phe494Asn The denaturation of hNBD1-D(RI,RE)-F508del is compared to that of the same construct containing in addition the Teem suppressor triplet37 (left), V510D39 (center), or F494N/Q637R15 (right). Login to comment 160 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg F494N/Q637R mutations were identified in a screen for surface substitutions that improve the solubility of purified hNBD1 in vitro.15 This screen focused on replacement of hydrophobic residues with more hydrophilic residues present at equivalent sites in other vertebrate CFTR sequences. Login to comment 161 ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg These mutations also show significant efficacy in suppressing the trafficking defect caused by the F508del mutation in vivo in tissue culture cells,32 although they are less effective that the Teem suppressors,37 the V510D mutation,39,40 or deletion of the RI.41 The Teem suppressor triplet (Fig. 4A-C), the V510D mutation (Fig. 4D-F), and the F494N/Q637R mutations (Fig. 4G-I) all stabilize hNBD1 against the initial unfolding transition, shifting its midpoint 0.25-0.50 M higher in urea concentration. Login to comment 162 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg The magnitude of the SLS increase following the initial unfolding transition is greatly reduced by the Teem suppressor triplet and the V510D mutation and significantly reduced by the F494N/Q637R mutations. Login to comment 165 ABCC7 p.Val510Asp In the presence of the Teem suppressor triplet or the V510D mutation, which significantly stabilize the domain, the urea-induced increase in trp fluorescence (Supporting Information Fig. S8B,E) precedes the initial unfolding transition as monitored by CD spectroscopy (Supporting Information Fig. S8A,D). Login to comment 168 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg First, the SLS data in Figure 4I demonstrate that the F494N/Q637R mutation pair in the F508del domain is unique in suppressing the self-association that occurs without an increase in trp fluorescence before the onset of the initial unfolding transition. Login to comment 170 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Second, the F494N/Q637R mutation pair appears to decrease the secondary structure content in the partially unfolded intermediate formed by both the F508del (Fig. 4G) and F508 (Supporting Information Fig. S8G) domains, while the Teem suppressor triplet may increase the secondary structure content in this state just in the F508del domain (Fig. 4A). Login to comment 179 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys 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 189 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg See Lewis et al.39 for a detailed description of the subdomain organization of hNBD1 and the stereochemical effects of the F508del mutation, the Teem suppressor mutation triplet,37 and the F494N/Q637R solubilizing mutations.15 reveals that hNBD1 unfolds via two sequential transitions which have similar spectroscopic and aggregation properties whether unfolding is driven by urea or by heat. Login to comment 199 ABCC7 p.Gln637Arg This inference is supported by the location in the ABCa subdomain of the F508del mutation that strongly facilitates the initial unfolding transition and all but Q637R among the second-site suppressor mutations demonstrated in Figures 4 and Supporting Information Figure S8 to inhibit this transition. Login to comment 200 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn The Teem suppressor triplet and the F494N mutation are located on the opposite face of the ABCa subdomain from the V510D mutation, which is adjacent to the F508del site, and crystallographic studies indicate that the direct stereochemical effects of these different mutations are likely to be unrelated.18 The influence of all of these mutations on the initial unfolding transition suggests that it involves a global change in ABCa subdomain conformation. Login to comment