ABCMdb

PMID: 26149808

Chong PA, Farber PJ, Vernon RM, Hudson RP, Mittermaier AK, Forman-Kay JD
Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.
J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Ile539Thr Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Login to comment 27 ABCC7 p.Ile539Thr WT, I539T and F508del NBD1 assignments have been deposited in the BMRB. Login to comment 38 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Evidence that NBD1 destabilization is problematic for proper processing was provided by NBD1-thermostabilizing mutations distant from the F508del site, including G550E, R553Q, R555K, and deletion of the RI. Login to comment 47 ABCC7 p.Ile539Thr Completion of b0e;82% of assignments for WT, F508del, and I539T NBD1 èc;RIèc;RE allowed extensive characterization of NBD1. Login to comment 50 ABCC7 p.Ile539Thr Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that isolated F508del NBD1 èc;RIèc;RE tumbles more rapidly in solution than WT or I539T NBD1 èc;RIèc;RE. Login to comment 55 ABCC7 p.Ile539Thr Experimental Procedures Protein Expression and Purification-WT, F508del, and I539T variants of human NBD1 èc;RIèc;RE (387-646, èc;405-436) were expressed and purified as described previously (32). Login to comment 56 ABCC7 p.Cys592Val ABCC7 p.Cys590Val ABCC7 p.Glu402Cys ABCC7 p.Cys491Val ABCC7 p.Cys524Thr A single cysteine mutant of NBD1, E402C NBD1 èc;RIèc;RE, was generated on a Cys-less NBD1 (C491V, C524T, C590V, and C592V) background by site-directed mutagenesis and confirmed by DNA sequencing. Login to comment 60 ABCC7 p.Glu402Cys The tempo-maleimide spin label was conjugated to E402C, by first reducing the cysteine with 5 mM of freshly added DTT followed by extensive buffer exchange into a buffer with no DTT and subsequent incubation with a 5-fold excess of tempo-maleimide. Login to comment 71 ABCC7 p.Glu402Cys Paramagnetic relaxation enhancement experiments were measured using standard HSQC experiments on a sample containing 525 òe;M tempo-maleimide-labeled natural abundance E402C NBD1 èc;RIèc;RE and 175 òe;M 15 N-labeled WT NBD1 èc;RIèc;RE in the absence of reductant. Login to comment 105 ABCC7 p.Ile539Thr NBD1 èc;RIèc;RE with the I539T-stabilizing mutation (23) was assigned first, with 91% completion of backbone 15 N, 13 Cb18;, and 1 HN resonances. Login to comment 106 ABCC7 p.Ile539Thr Full assignment experiments were also recorded for WT and, together with the I539T assignments, enabled us to complete assignment of 89% of backbone 15 N, 13 Cb18;, and 1 HN resonances for WT. Login to comment 108 ABCC7 p.Ile539Thr Comparison of WT, I539T, and F508del Spectra-Human NBD1 èc;RIèc;RE spectra, while significantly better than full-length mouse NBD1 spectra with the RI (13), still exhibit inhomogeneous peak intensities (Fig. 1). Login to comment 121 ABCC7 p.Ile539Thr Comparison of WT, I539T, and F508del demonstrates overlapping unassigned regions clustered on or near the ॷ-subdomain, including most of the Q-loop, portions of helix 5 (H5), and the adjacent ABC signature sequence and residues immediately following the Walker B motif (Fig. 2). Login to comment 126 ABCC7 p.Ile539Thr Comparison of WT, I539T, and F508del spectra demonstrate that the two mutations do not cause any major structural changes. Login to comment 128 ABCC7 p.Ile539Thr We used the set of I539T chemical shift assignments for our prediction, because it was the most complete. Login to comment 136 ABCC7 p.Ile539Thr FIGURE 2. a-c, ribbon diagrams of WT, I539T, and F508del NBD1 èc;RIèc;RE. Login to comment 144 ABCC7 p.Ile539Thr F508del Increases Exchange and Reduces Dimerization 22866 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 38ߦSEPTEMBER 18, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 4, The similarity between WT, I539T, and F508del spectra strongly supports the conclusion that all share the same fold. Login to comment 152 ABCC7 p.Ile539Thr NBD1 15 N Relaxation Studies-Because changes in NBD1 thermostability underlie F508del defects, we probed the changes in dynamics resulting either from deletion of Phe-508 or the I539T stabilizing mutation. Login to comment 153 ABCC7 p.Ile539Thr We measured T1, T2, and heteronuclear NOE values, which are responsive to fast time scale (nanosecond to picosecond) motions, for WT, I539T, and F508del NBD1 èc;RIèc;RE. Login to comment 155 ABCC7 p.Ile539Thr Plot of TALOSd19; chemical shift derived secondary structure for I539T NBD1 èc;RIèc;RE as a function of residue number. Login to comment 166 ABCC7 p.Ile539Thr In contrast, the WT and I539T protein appear to be in exchange between the monomeric form and dimeric or higher order oligomeric forms resulting in higher ঄c values. Login to comment 170 ABCC7 p.Ile539Thr The ঄c differences also complicate the quantitative interpretation of T1 and T2 in terms of fast time scale dynamics because it is likely that the WT and I539T samples, in particular, contain a mixture of monomers, dimers, and possibly higher order oligomers. Login to comment 190 ABCC7 p.Ile539Thr Variant Sample concentration ঄c mM ns Predicted for monomer 16-20 Predicted for dimer 32-38 WT 0.6 29.4 afe; 2.5 F508del 0.9 19.9 afe; 1.1 I539T 1.5 27.3 afe; 1.5 F508del Increases Exchange and Reduces Dimerization 22868 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 38ߦSEPTEMBER 18, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , 547) (Fig. ). Login to comment 211 ABCC7 p.Ile539Thr 87ób;H) ribbon diagram plots for WT (a), F508del (b), and I539T NBD1 èc;RIèc;RE (c) are shown. Login to comment 213 ABCC7 p.Ile539Thr d, spectral density functions at three different frequencies as a function of NBD1 residue number for WT (green), F508del (red), and I539T (blue). Login to comment 218 ABCC7 p.Tyr577Lys b, overlay of spectra of Y577K NBD1 èc;RIèc;RE recorded at 1.7 mM (black) and 0.1 mM (red). Login to comment 220 ABCC7 p.Tyr577Glu c, overlay of spectra of Y577E NBD1 èc;RIèc;RE recorded at 1.9 mM (black) and 0.1 mM (red). Login to comment 230 ABCC7 p.Ile539Thr Notably, for I539T NBD1 èc;RIèc;RE, similar shift perturbations were confirmed for the indole of Trp-496 and the amides of Cys-491, Leu-571, and Glu-403 (Fig. 6e). Login to comment 231 ABCC7 p.Ile539Thr For both WT and I539T, these chemical shift perturbations are modulated by concentration causing the peaks to "move" along a straight vector across the concentration series. Login to comment 234 ABCC7 p.Tyr577Lys Peaks corresponding to the amide groups of Gly-486, Cys-491, Gly-509, and Gly-551 and the Trp-496 indole NঈH, which have the largest concentration-dependent chemical shift perturbations in the WT, become invisible at high concentrations and nearly so at low concentrations for the Y577K NBD1 mutant (Fig. 6b). Login to comment 235 ABCC7 p.Tyr577Lys Although the effect of Y577K on the amount of dimer is unclear from these experiments, this mutation in the dimer interface has a pronounced effect (i.e. extensive broadening) on Gly-486, Cys-491, Trp-496, and Gly-509 confirming that it is reasonable to interpret the concentration-dependent chemical shift perturbations at these residues in the WT as a response to dimerization at the expected interface. Login to comment 236 ABCC7 p.Tyr577Glu Interestingly, a Y577E mutant did not result in significant broadening of Cys-491 and Trp-496 resonances relative to WT or to significant concentration-dependent chemical shift perturbations (Fig. 6c). Login to comment 238 ABCC7 p.Tyr577Glu Visual inspection indicates changes in linewidth and relative peak intensity throughout the Y577E spectra at 0.1 and 1.9 mM, strongly hinting that significant dimerization is occurring in this mutant. Login to comment 239 ABCC7 p.Tyr577Lys Combined with observations seen in Y577K, these data indicate that chemical shift perturbations at Ser-485, Gly-486, Cys-491, Trp-496, and Gly-509 are linked to changes at the presumed heterodimer interface. Login to comment 244 ABCC7 p.Arg487Ser To determine whether we were observing intermolecular interactions between Arg-487 and Asp-565 and Asp-567, which form the di-acidic motif, we introduced the R487S mutation to remove the positive charge and disrupt this interface. Login to comment 245 ABCC7 p.Arg487Ser R487S NBD1 èc;RIèc;RE still exhibits FIGURE 7. a, mapping of the concentration-dependent chemical shift perturbations for NBD1 on a ribbon diagram of NBD1 using spheres colored according to the legend. Login to comment 268 ABCC7 p.Tyr577Lys ABCC7 p.Tyr577Glu Mutations at the dimer interface (Y577K and Y577E) also affect the Q-loop segment conformational sampling. Login to comment 284 ABCC7 p.Cys592Val ABCC7 p.Cys590Val ABCC7 p.Glu402Cys ABCC7 p.Cys491Val ABCC7 p.Cys524Thr The single cysteine residue, E402C, was introduced on a Cys-less NBD1 èc;RIèc;RE (C491V, C524T, C590V, and C592V). Login to comment 287 ABCC7 p.Glu402Cys E402C NBD1 èc;RIèc;RE could be expressed and purified in the amounts required for NMR. Login to comment 288 ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser ABCC7 p.Cys592Val ABCC7 p.Cys590Val Of note, we were not able to express or purify NBD1 using the previously published mutations C491S, C524S, C590V, and C592V (12), probably because these mutations destabilize NBD1. Login to comment 289 ABCC7 p.Glu402Cys To ensure that E402C NBD1 èc;RIèc;RE is folded similarly to WT NBD1 èc;RIèc;RE, spectra of the mutant were recorded and compared with the WT (Fig. 9a). Login to comment 295 ABCC7 p.Glu402Cys c, overlay of spectra of 15 N WT NBD1 èc;RIèc;RE recorded in the presence of a 3-fold excess of isotopically natural abundance tempo-maleimide-labeled E402C NBD1 èc;RIèc;RE recorded before (red) and after (black) reduction of the spin label with TCEP. Login to comment 296 ABCC7 p.Glu402Cys d, ribbon diagram of an NBD1 dimer with 15 N WT NBD1 èc;RIèc;RE (teal) bound to E402C WT NBD1 èc;RIèc;RE (gray, not observable in this experiment). Login to comment 303 ABCC7 p.Glu402Cys F508del Increases Exchange and Reduces Dimerization SEPTEMBER 18, 2015ߦVOLUME 290ߦNUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 22873 E402C NBD1 èc;RIèc;RE. Login to comment 307 ABCC7 p.Glu402Cys To detect dimers, a sample containing 525 òe;M tempo-maleimide-labeled, isotopically natural abundance E402C NBD1 èc;RIèc;RE and 175 òe;M 15 N-labeled WT NBD1 èc;RIèc;RE was prepared. Login to comment 319 ABCC7 p.Glu402Cys Note that no broadening is expected or observed near Cys-402, because the tempo-maleimide-labeled E402C NBD1 èc;RIèc;RE is not 15 N-labeled and thus not visible in these spectra. Login to comment 334 ABCC7 p.Tyr577Lys Evidence supporting the connection between dimerization and the Phe-508 position comes from the large concentration-dependent chemical shift change of the adjacent residue Gly-509 (b03;30 Hz) seen in WT spectra (Fig. 6a) and the concentration-dependent broadening of this signal in the Y577K spectra (Fig. 6b). Login to comment 336 ABCC7 p.Phe508Trp Collectively, the data indicate that F508del reduces the ability of NBD1 èc;RIèc;RE to homodimerize, probably by disrupting the Phe-508/Trp-496 side chain interaction that stabilizes the Q-loop. Login to comment 362 ABCC7 p.Asn1303Lys Supporting this contention, at least two mutations in NBD2 (N1303K, which interacts with the Q-loop of NBD2, and p.Ile1234_Arg1239del) are known to reduce the amount of mature CFTR (52, 57). Login to comment 363 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Interestingly, the combined suppressor mutations I539T, G550E, R553M, and R555K have a bigger positive effect on F508del CFTR when NBD2 is present (58), suggesting the importance of the NBD interaction and hinting that these NBD1-stabilizing mutations may also improve the ability of F508del NBD1 to dimerize with NBD2. Login to comment 364 ABCC7 p.Gly550Glu Structural models have led to the prediction that the suppressor mutation G550E enhances dimerization through an electrostatic interaction with basic surfaces on NBD2 (59). Login to comment 370 ABCC7 p.Ile539Thr This conclusion is strongly supported by our NMR structural data, as well as studies of fast time scale dynamics in NBD1 èc;RIèc;RE indicating that overall patterns of flexibility are shared by the ground states of WT, F508del, and I539T NBD1. Login to comment 373 ABCC7 p.Tyr577Lys ABCC7 p.Tyr577Glu Furthermore, mutations in the dimer interface (Y577K and Y577E) clearly alter the Q-loop segment equilibrium, with changes observed as far away from the dimer interface as Gly-509, which is adjacent to the Phe-508 position. Login to comment 386 ABCC7 p.Ser492Pro ABCC7 p.Ser495Pro Other published Q-loop segment suppressor mutations such as S492P and S495P (21, 29) are also likely to modulate NBD dimerization. Login to comment