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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 T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of WT and mutant CFTR macroscopic currents after the removal of 2 mM ATP (gray). Solid colored lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-L1353 built on nonhydrolytic closing rates (1/τrelaxation). (C) Noise analysis was performed on 2–3-min records from patches containing <100 channels. Each point represents one patch. Po was calculated for each patch; mean ± SEM Po values are shown in the inset. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on Keq = Po/(1−Po) values under nonhydrolytic conditions.
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fig5: The T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of WT and mutant CFTR macroscopic currents after the removal of 2 mM ATP (gray). Solid colored lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-L1353 built on nonhydrolytic closing rates (1/τrelaxation). (C) Noise analysis was performed on 2–3-min records from patches containing <100 channels. Each point represents one patch. Po was calculated for each patch; mean ± SEM Po values are shown in the inset. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on Keq = Po/(1−Po) values under nonhydrolytic conditions.

Mentions: Hydrolysis can be inhibited by mutating key catalytic residues at composite site 2. In other ABC transporters, mutating the Walker A lysine to arginine greatly reduced or abolished hydrolysis (e.g., Urbatsch et al., 1998; Lerner-Marmarosh et al., 1999). In CFTR, the equivalent mutation, K1250R, caused an increase in open burst duration (Vergani et al., 2005; Csanády et al., 2006), consistent with blocking of the fast hydrolytic closure pathway. To determine if the mutations T460S, L1353M, and T460S/L1353M increased the rate of nonhydrolytic closure from an open state with ATP bound at both composite sites, we introduced the above site-1 mutations in a K1250R background. Closing rates were determined from the rates of macroscopic current decay upon ATP removal, which followed single-exponential time courses (Fig. 5 A; colored lines are fitted exponentials). The fitted time constant for current decay, τrelaxation (Fig. 5 A, inset), provided an estimate for the average lifetime of the open state, which was 5.9 ± 0.5 s (n = 13) for K1250R (black bar) and unchanged in L1353M/K1250R (7.2 ± 0.8 s; n = 10; P = 0.11; blue bar), but significantly reduced in T460S/K1250R (4.2 ± 0.3 s; n = 13; P < 0.01; red bar). Because τrelaxation was additively affected in T460S/L1353M/K1250R (4.5 ± 0.6 s; n = 10; P < 0.05; green bar), ΔΔG‡int(closing) was not significantly different from zero (Fig. 5 B), indicating that the coupling between the two residues on opposite sides of composite site 1 was not changed along the nonhydrolytic closure pathway between the ATP-bound open state and the transition state.


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 T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of WT and mutant CFTR macroscopic currents after the removal of 2 mM ATP (gray). Solid colored lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-L1353 built on nonhydrolytic closing rates (1/τrelaxation). (C) Noise analysis was performed on 2–3-min records from patches containing <100 channels. Each point represents one patch. Po was calculated for each patch; mean ± SEM Po values are shown in the inset. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on Keq = Po/(1−Po) values under nonhydrolytic conditions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105517&req=5

fig5: The T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. (A) Representative normalized decay time courses of WT and mutant CFTR macroscopic currents after the removal of 2 mM ATP (gray). Solid colored lines are fitted exponentials; mean ± SEM relaxation time constants (τrelaxation) are shown in the inset. (B) Thermodynamic mutant cycle for target pair T460-L1353 built on nonhydrolytic closing rates (1/τrelaxation). (C) Noise analysis was performed on 2–3-min records from patches containing <100 channels. Each point represents one patch. Po was calculated for each patch; mean ± SEM Po values are shown in the inset. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on Keq = Po/(1−Po) values under nonhydrolytic conditions.
Mentions: Hydrolysis can be inhibited by mutating key catalytic residues at composite site 2. In other ABC transporters, mutating the Walker A lysine to arginine greatly reduced or abolished hydrolysis (e.g., Urbatsch et al., 1998; Lerner-Marmarosh et al., 1999). In CFTR, the equivalent mutation, K1250R, caused an increase in open burst duration (Vergani et al., 2005; Csanády et al., 2006), consistent with blocking of the fast hydrolytic closure pathway. To determine if the mutations T460S, L1353M, and T460S/L1353M increased the rate of nonhydrolytic closure from an open state with ATP bound at both composite sites, we introduced the above site-1 mutations in a K1250R background. Closing rates were determined from the rates of macroscopic current decay upon ATP removal, which followed single-exponential time courses (Fig. 5 A; colored lines are fitted exponentials). The fitted time constant for current decay, τrelaxation (Fig. 5 A, inset), provided an estimate for the average lifetime of the open state, which was 5.9 ± 0.5 s (n = 13) for K1250R (black bar) and unchanged in L1353M/K1250R (7.2 ± 0.8 s; n = 10; P = 0.11; blue bar), but significantly reduced in T460S/K1250R (4.2 ± 0.3 s; n = 13; P < 0.01; red bar). Because τrelaxation was additively affected in T460S/L1353M/K1250R (4.5 ± 0.6 s; n = 10; P < 0.05; green bar), ΔΔG‡int(closing) was not significantly different from zero (Fig. 5 B), indicating that the coupling between the two residues on opposite sides of composite site 1 was not changed along the nonhydrolytic closure pathway between the ATP-bound open state and the transition state.

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