ABCMdb

PMID: 15619636

Thibodeau PH, Brautigam CA, Machius M, Thomas PJ
Side chain and backbone contributions of Phe508 to CFTR folding.
Nat Struct Mol Biol. 2005 Jan;12(1):10-6. Epub 2004 Dec 26., [PubMed]

Sentences

No. Mutations Sentence Comment

17 ABCC7 p.Phe508Ser Finally, to assess the specific structural consequences of these mutations, the crystal structures of two mutant NBD1 proteins, F508S, a previously identified non-CF-causing mutation (http://www. Login to comment 18 ABCC7 p.Phe508Arg genet.sickkids.on.ca/cftr), and F508R, a maturation-deficient mutation5, were determined. Login to comment 28 ABCC7 p.Phe508Trp With the exception of F508W, all measured NBD1 proteins were capable of folding at 4 °C at near-100% efficiency as measured by the production of soluble conformers that were quantified by tryptophan fluorescence intensity or western blotting.In addition,all of the domains exhibited a temperature-dependence of refolding efficiency where overall yield in the soluble fraction decreased as temperature increased. Login to comment 33 ABCC7 p.Phe508Asp ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Met ABCC7 p.Phe508Pro ABCC7 p.Phe508Gln The F508A,F508M,F508P,F508D,F508Q,F508R and F508S mutant proteins were more similar to the wild type than the ∆F508 protein in their temperature-dependence of refolding (Fig. 1b,c). Login to comment 36 ABCC7 p.Trp496Phe A tryptophan was also introduced at position 508 to assess the effects of substitution of a larger hydrophobic residue and to act as a spectral probe to track the folding of the NBD both in the wild-type domain and in a mutant background of W496F. Login to comment 38 ABCC7 p.Phe508Trp ABCC7 p.Trp496Phe However, when the F508W mutation was introduced onto the background of W496F, folding of the NBD1 was partially restored and the protein was capable of refolding with higher efficiency (>90% soluble) at 4 °C (Fig.1b). Login to comment 43 ABCC7 p.Phe508Asp ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Met ABCC7 p.Phe508Pro ABCC7 p.Phe508Gln The missense mutant proteins F508A, F508M,F508P,F508D,F508Q,F508R and F508S had similar ∆Gunfolding and m-values, 3.4-3.8 kcal mol-1 and 1.5-1.7 kcal mol-1 M-1 denaturant, respectively, highlighting the fact that changes in the bulk or chemical properties of the substituted side chain had little effect on the native-state stabilities of these domains as measured by denaturation with GuHCl (Table 1). Login to comment 46 ABCC7 p.Phe508Asp ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Trp ABCC7 p.Phe508Trp ABCC7 p.Phe508Met ABCC7 p.Phe508Pro ABCC7 p.Phe508Gln ABCC7 p.Trp496Phe How does the isolated NBD accommodate such Temperature (ºC) 4 10 16 22 Fractionalyield 0.0 0.5 1.0 Temperature (ºC) 4 10 16 22 Temperature (ºC) 4 10 16 22 Wild type ∆F508 Wild type ∆F508 ̄ F508A ̄ F508M F508P F508W ͷ F508W W496F Wild type ∆F508 F508Q F508R F508D F508S a b c Figure 1 NBD1 folding efficiency as a function of folding temperature. Login to comment 52 ABCC7 p.Phe508Trp ABCC7 p.Trp496Phe (b) The F508W mutant, the only mutant that deviated markedly from the wild type, was rescued by the introduction of a second missense mutation, W496F. Login to comment 53 ABCC7 p.Phe508Asp ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Met ABCC7 p.Phe508Pro ABCC7 p.Phe508Gln Table 1 Stability of wild-type and mutant NBD proteins Protein ∆Gunfolding ∆∆Gunfolding m-value (kcal mol-1) (kcal mol-1) (kcal mol-1 M-1) Wild type 3.7 ± 0.1 0 1.7 ∆F508 3.6 ± 0.1 0.1 1.7 F508A 3.6 ± 0.2 0.1 1.6 F508M 3.5 ± 0.1 0.1 1.6 F508P 3.5 ± 0.3 0.2 1.6 F508D 3.6 ± 0.1 0.1 1.6 F508Q 3.5 ± 0.2 0.2 1.6 F508R 3.4 ± 0.3 0.3 1.6 F508S 3.8 ± 0.2 -0.1 1.6 considerable changes in amino acid character at position 508 when this position is critical to the proper biogenesis of the full-length protein, and what are the underlying structural changes associated with these substitutions? Login to comment 54 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg NBD1 structure To directly assess the structural changes associated with the substitution of residues at position 508, crystal structures of two missense-mutant proteins were determined for the highly similar murine NBD1: F508S, a previously identified non-CF-causing variant, and F508R,a previously described maturation- deficientmutation.Theproteinswereexpressed and purified essentially as described for the wild-type protein and crystallized under conditions similar to the wild-type protein in the presence of Mg2+ andATP with sodium acetate as the precipitant26. Login to comment 55 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Tetragonal bipyramidal crystals grew for the F508R proteins, whereas the F508S protein spontaneously crystallized as large tetragonal plates. Login to comment 56 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg The F508S and F508R crystals diffracted to 2.7 and 3.1 Å,respectively and structures were determined with final R/Rfree valuesof 0.207/0.262and0.254/0.266, respectively (Table 2). Login to comment 59 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg All of the major structural elements are conserved and the r.m.s. deviations between the Cα atoms of the wild type and F508S or F508R structures were <0.33 Å (Fig.2a)26. Login to comment 63 ABCC7 p.Phe508Arg This rotation is observed in only one of two monomers in the asymmetric unit when the phenylalanine is replaced by the larger arginine side chain in the F508R structure (Fig.2c).In both mutants,like the wild type,the side chain of the residue at position 508 is largely surface-exposed and accessible (Fig.2d). Login to comment 67 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg The conformation of ATP in the wild-type26 and F508S structures is very similar, with a noncanonical interaction between the NBD and ATP and unusual torsional angles in the ATP molecule (SupplementaryFig.1online).In the F508R structure,the two monomers that occur in the asymmetric unit contain ATP in different conformations. Login to comment 68 ABCC7 p.Phe508Ser In one monomer, the ATP is bound in an orientation similar to that observed in the wild-type and F508S structures.The ATP bound to the other monomer,however,adopts a more conventional orientation (Supplementary Fig. 1 online). Login to comment 71 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Examination of the calculated molecular surface of the wild-type, F508S and F508R proteins is revealing. Login to comment 73 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Quantification of the surface-accessibility of position 508 reveals that the wild-type and F508S side chains are very similar at 8.5 and 9.6 Å2 respectively.The F508R protein has greater average accessible surface area at position 508, with a value of 16.8 Å2. Login to comment 78 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Wild type, green; F508S variant, orange; F508R variant, blue. Login to comment 79 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg The r.m.s. deviations between the wild-type and F508S or F508R structures are ~0.3 Å. Login to comment 84 ABCC7 p.Phe508Ser ABCC7 p.Phe508Ser The 2Fo - Fc electron density map (contoured at 1 σ) calculated with the F508S data at a resolution of 2.7 Å superposed on the final F508S model. Login to comment 85 ABCC7 p.Phe508Arg ABCC7 p.Phe508Arg (c) The 2Fo - Fc map (contoured at 1 σ) calculated from the F508R data at a resolution of 3.1 Å superposed on the final F508R model. Login to comment 90 ABCC7 p.Phe508Cys ABCC7 p.Phe508Met The steady-state band C levels of F508C and F508M were reduced, but closest to those of wild type. Login to comment 91 ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Gln ABCC7 p.Phe508Gly ABCC7 p.Phe508Leu ABCC7 p.Phe508Asn ABCC7 p.Phe508Thr ABCC7 p.Phe508Val Band C levels in F508A, F508G, F508L and F508V as well as the polar amino acid substitutions F508S, F508T, F508N and F508Q were evident, but substantially reduced relative to wild-type band C levels. Login to comment 92 ABCC7 p.Phe508Cys ABCC7 p.Phe508Asp ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Trp ABCC7 p.Phe508Glu ABCC7 p.Phe508His ABCC7 p.Phe508Ile ABCC7 p.Phe508Lys ABCC7 p.Phe508Tyr The known polymorphism F508C and the non-CF-causing variant F508S both showed measurable quantities of band C at steady-state levels, as would be expected for non-CF-causingsubstitutions.Thehydrophobicaminoacidsubstitutions F508I,F508W and F508Y did not produce substantial steady-state levels of band C as measured by western blotting, nor did the ionizable amino acid substitutions F508D, F508E, F508K, F508H or F508R. Login to comment 93 ABCC7 p.Phe508Pro No band C was seen for the F508P substitution.No endogenous CFTR was detected in HEK 293 cells transfected with a pCMV-GFP expression plasmid. Login to comment 101 ABCC7 p.Phe508Trp Notably, missense mutations other than F508W had little effect on either the folding or stability of the isolated NBD, suggesting that the peptide backbone at this locus is critical to NBD1 folding efficiency, whereas the side chain character is largely unimportant. Login to comment 103 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Most notably, the crystal structures of F508S and F508R both indicate that substitutions for Phe508 do not substantially impact the structure of NBD1,providing further evidence for the high tolerance for substitution at this position in the isolated domain. Login to comment 104 ABCC7 p.Phe508Trp ABCC7 p.Trp496Phe The structures of NBD1 proteins also suggest a potential mechanism for the deleterious effects of the F508W substitution,as the phenylalanine side chain, although partially surface-exposed and accessible, interacts with surrounding residues.The nearest atom distances from both Trp496 and Met498 to Phe508 are ~4 Å.The additional physical size of the tryptophan side chain thus may not be accommodated by the local protein structure.However,when a second substitution,W496F,was introduced, the folding of the domain was rescued.Given the close proximity of both residues,theW496F substitution probably resolves a steric clash between the substituted tryptophan at position 508 and other local residues,consistent with the refolded protein reaching a native or near-native-state structure in vitro. Login to comment 113 ABCC7 p.Phe508Cys ABCC7 p.Phe508Asp ABCC7 p.Phe508Ala ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Trp ABCC7 p.Phe508Met ABCC7 p.Phe508Pro ABCC7 p.Phe508Gln ABCC7 p.Phe508Gly ABCC7 p.Phe508Leu ABCC7 p.Phe508Asn ABCC7 p.Phe508Thr ABCC7 p.Phe508Val ABCC7 p.Phe508Glu ABCC7 p.Phe508His ABCC7 p.Phe508Ile ABCC7 p.Phe508Lys ABCC7 p.Phe508Tyr W ild type ∆∆F508 F508 F508D F508K F508E F508R F508H F508S F508T F508N F508Q C B Charged Polar F508A F508C F508I F508L ∆F508 F508 W ild type C B F508W F508Y F508G F508P Hydrophobic F508M F508V ̅̆ ̆ ̅ Figure 3 Maturation of full-length CFTR mutants. Login to comment 117 ABCC7 p.Phe508Ser (F508S has been associated with congenital bilateral absence of (the vas deferens, but not CF.) Login to comment 148 ABCC7 p.Arg553Gln ABCC7 p.His667Arg ABCC7 p.His667Arg ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Phe508Ala ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe409Leu ABCC7 p.Phe433Leu Note added in proof: Crystal structures of the human F508A missense NBD1 (with solublizing mutations F429S and H667R) and the corrected ∆F508 NBD1 (with three known suppressor mutations G550E, R553Q and R555K, and the solublizing mutations F409L, F429S, F433L and H667R) have been reported51. Login to comment 150 ABCC7 p.Phe508Ala The in vivo yield of soluble ∆F508 protein is decreased relative to both the wild-type and F508A proteins with both solublizing and suppressor mutations, consistent with a decrease in the efficiency of domain folding as described in this study. Login to comment 178 ABCC7 p.Phe508Arg The structure of F508R was determined using the molecular replacement protocols available in CNS version 1.1 (ref. 48). Login to comment 180 ABCC7 p.Phe508Ser The structure of F508S was determined using the mNBD1 structure (PDB entry 1R0W)26 stripped of water molecules, ions and ATP as the starting model. Login to comment 186 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg The atomic coordinates and structure factors for the F508S and F508R NBD1 structures have been deposited in the Protein Data Bank (accession codes 1XF9 and 1XFA, respectively). Login to comment 188 ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg Table 2 Data collection and refinement statistics F508S F508R Data collection Space group P4212 I4122 Cell dimensions (Å) a 170.45 139.99 b 170.45 139.99 c 109.07 278.72 Resolution (Å) 40.4-2.7 (2.75-2.7) 44.3-3.1 (3.15-3.1) Rsym 0.078 (0.431) 0.071 (0.987) I / σI 22.9 (2.3) 34.8 (2.5) Completeness (%) 98.8 (95.6) 99.9 (100) Redundancy 7.7 (3.3) 11.9 (10.7) Refinement Resolution (Å) 40.4-2.7 44.3-3.1 No. Login to comment 336 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Xiong, X., Bragin, A., Widdicombe, J.H., Cohn, J. & Skach, W.R. Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. Login to comment 337 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Xiong, X., Bragin, A., Widdicombe, J.H., Cohn, J. & Skach, W.R. Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator. J. Clin. 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