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Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

Szollosi A, Muallem DR, Csanády L, Vergani P - J. Gen. Physiol. (2011)

Bottom Line: Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates.Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle.These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

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The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Solid blue and green lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-H1348 built on nonhydrolytic closing rates (1/τrelaxation). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/H1348A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on Keq = Po/(1−Po) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.
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fig7: The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Solid blue and green lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-H1348 built on nonhydrolytic closing rates (1/τrelaxation). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/H1348A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on Keq = Po/(1−Po) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.

Mentions: Following a similar methodology, we proceeded to study changes in coupling between positions 460 and 1348 during gating, using perturbations T460S and H1348A. In these mutant cycles, two of the corners (WT and single-mutant T460S) are identical to the corresponding corners of the respective T460-L1353 mutant cycle. To rigorously compare the effects of the H1348A mutation onto the T460S versus WT backgrounds, the gating parameters for the latter two constructs should have been repeatedly measured in experiments side by side with those conducted on H1348A and T460S/H1348A. However, because ΔΔGint can be calculated using any two parallel sides of a mutant cycle, we did not repeat experiments for WT and T460S; instead, we calculated ΔΔGint using the two horizontal sides of each cycle, i.e., by comparing the effects of the T460S mutation onto the H1348A versus WT backgrounds. For this reason, we refrain from providing absolute ΔG values for the vertical sides of the T460-H1348 mutant cycles (Figs. 6, B and D, and 7, B and D); and the same applies for the T460-H1375 mutant cycles (see below; Figs. 8, B and D, and 9, B and D).


Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

Szollosi A, Muallem DR, Csanády L, Vergani P - J. Gen. Physiol. (2011)

The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Solid blue and green lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-H1348 built on nonhydrolytic closing rates (1/τrelaxation). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/H1348A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on Keq = Po/(1−Po) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.
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fig7: The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Solid blue and green lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-H1348 built on nonhydrolytic closing rates (1/τrelaxation). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/H1348A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on Keq = Po/(1−Po) values under nonhydrolytic conditions. The top two corners of the mutant cycle were taken from Fig. 5 D.
Mentions: Following a similar methodology, we proceeded to study changes in coupling between positions 460 and 1348 during gating, using perturbations T460S and H1348A. In these mutant cycles, two of the corners (WT and single-mutant T460S) are identical to the corresponding corners of the respective T460-L1353 mutant cycle. To rigorously compare the effects of the H1348A mutation onto the T460S versus WT backgrounds, the gating parameters for the latter two constructs should have been repeatedly measured in experiments side by side with those conducted on H1348A and T460S/H1348A. However, because ΔΔGint can be calculated using any two parallel sides of a mutant cycle, we did not repeat experiments for WT and T460S; instead, we calculated ΔΔGint using the two horizontal sides of each cycle, i.e., by comparing the effects of the T460S mutation onto the H1348A versus WT backgrounds. For this reason, we refrain from providing absolute ΔG values for the vertical sides of the T460-H1348 mutant cycles (Figs. 6, B and D, and 7, B and D); and the same applies for the T460-H1375 mutant cycles (see below; Figs. 8, B and D, and 9, B and D).

Bottom Line: Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates.Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle.These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

Show MeSH
Related in: MedlinePlus