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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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Mutations at positions 460 and 1353 do not affect apparent affinity for ATP. (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Different [ATP] were tested several times within one recording. (B) [ATP] dose–response relationships for WT and mutant CFTR channels; currents in test [ATP] were normalized to the average of the currents observed in bracketing segments in the presence of 2 mM ATP. Solid lines show fits of the Michaelis-Menten equation; KPo values are plotted in the panel. Between 5 and 14 measurements were made for each concentration tested. (C) Estimates of KrCO for each construct, calculated (see Results) using KPo from B and Po;max from Fig. 3 B. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on KrCO values.
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fig4: Mutations at positions 460 and 1353 do not affect apparent affinity for ATP. (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Different [ATP] were tested several times within one recording. (B) [ATP] dose–response relationships for WT and mutant CFTR channels; currents in test [ATP] were normalized to the average of the currents observed in bracketing segments in the presence of 2 mM ATP. Solid lines show fits of the Michaelis-Menten equation; KPo values are plotted in the panel. Between 5 and 14 measurements were made for each concentration tested. (C) Estimates of KrCO for each construct, calculated (see Results) using KPo from B and Po;max from Fig. 3 B. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on KrCO values.

Mentions: To test for possible effects on ATP binding, a dose–response for ATP (Fig. 4 B) was obtained by measuring relative current in macro-patches containing >40 channels. All exposures to test [ATP] were bracketed with periods of exposure to 2 mM (approximately saturating) ATP used for normalizing (Fig. 4 A). There was no significant difference in relative current at any [ATP] between WT and mutants. The apparent affinities, KPo, determined from fitting the Michaelis-Menten equation to the dose–response of the normalized current were between 68 and 77 µM. For WT, the decrease in steady-state current at low [ATP] is a result of a reduced opening rate without any change in closing rate (Gunderson and Kopito, 1994; Venglarik et al., 1994; Winter et al., 1994). We confirmed this was also the case for T460S and L1353M using multichannel analysis on patches containing <10 channels (not depicted), which showed that when [ATP] was reduced from 2 mM to 50 µM, burst duration was not significantly affected, and the fractional Po supported by 50 µM ATP (0.39 ± 0.07 and n = 6 for T460S, and 0.51 ± 0.08 and n = 5 for L1353M) could be accounted for by the fractional opening rate observed under the same conditions (0.39 ± 0.06 and n = 6 for T460S, and 0.46 ± 0.07 and n = 5 for L1353M). Thus, we could estimate for each construct the apparent affinity of ATP for affecting channel opening rate (KrCO; Fig. 4 C) using the relationship KrCO = KPo/(1−Po,max) (Csanády et al., 2000; Po,max values were taken from Fig. 3 B). Because ATP binding to the NBD2 site is likely a rapid equilibrium process relative to the slow (∼1-s−1) channel opening step (Zeltwanger et al., 1999), KrCO approximates the Kd of ATP from the NBD2 head of closed channels and therefore reflects ΔG between closed states with or without ATP bound to NBD2 (compare Szollosi et al., 2010). However, because none of our mutations affected KrCO (Fig. 4 C), a mutant cycle built on this parameter (Fig. 4 D) yielded a ΔΔGint ≈ 0.


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)

Mutations at positions 460 and 1353 do not affect apparent affinity for ATP. (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Different [ATP] were tested several times within one recording. (B) [ATP] dose–response relationships for WT and mutant CFTR channels; currents in test [ATP] were normalized to the average of the currents observed in bracketing segments in the presence of 2 mM ATP. Solid lines show fits of the Michaelis-Menten equation; KPo values are plotted in the panel. Between 5 and 14 measurements were made for each concentration tested. (C) Estimates of KrCO for each construct, calculated (see Results) using KPo from B and Po;max from Fig. 3 B. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on KrCO values.
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fig4: Mutations at positions 460 and 1353 do not affect apparent affinity for ATP. (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Different [ATP] were tested several times within one recording. (B) [ATP] dose–response relationships for WT and mutant CFTR channels; currents in test [ATP] were normalized to the average of the currents observed in bracketing segments in the presence of 2 mM ATP. Solid lines show fits of the Michaelis-Menten equation; KPo values are plotted in the panel. Between 5 and 14 measurements were made for each concentration tested. (C) Estimates of KrCO for each construct, calculated (see Results) using KPo from B and Po;max from Fig. 3 B. (D) Thermodynamic mutant cycle for target pair T460-L1353 built on KrCO values.
Mentions: To test for possible effects on ATP binding, a dose–response for ATP (Fig. 4 B) was obtained by measuring relative current in macro-patches containing >40 channels. All exposures to test [ATP] were bracketed with periods of exposure to 2 mM (approximately saturating) ATP used for normalizing (Fig. 4 A). There was no significant difference in relative current at any [ATP] between WT and mutants. The apparent affinities, KPo, determined from fitting the Michaelis-Menten equation to the dose–response of the normalized current were between 68 and 77 µM. For WT, the decrease in steady-state current at low [ATP] is a result of a reduced opening rate without any change in closing rate (Gunderson and Kopito, 1994; Venglarik et al., 1994; Winter et al., 1994). We confirmed this was also the case for T460S and L1353M using multichannel analysis on patches containing <10 channels (not depicted), which showed that when [ATP] was reduced from 2 mM to 50 µM, burst duration was not significantly affected, and the fractional Po supported by 50 µM ATP (0.39 ± 0.07 and n = 6 for T460S, and 0.51 ± 0.08 and n = 5 for L1353M) could be accounted for by the fractional opening rate observed under the same conditions (0.39 ± 0.06 and n = 6 for T460S, and 0.46 ± 0.07 and n = 5 for L1353M). Thus, we could estimate for each construct the apparent affinity of ATP for affecting channel opening rate (KrCO; Fig. 4 C) using the relationship KrCO = KPo/(1−Po,max) (Csanády et al., 2000; Po,max values were taken from Fig. 3 B). Because ATP binding to the NBD2 site is likely a rapid equilibrium process relative to the slow (∼1-s−1) channel opening step (Zeltwanger et al., 1999), KrCO approximates the Kd of ATP from the NBD2 head of closed channels and therefore reflects ΔG between closed states with or without ATP bound to NBD2 (compare Szollosi et al., 2010). However, because none of our mutations affected KrCO (Fig. 4 C), a mutant cycle built on this parameter (Fig. 4 D) yielded a ΔΔGint ≈ 0.

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