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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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Effect of mutations at positions 460 and 1353 on channel closing rates. (A) Representative traces of multichannel recordings of prephosphorylated WT and mutant CFTR channels, used to determine burst duration. Downward deflection indicates inward current. (B) Closing rates of WT and mutant CFTR channels, defined as the inverse of the mean burst duration (see Materials and methods). (C) Thermodynamic mutant cycle for target pair T460-L1353 built on the closing rates from B; each corner is represented by the side chains at positions 460 and 1353, respectively. ΔΔG0 values (mean ± SEM) on arrows show mutation-induced changes in the stability of the transition state for closure with respect to the open ground state and were used to calculate (see Materials and methods) the coupling energy for the 460–1353 interaction (ΔΔG‡int (closing)).
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fig2: Effect of mutations at positions 460 and 1353 on channel closing rates. (A) Representative traces of multichannel recordings of prephosphorylated WT and mutant CFTR channels, used to determine burst duration. Downward deflection indicates inward current. (B) Closing rates of WT and mutant CFTR channels, defined as the inverse of the mean burst duration (see Materials and methods). (C) Thermodynamic mutant cycle for target pair T460-L1353 built on the closing rates from B; each corner is represented by the side chains at positions 460 and 1353, respectively. ΔΔG0 values (mean ± SEM) on arrows show mutation-induced changes in the stability of the transition state for closure with respect to the open ground state and were used to calculate (see Materials and methods) the coupling energy for the 460–1353 interaction (ΔΔG‡int (closing)).

Mentions: To determine if the mutations T460S and L1353M, individually or together, had any effect on channel gating in saturating 2 mM ATP, burst durations were determined from patches containing 1–10 channels (Fig. 2 A). There was a small increase in closing rate (defined as inverse of the mean burst duration; Fig. 2 B) for T460S (3.6 ± 0.3 s−1; n = 20; Fig. 2 B, red bar) and L1353M (3.3 ± 0.4 s−1; n = 8; Fig. 2 B, blue bar) compared with WT (2.6 ± 0.3 s−1; n = 13; Fig. 2 B, black bar), whereas there was no significant change for T460S/L1353M (n = 9; Fig. 2 B, green bar). Accordingly, a thermodynamic mutant cycle built on closing rates (Fig. 2 C) yielded an interaction free energy (ΔΔGint(closing)), which was significantly different from zero (P = 0.007) but rather small (0.63 ± 0.19 kT). To obtain correct estimates of opening rates, we first determined Po using noise analysis (Fig. 3 A; see Materials and methods). There was no significant difference between Po in WT and mutants (Fig. 3 B), with values ranging between 0.23 and 0.25. Accordingly, the calculated (see Materials and methods) opening rates (Fig. 3 C) were slightly increased for the single mutants, but for this parameter the resulting mutant cycle (Fig. 3 D) yielded a ΔΔGint not significantly different from zero (P = 0.1).


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)

Effect of mutations at positions 460 and 1353 on channel closing rates. (A) Representative traces of multichannel recordings of prephosphorylated WT and mutant CFTR channels, used to determine burst duration. Downward deflection indicates inward current. (B) Closing rates of WT and mutant CFTR channels, defined as the inverse of the mean burst duration (see Materials and methods). (C) Thermodynamic mutant cycle for target pair T460-L1353 built on the closing rates from B; each corner is represented by the side chains at positions 460 and 1353, respectively. ΔΔG0 values (mean ± SEM) on arrows show mutation-induced changes in the stability of the transition state for closure with respect to the open ground state and were used to calculate (see Materials and methods) the coupling energy for the 460–1353 interaction (ΔΔG‡int (closing)).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105517&req=5

fig2: Effect of mutations at positions 460 and 1353 on channel closing rates. (A) Representative traces of multichannel recordings of prephosphorylated WT and mutant CFTR channels, used to determine burst duration. Downward deflection indicates inward current. (B) Closing rates of WT and mutant CFTR channels, defined as the inverse of the mean burst duration (see Materials and methods). (C) Thermodynamic mutant cycle for target pair T460-L1353 built on the closing rates from B; each corner is represented by the side chains at positions 460 and 1353, respectively. ΔΔG0 values (mean ± SEM) on arrows show mutation-induced changes in the stability of the transition state for closure with respect to the open ground state and were used to calculate (see Materials and methods) the coupling energy for the 460–1353 interaction (ΔΔG‡int (closing)).
Mentions: To determine if the mutations T460S and L1353M, individually or together, had any effect on channel gating in saturating 2 mM ATP, burst durations were determined from patches containing 1–10 channels (Fig. 2 A). There was a small increase in closing rate (defined as inverse of the mean burst duration; Fig. 2 B) for T460S (3.6 ± 0.3 s−1; n = 20; Fig. 2 B, red bar) and L1353M (3.3 ± 0.4 s−1; n = 8; Fig. 2 B, blue bar) compared with WT (2.6 ± 0.3 s−1; n = 13; Fig. 2 B, black bar), whereas there was no significant change for T460S/L1353M (n = 9; Fig. 2 B, green bar). Accordingly, a thermodynamic mutant cycle built on closing rates (Fig. 2 C) yielded an interaction free energy (ΔΔGint(closing)), which was significantly different from zero (P = 0.007) but rather small (0.63 ± 0.19 kT). To obtain correct estimates of opening rates, we first determined Po using noise analysis (Fig. 3 A; see Materials and methods). There was no significant difference between Po in WT and mutants (Fig. 3 B), with values ranging between 0.23 and 0.25. Accordingly, the calculated (see Materials and methods) opening rates (Fig. 3 C) were slightly increased for the single mutants, but for this parameter the resulting mutant cycle (Fig. 3 D) yielded a ΔΔGint not significantly different from zero (P = 0.1).

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