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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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Effects of mutations at positions 460 and 1375 on nonhydrolytic gating in the K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Fitted parameters were τ1 = 2.8 s, τ2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/K1250R trace, and τ1 = 2.8 s, τ2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/K1250R trace. Average steady-state burst durations (τ*; inset) were estimated from the two fitted time constants (τ1 and τ2) and their fractional amplitudes (A1 and A2) as τ* = (A1+A2)τ1τ2/(A1τ2+A2τ1). (B) Thermodynamic mutant cycle for target pair T460-H1375 built on average nonhydrolytic closing rates (1/τ*). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1375 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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fig9: Effects of mutations at positions 460 and 1375 on nonhydrolytic gating in the K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Fitted parameters were τ1 = 2.8 s, τ2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/K1250R trace, and τ1 = 2.8 s, τ2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/K1250R trace. Average steady-state burst durations (τ*; inset) were estimated from the two fitted time constants (τ1 and τ2) and their fractional amplitudes (A1 and A2) as τ* = (A1+A2)τ1τ2/(A1τ2+A2τ1). (B) Thermodynamic mutant cycle for target pair T460-H1375 built on average nonhydrolytic closing rates (1/τ*). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1375 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: Macroscopic current decay time courses were fit with single- or bi-exponential equations (pCLAMP 10.2; Molecular Devices). Fitting the relaxation time course after ATP removal for the nonhydrolytic H1375A/K1250R and T460S/H1375A/K1250R constructs consistently required a double exponential with two slow time constants (each in the seconds range), suggesting two populations of open-channel bursts (see Fig. 9 A). For these two constructs, average steady-state burst duration (τ*) was estimated from the two fitted time constants (τ1 and τ2) and their fractional amplitudes (A1 and A2) as τ* = (A1+A2)τ1τ2/(A1τ2+A2τ1), and average closing rate for the mutant cycle in Fig. 9 B was defined as 1/τ*.


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 1375 on nonhydrolytic gating in the K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Fitted parameters were τ1 = 2.8 s, τ2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/K1250R trace, and τ1 = 2.8 s, τ2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/K1250R trace. Average steady-state burst durations (τ*; inset) were estimated from the two fitted time constants (τ1 and τ2) and their fractional amplitudes (A1 and A2) as τ* = (A1+A2)τ1τ2/(A1τ2+A2τ1). (B) Thermodynamic mutant cycle for target pair T460-H1375 built on average nonhydrolytic closing rates (1/τ*). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1375 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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fig9: Effects of mutations at positions 460 and 1375 on nonhydrolytic gating in the K1250R background. (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Fitted parameters were τ1 = 2.8 s, τ2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/K1250R trace, and τ1 = 2.8 s, τ2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/K1250R trace. Average steady-state burst durations (τ*; inset) were estimated from the two fitted time constants (τ1 and τ2) and their fractional amplitudes (A1 and A2) as τ* = (A1+A2)τ1τ2/(A1τ2+A2τ1). (B) Thermodynamic mutant cycle for target pair T460-H1375 built on average nonhydrolytic closing rates (1/τ*). The top two corners of the mutant cycle were taken from Fig. 5 B. (C) Noise analysis for estimation of Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). (Right) Thermodynamic mutant cycle for target pair T460-H1375 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: Macroscopic current decay time courses were fit with single- or bi-exponential equations (pCLAMP 10.2; Molecular Devices). Fitting the relaxation time course after ATP removal for the nonhydrolytic H1375A/K1250R and T460S/H1375A/K1250R constructs consistently required a double exponential with two slow time constants (each in the seconds range), suggesting two populations of open-channel bursts (see Fig. 9 A). For these two constructs, average steady-state burst duration (τ*) was estimated from the two fitted time constants (τ1 and τ2) and their fractional amplitudes (A1 and A2) as τ* = (A1+A2)τ1τ2/(A1τ2+A2τ1), and average closing rate for the mutant cycle in Fig. 9 B was defined as 1/τ*.

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