ABCMdb

PMID: 15209503

Choi MY, Cardarelli L, Therien AG, Deber CM
Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations.
Biochemistry. 2004 Jun 29;43(25):8077-83., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Val232Asp Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G194LALAHFVWIAPLQ207VALLMGLIWELLQASAFAGLGFLIV232LALFQ237AGLG241) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. Login to comment 28 ABCC7 p.Val232Asp 1 Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; TM, transmembrane; VD, V232D; wt, wild type; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; CD, circular dichroism; H-bond, hydrogen bond; RP-HPLC, reversed-phase high-performance liquid chromatography; LB, Luria-Bertani; MW, molecular weight; TFE, trifluoroethanol. Login to comment 33 ABCC7 p.Val232Asp With the use of this "gel-shift assay" on SDS-PAGE, we observed that the construct derived from the phenotypic mutant Val232-Aspswhich induces a mild form of CFsmigrated significantly faster than the wt construct (22), indicating that V232D adopts a more compact conformation likely imposed by a tight interaction between the mutant Asp 232 in TM helix 4 and its polar partner Gln 207 in TM helix 3 (22). Login to comment 81 ABCC7 p.Val232Asp Previous studies have shown that the V232D mutant migrates at a rate faster than that of the wt in NuPAGE MES gels (22). Login to comment 82 ABCC7 p.Val232Asp ABCC7 p.Val232Asp This migration pattern was shown to be associated with a more compact conformation of the V232D construct, imposed by a tight interaction between polar partners Asp 232 and Gln 207 of the V232D TM3/4 construct. Login to comment 87 ABCC7 p.Ile231Asp When these results are quantitated for the full TM4 D mutant library (Figure 3b), we find that the I231D construct has the largest negative percentage molecular weight decrease relative to the wt (-11.65% ( 0.61%), and hence migrated the furthest among all the mutants in TM4 on SDS-PAGE. Login to comment 89 ABCC7 p.Val232Asp ABCC7 p.Ile231Asp ABCC7 p.Phe236Asp ABCC7 p.Leu233Asp ABCC7 p.Gly228Asp Interestingly, mutants G228D through I231D, and L233D through F236D, have a more negative value of the percent of MW decrease relative to the wt than the CF-phenotypic mutant V232D (Figure 3b) (see the Discussion). Login to comment 90 ABCC7 p.Val232Asp The remaining Asp mutants toward either terminus of the TM4 helix migrated slower than the V232D construct on SDS-PAGE. Login to comment 97 ABCC7 p.Val232Asp V232D is the original CF-phenotypic mutant studied by Therien et al. (22). Login to comment 104 ABCC7 p.Val232Asp V232D is the CF-phenotypic mutant in TM4. Login to comment 111 ABCC7 p.Val232Asp Double asterisks denote values statistically different from that of the construct V232D on SDS-PAGE gel (p < 0.01). Login to comment 112 ABCC7 p.Gln207Leu To confirm the role of Q207 as the "unique" polar partner for Asp side chains along TM4, we prepared a library of 21 TM3/4 mutants designated XD/Q207L, which has the same net charge as XD (where X is any positional D mutant in TM4) but eliminates Q207 as a possible polar partner for D mutants in TM4. Login to comment 113 ABCC7 p.Gln207Leu These mutants were found uniformly to display the same migration rate as the wt, as shown for selected XD/Q207L constructs in Figure 4. Login to comment 118 ABCC7 p.Gln237Leu Figure 5 displays the comparison of the migration differences on SDS-PAGE between six X f D mutants and their Q237L counterparts in the CFTR TM3/4 construct. Login to comment 119 ABCC7 p.Gln237Leu ABCC7 p.Gln237Leu ABCC7 p.Gly239Asp ABCC7 p.Phe224Asp ABCC7 p.Ala238Asp Our results indicate a discrete role for Q237, viz., the double mutants F224D/ Q237L, A238D/Q237L and G239D/Q237Lsin which the D locus is nearer to the Nor C-terminus of the TM4 helixs returned to the wt migration rate. Login to comment 120 ABCC7 p.Val232Asp ABCC7 p.Gln237Leu ABCC7 p.Gln237Leu ABCC7 p.Leu230Asp D mutants in mid-TM4, i.e., V232D/Q237L and L230D/Q237L, continued to migrate faster than the wt, but were clearly retarded vs their D counterparts. Login to comment 121 ABCC7 p.Val232Asp DISCUSSION In a helical hairpin construct derived from the first transmembrane domain of CFTR, the CF-phenotypic mutant V232D in TM4 was previously found to form a non-native side chain-side chain H-bond with the wild-type residue Q207 in TM3 (22) which was proposed to be responsible, in part, for the disruption of the functional CFTR protein. Login to comment 131 ABCC7 p.Ile231Asp Molecular Modeling Studies on the I231D Construct. Login to comment 137 ABCC7 p.Gln207Leu Our observations that interhelical H-bonds are strong determinants of helical hairpin folding can be viewed in the context of studies by Faham et al. which indicate that TM-TM packing forces are dominant over H-bonds in a series FIGURE 4: SDS-PAGE analysis for selected knockout XD/Q207L mutants. Login to comment 138 ABCC7 p.Gln207Leu The D mutants in TM4 migrate faster than the wild-type construct, while the knockout mutants (with Q207L) retain the wt migration rate due to the fact that the polar partner in TM3 has been eliminated. Login to comment 140 ABCC7 p.Gln207Leu All 21 TM4 D mutants with Q207L retained the wt migration rate. Login to comment 141 ABCC7 p.Gln237Leu FIGURE 5: Comparison of relative migration rates on SDS-PAGE (NuPAGE MES gel system) for X f D mutants and selected XD/ Q237L mutants in CFTR TM3/4 constructs. Login to comment 162 ABCC7 p.Ile231Asp ABCC7 p.Ile231Asp ABCC7 p.Gly241Asp Thus, for example, if one assumes that I231D in TM4 forms the closest contacts with Q207 in TM3, there are 10 residues ()2.7 turns of helix) from I231D for G241D. Login to comment 165 ABCC7 p.Gly241Asp Thus, the total vertical distance for the formation of an H-bond between Q207 in TM3 and G241D in TM4 will be about 15 Å (7.5 Å between the main chain carbon and the carboxamide in Q207 + 3 Å (average distance for H-bond formation) + 4.5 Å between the main chain carbon and the Asp carboxylate). Login to comment 166 ABCC7 p.Gly241Asp Using standard side chain torsional angles, models indicate that such H-bond formation is physically realistic between Q207 in TM3 and G241D in TM4. Login to comment 170 ABCC7 p.Ile231Asp The present model TM3/4 systems also assume that sequentially consecutive helices will be adjacent and aligned in the native proteins FIGURE 6: Energy-minimized structural models of antiparallel CFTR transmembrane helical segments 3 and 4 in the I231D mutant. Login to comment 171 ABCC7 p.Ile231Asp Space-filling model of the I231D helical hairpin (intervening loop omitted). Login to comment 173 ABCC7 p.Ile231Asp (a) View of the I231D model showing the side chain carboxamide from Q207 in TM3 interacting with the side chain carboxylate of D231 in TM4. Login to comment 178 ABCC7 p.Val232Asp ABCC7 p.Gln237Leu ABCC7 p.Gln237Leu ABCC7 p.Gln237Leu ABCC7 p.Gln237Leu ABCC7 p.Gln237Leu ABCC7 p.Gly239Asp ABCC7 p.Phe224Asp ABCC7 p.Ala238Asp ABCC7 p.Leu230Asp From the results (Figures 5 and 7), we found that double mutants where D is relatively distant from Q207 in TM3 (F224D/ Q237L, A238D/Q237L, and G239D/Q237L) returned to the wt migration rate, whereas the other double mutants (V232D/ Q237L and L230D/Q237L) migrated more slowly than the corresponding D mutants, but still significantly faster than the wt construct. Login to comment 180 ABCC7 p.Gly241Asp ABCC7 p.Gly228Asp ABCC7 p.Gln237Asp ABCC7 p.Ala221Asp Therefore, without Q237 (i.e., with Q237 mutated to Leu), the X f D mutants in TM4 become more open through regions encompassing A221D to G228D, and Q237D to G241D, and are ultimately unable to form an H-bond with Q207. Login to comment 182 ABCC7 p.Val232Asp The CF-phenotypic Mutant V232D Is Not the Optimal Position for Interhelix H-Bond Formation. Login to comment 183 ABCC7 p.Val232Asp As elaborated in Figure 3b, the CF-phenotypic mutant V232D does not form the tightest H-bond with Q207 in TM3 in comparison to the several X f D mutants on TM4. Login to comment 184 ABCC7 p.Val232Asp In fact, the migration rate of V232D is statistically slower vs those of D mutants at 229, 230, 231, and 233 (double asterisks, Figure 3b). Login to comment 185 ABCC7 p.Ile231Asp In fact, the I231D construct has the greatest percentage molecular weight decrease relative to the wt, and ostensibly forms the tightest H-bond with Q207 (illustrated with energy-minimized helical dimers in Figure 6). Login to comment 186 ABCC7 p.Val232Asp As V232D is the CF-phenotypic mutant in this series, this result initially appeared counterintuitive. Login to comment 188 ABCC7 p.Gly241Asp ABCC7 p.Gly228Asp ABCC7 p.Gly239Asp ABCC7 p.Ala238Asp ABCC7 p.Ala221Asp ABCC7 p.Ala234Asp ABCC7 p.Ala223Asp ABCC7 p.Gly226Asp Nevertheless, for any A f D or G f D mutants (in this case, A221D, A223D, G226D, G228D, A234D, A238D, G239D, and G241D), only a single-base change is required, and therefore, it is possible these mutants represent potential phenotypic CF mutants, which have yet to be discovered. Login to comment 189 ABCC7 p.Val232Asp ABCC7 p.Gly226Asp ABCC7 p.Ser222Asp It is also interesting to note that several mutantssincluding S222D, G226D, V232D, and Q237Dsdisplay locally slower migration rates. Login to comment 191 ABCC7 p.Val232Asp One might speculate that this is the case for CF-phenotypic mutant V232D, and therefore, its capture by Q207 necessitates major reorientation of the wild-type TM4 axissand accordingly the greatest disruption of the native folded domain structure. Login to comment