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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.

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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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Effects of mutations at positions 460 and 1348 on normal hydrolytic channel gating. (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on closing rates. The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute ΔΔG values are not printed for the vertical sides of the cycle. (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on opening rates. The top two corners of the mutant cycle were taken from Fig. 3 D.
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fig6: Effects of mutations at positions 460 and 1348 on normal hydrolytic channel gating. (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on closing rates. The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute ΔΔG values are not printed for the vertical sides of the cycle. (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on opening rates. The top two corners of the mutant cycle were taken from Fig. 3 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)

Effects of mutations at positions 460 and 1348 on normal hydrolytic channel gating. (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on closing rates. The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute ΔΔG values are not printed for the vertical sides of the cycle. (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on opening rates. The top two corners of the mutant cycle were taken from Fig. 3 D.
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fig6: Effects of mutations at positions 460 and 1348 on normal hydrolytic channel gating. (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on closing rates. The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute ΔΔG values are not printed for the vertical sides of the cycle. (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). (Right) Thermodynamic mutant cycle for target pair T460-H1348 built on opening rates. The top two corners of the mutant cycle were taken from Fig. 3 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