ABCMdb

PMID: 24058550

Dawson JE, Farber PJ, Forman-Kay JD
Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR.
PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Login to comment 6 ABCC7 p.Gln637Arg Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. Login to comment 25 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg The maturation defects can be partially suppressed by mutations in NBD1 or the CL4 coupling helix, such as V510D and F494N/Q637R [11,16-21], and by the drug VX-809 [22,23], now in clinical trials. Login to comment 27 ABCC7 p.Phe494Asn Many of the F508del-suppressor mutations, such as F494N, as well as the RI deletion, increase the solubility of the NBD1 domain [20,24]. Login to comment 29 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Both the solubilizing double mutant F494N/Q637R and the deletion of the RI increase the locked open time of WT and F508del CFTR binding pyrophosphate [27]. Login to comment 46 ABCC7 p.Gln637Arg A mutation between C-terminal helices H8 and H9, Q637R, alters 15 N-1 H NMR HSQC spectral peak intensities that correspond to residues in the CL4-binding and RI-deletion sites, consistent with a change in dynamics within the spatially remote regions. Login to comment 50 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Similar titration patterns were observed in NBD1 constructs containing Q637R and mutations near the CL4-binding site (F508del, F494N, and V510D), as well as in a CL4-NBD1 fusion. Login to comment 56 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg Mutant NBD1 constructs with F494N, F508del, V510D, Q637R or F494N/ Q637R were constructed with a Stratagene QuikChange site-directed mutagenesis kit. Login to comment 74 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg (The 1.0 mM F494N and F494N/Q637R NBD1 samples used 50 mM sodium phosphate to maintain consistent pH conditions.) Login to comment 77 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg 1.0 mM F494N and F494N/Q637R NBD1 samples were used for peak intensity comparison. Login to comment 86 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg TROSY 15 N-1 H HSQC [40], T1 [41], and T1r [41] data were collected on 1 mM F494N, Q637R, and F494N/Q637R NBD1 samples using a cryoprobe-equipped 600 MHz Varian spectrometer. Login to comment 100 ABCC7 p.Gln637Arg Results Q637R Mutation Alters Protein Dynamics in the RI-deletion Site and CL4-binding Site In order to probe the molecular mechanism of allostery in CFTR NBD1, we monitored effects on NMR spectra of NBD1 DRI DRE (residues 387-646, D405-436) upon perturbation by mutagenesis and ligand binding. Login to comment 115 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Q637R is a solubilizing mutant that, in combination with F494N, partially corrects the folding defects induced by F508del in CFTR. Login to comment 116 ABCC7 p.Gln637Arg Like WT NBD1, Q637R NBD1 has heterogeneous peak lineshapes (Figure 2A). Login to comment 119 ABCC7 p.Gln637Arg At 1 mM, WT and Q637R NBD1 tumble in solution like objects that are larger than a monomer, with a rotational correlation time of tc = 2765 ns (A. Chong, unpublished data) and 2764 ns, respectively. Login to comment 121 ABCC7 p.Phe494Asn The solubilizing mutation, F494N, reduces this association tendency. Login to comment 122 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Both F494N NDB1 and F494N/Q637R NBD1 have a rotational correlation time of 2262 ns. Login to comment 124 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg The experimental equivalence of F494N and F494N/Q637R NBD1 tc values reduces the probability that any difference in lineshape may be caused by a difference in association states. Login to comment 125 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Both decreases and increases in peak intensity, quantified by ratios of the intensities of the F494N/Q637R NBD1 and F494N NBD1 resonances (Figure 2B), are indicative of changes in dynamics. Login to comment 127 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg y~ I(F494N Q637R) I(F494N) {1 &#f0;2&#de; These changes in intensity point to the Q637R mutation affecting the dynamics of F494N NBD1 in multiple locations. Login to comment 128 ABCC7 p.Gln637Arg The site of the Q637R mutations is located between the C-terminal helices H8 and H9 (residues 630-646), which are adjacent to a sheet containing b-strands S3/S9/S10 (residues 453-458, 615-629). Login to comment 136 ABCC7 p.Gln637Arg While the Q637R mutation only changes the chemical shifts of neighboring residues (Figure S2), it perturbs the intensities of resonances in the C-terminal site nearby the mutation position, as well as for remote peaks near the site of the RI deletion and for some residues within the predicted CL4-binding site [8-10] (Figure 1A). Login to comment 144 ABCC7 p.Gln637Arg Change in peak intensity due to H8/H9 mutation Q637R. Login to comment 146 ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Overlay of F494N (black) and F494N/Q637R (orange) NBD1 HSQC spectra. B. Login to comment 147 ABCC7 p.Gln637Arg Ratio of peak intensities for NBD1 containing the Q637R mutation to those of NBD1 without this mutation, reflective of changes in dynamics, as a function of residue. Login to comment 154 ABCC7 p.Gln637Arg Dynamics changes (absolute value of the intensity ratio minus one) due to Q637R mapped onto the structure of NBD1, highlighting long-range effects in the RI-deletion site and CL4-binding site. Login to comment 155 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Q637R effects were probed using F494N NBD1 for improved solubility and spectral quality. Login to comment 160 ABCC7 p.Gln637Arg Q637R, located between C-terminal helices H8 and H9, is marked with a red star due to lack of electron density in the structure. Login to comment 175 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg CL4 also binds to NBD1 proteins containing single F508del-suppressor mutations, V510D and F494N, located near the predicted CL4-binding site, and Q637R, which lies between H8 and H9 (Figures 4 and S4). Login to comment 178 ABCC7 p.Val510Asp In contrast, V510D NBD1 has greater chemical shift changes in the CL4-binding site upon CL4 titration than WT NBD1, relative to changes in other residues (Figures 4E and 4F). Login to comment 179 ABCC7 p.Gln637Arg Other differences are due to the lack of assignment for some residues near the site of the Q637R mutation (Figures 4G and 4H) and due to low spectral signal-to-noise. Login to comment 201 ABCC7 p.Val510Asp Correlation Analysis of Titration Data Improves the Detection of Weak but Significant Changes due to CL4 Binding CL4 binding causes relatively small Dvobs, the greatest of which is less than 0.14 ppm (Figure 4E; binding to V510D NBD1). Login to comment 218 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg A, C, E, G. Dvobs between spectra of apo and CL4 peptide:NBD1 are shown in green for F508del, F494N, V510D, and Q637R NBD1, respectively. Login to comment 220 ABCC7 p.Gln637Arg Purple triangles in chart G indicate residues that could not be assigned for Q637R NBD1. Login to comment 221 ABCC7 p.Gln637Arg The CL4:NBD1 proportions are 10:1 for Q637R NBD1 and its CL4:WT NBD1 control, but is 12.5:1 for the rest of the data. Login to comment 223 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Dvobs values are mapped onto NBD1 structure as in Figure 2: B. F508del, D. F494N, F. V510D, and H. Q637R. Login to comment 224 ABCC7 p.Phe494Asn F508del (position of F508 indicated as black sticks), F494N (orange space-filling representation), and V510D (purple space-filling representation) are near the predicted CL4-binding site. Login to comment 225 ABCC7 p.Gln637Arg Q637R (red star) is between C-terminal helices H8 and H9. Login to comment 265 ABCC7 p.Asn1303Gln ABCC7 p.Arg1358Ala ABCC7 p.Phe1296Ser ATP-independent channel opening has been enhanced by Cys, Ser, and Pro mutations of K978 in the ICDs [15] and F1296S/N1303Q and R1358A in NBD2 [60]. Login to comment 298 ABCC7 p.Gln637Arg We found evidence for an allosteric network revealed by both the Q637R mutation and CL4 peptide binding to NBD1 that connects the CL4-binding site predicted in CFTR homology models to two regulatory sites in NBD1, near to the RI and to where the RE/R regions are located in the full-length channel. Login to comment 299 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Chemical shift changes upon CL4 binding provide evidence for this allosteric network observed with minor variations in five variants of isolated NBD1-WT, F508del, F494N, V510D, and Q637R, in a CL4-NBD1 fusion protein with slightly different CL4 boundaries and in different buffer conditions. Login to comment 309 ABCC7 p.Ile539Thr ABCC7 p.Val510Asp ABCC7 p.Gln637Arg The mutations that can partially suppress the folding defects of F508del NBD1 are scattered across NBD1- V510D near CL4 in the channel, I539T directly opposite the NBD1:NBD2 interface, and Q637R near the start of the R region [12], hinting at the complex allosteric nature of the NBD1 folding landscape. Login to comment 310 ABCC7 p.Gly1069Arg ABCC7 p.Ser492Phe As with protein folding, mutations such as S492F and F508del in NBD1 and G1069R in the CL4 coupling helix perturb the gating of the channel [6,7,75]. Login to comment 312 ABCC7 p.Gln637Arg The deletion site is perturbed by both CL4 binding and the Q637R mutation between C-terminal helices H8 and H9. Login to comment 313 ABCC7 p.Gln637Arg The heterogeneous lineshapes observed in WT and Q637R NBD1 spectra indicate that there is exchange between conformations at different timescales. Login to comment 314 ABCC7 p.Gln637Arg The Q637R mutation has far-reaching effects on the dynamics within the ensemble, altering dynamics in several distal locations, including in the RI-deletion site and in the CL4-binding site. Login to comment 325 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg F508del suppressor modifications are found in this region as well, including the substitution of mouse RE into human CFTR [77] and Q637R, which in combination with F494N helps suppress folding defects in CFTR [20]. Login to comment 326 ABCC7 p.Gln637Arg The compound binding also caused small chemical shift changes in the CL4-binding site [35], as expected for sites that are allosterically linked, demonstrating the reciprocal nature of the linkage and supporting our observation that a mutation between H8 and H9, Q637R, changes the dynamics of NBD1 residues at the CL4-binding site, as well as the RI-deletion site. Login to comment 337 ABCC7 p.Gln637Arg (TIF) Figure S2 The effects of Q637R on NBD1 chemical shifts. Login to comment 338 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Chemical shift changes in F494N NBD1 due to H8/H9 mutation Q637R limited to neighboring residues in S3/S9/S10 and H8/H9. Login to comment 339 ABCC7 p.Phe494Asn The F494N mutation was used to improve solubility. Login to comment 357 ABCC7 p.Val510Asp ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Overlay of apo (black) and 12.5:1 CL4:NBD1 (red) spectra for A. F508del NBD1, B. F494N NBD1, C. V510D NBD1, and D. Q637R NBD1 mutants. Login to comment