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J Gen Physiol
2013 Jul 01;1421:61-73. doi: 10.1085/jgp.201210954.
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Conformational changes in the catalytically inactive nucleotide-binding site of CFTR.
Csanády L
,
Mihályi C
,
Szollosi A
,
Töröcsik B
,
Vergani P
.
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A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N(6)-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.
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Figure 1. Effects of P-ATP on rough channel gating parameters of WT CFTR. (A and B) Macroscopic currents of prephosphorylated CFTR channels elicited by step applications (bars) of various concentrations of ATP (A) or P-ATP (B). (C) Doseâresponse curves for current stimulation by ATP (blue symbols) and P-ATP (red symbols). Mean steady currents in the presence of test nucleotide concentrations were normalized to the average of those measured in bracketing segments in the presence of 2 mM (ATP) or 32 µM (P-ATP) nucleotide. Fits to the MichaelisâMenten equation (solid lines) yielded Km values shown. (D) Steady-state current recording from a patch containing two active WT CFTR channels gating in 2 mM ATP (blue segments) or 10 µM P-ATP (red segment). (EâG) Steady-state open probabilities (E), mean burst durations (F), and mean interburst durations (G) of WT CFTR in the presence of 2 mM ATP (blue bars) or 10 µM P-ATP (red bars), extracted from records with â¤2 active channels, as described in Materials and methods.
Figure 2. Steady-state mean burst duration of H1348A CFTR is insensitive to elevation of [ATP]. (A) Steady-state current recording from a patch containing two active H1348A CFTR channels gating in 2 mM (green segment) or 10 mM (dark blue segment) ATP. (BâD) Steady-state open probabilities (B), mean burst durations (C), and mean interburst durations (D) of H1348A CFTR in the presence of 2 mM (green bars) or 10 mM (dark blue bars) ATP, extracted from records with â¤5 active channels, as described in Materials and methods.
Figure 3. P-ATP and the H1348A mutation slow nonhydrolytic CFTR closure. (A and D) Macroscopic currents of prephosphorylated K1250A (A) and E1371S (D) CFTR channels elicited by exposure (bars) to either 10 mM ATP alternating with 50 µM P-ATP (A) or 2 mM ATP alternating with 10 µM P-ATP (D); the fivefold higher nucleotide concentrations for the K1250A constructs were used to compensate for the large decrease in apparent ATP affinity caused by this mutation (Vergani et al., 2003). Current decay time courses after sudden removal of the nucleotide were fitted with single exponentials (colored solid lines); colored numbers are time constants (in milliseconds), which reflect mean burst durations. (B and E) Macroscopic currents of prephosphorylated K1250A/H1348A (B) and E1371S/H1348A (E) CFTR channels elicited by transient exposure (bars) to either 10 mM (B) or 2 mM (E) ATP. Current decay time courses after sudden ATP removal were fitted with double exponentials (colored solid lines); colored numbers are calculated average burst durations (Ï*, in milliseconds; see Materials and methods). The fit parameters were Ï1 = 37 s, Ï2 = 165 s, A1 = 0.34, and A2 = 0.66 for B, and Ï1 = 24 s, Ï2 = 179 s, A1 = 0.07, and A2 = 0.93 for E. (C and F) Nonhydrolytic closing rates of channels opened by ATP (blue bars) or P-ATP (red bars), or of channels bearing the H1348A mutation opened by ATP (green bars), measured in the K1250A (C) or E1371S (F) background. Closing rates were calculated as the inverse of the decay time constant after nucleotide removal; for traces that were not well fit by a single exponential, the average closing rate was calculated as 1/Ï*.
Figure 4. Mutation H1348A and deletion of segment 415â432 (ÎRI) abolish the effect of P-ATP on hydrolytic channel closure. (A and D) Steady-state recordings of single-channel currents in the presence of 2 mM ATP (green segments) or 10 µM P-ATP (brown segments) for H1348A (A) or ÎRI CFTR (D). Insets show at an expanded time scale 10-s intervals taken from the stable steady section of each segment (gray bars). In A, Vm was â40 mV. (B and E) Closing rates, obtained as the inverses of the steady-state mean burst duration, for H1348A (B) and ÎRI (E) CFTR channels gating in ATP (green bars) or P-ATP (brown bars). Steady-state closing rates of WT CFTR in ATP (blue bars) and P-ATP (red bars) were extracted from the data shown in Fig. 1 F. (C and F) Thermodynamic mutant cycles built on (hydrolytic) closing rates for the interaction of the P-ATP P group with residue 1348 (C) or the RI region (F). Each corner is represented by the particular site-1 protein structure (H or A at position 1348 [C]; maintained or deleted RI [F]) and the nucleotide driving gating (ATP or P-ATP), respectively. ÎÎGâ¡ values (mean ± SEM) on arrows show perturbation-induced changes in the stability of the closing transition state with respect to the open ground state and were used to calculate (see Materials and methods) the coupling energy for the P groupâ1348 (C) and the P groupâRI (F) interaction (ÎÎGint(closing)).
Figure 5. Mutation H1348A and deletion of segment 415â432 (ÎRI) potentiate the effect of P-ATP on nonhydrolytic channel closure. (A and D) Macroscopic currents from K1250A/H1348A (A) and K1250A/ÎRI (D) CFTR channels elicited by exposures to 10 mM ATP or 50 µM P-ATP (bars). Solid lines are single-exponential fits to the current relaxation time courses upon nucleotide removal, with time constants indicated. (B and E) Nonhydrolytic closing rates for K1250A/H1348A (B) and K1250A/ÎRI (E) CFTR channels, obtained as the inverses of the relaxation time constants upon removal of ATP (green bars) or P-ATP (brown bars); closing rates of K1250A CFTR upon removal of ATP (blue bars) and P-ATP (red bars) were replotted from Fig. 3 C. (C and F) Thermodynamic mutant cycles built on nonhydrolytic closing rates for the interaction of the P-ATP P group with residue 1348 (C) or the RI region (F). Each corner is represented by the particular site-1 protein structure (H or A at position 1348 [C]; maintained or deleted RI [F]) and the nucleotide driving gating (ATP or P-ATP), respectively. ÎÎGâ¡ values (mean ± SEM) on arrows show perturbation-induced changes in the stability of the transition state for nonhydrolytic closure with respect to the open ground state and were used to calculate the coupling energy for the P groupâ1348 (C) and the P groupâRI (F) interaction (ÎÎGint(closing)).
Figure 6. Relaxation time courses of macroscopic WT, H1348A, and ÎRI CFTR currents upon sudden nucleotide removal. (AâC) Macroscopic currents of prephosphorylated WT (A), H1348A (B), and ÎRI (C) CFTR channels elicited by exposure (bars) to either 2 mM ATP alternating with 10 µM P-ATP (A and C), or 2 mM ATP alternating with either 10 mM ATP (B, top) or 10 µM P-ATP (B, bottom). Current decay time courses upon sudden removal of the nucleotide were fitted with single exponentials (colored solid lines); colored numbers are time constants (in milliseconds). (D) Closing time constants of WT CFTR currents upon removal of 2 mM ATP (blue bar) or 10 µM P-ATP (red bar), of H1348A CFTR currents upon removal of 2 (left green bar) or 10 mM ATP (dark blue bar) or 10 µM P-ATP (left brown bar), and of ÎRI CFTR currents upon removal of 2 mM ATP (right green bar) or 10 µM P-ATP (right brown bar). As a comparison, striped bars replot mean burst durations of the respective constructs measured at steady state in the presence of the respective nucleotide (from Figs. 1 F, 2 C, and 4, B and E).
Figure 7. P-ATP and the H1348A mutation slow the O1âO2 transition of CFTR channels. (A and B) Histograms of open burst durations compiled from 621 open burst events of single WT CFTR channels gating in 10 µM P-ATP (A) and from 908 open burst events of single H1348A CFTR channels gating in 2 mM ATP (B). Both distributions were fitted by maximum likelihood to either a single exponential (blue dotted lines) or to the scheme in C, with rate k-1 fixed to zero (red lines); the latter fits proved significantly (P < 0.01 for A and P â 0.05 for B) better by the log-likelihood ratio test. Fit parameters, as well as calculated time constants and fractional amplitudes of the exponential components of the fitted distributions, are plotted in both panels. (Insets) 30-s segments of single-channel recordings. (C) Simplified cyclic gating scheme (from Csanády et al., 2010) used for maximum likelihood fitting of open burst distributions, depicting closed and open states with either ATP (states C1 and O1) or ADP (states C2 and O2) bound at site 2. Pore opening/closure is assumed quasi-simultaneous with formation/disruption of the NBD dimer. Cyan rectangles, TMDs; green, NBD1; blue, NBD2; yellow, ATP; red, ADP. (D) Summary of rates k1 and k2 obtained from the fits in A and B for WT CFTR gating in 10 µM P-ATP (red bars) and H1348A CFTR gating in 2 mM ATP (green bars); as a comparison, the values measured for WT CFTR gating in 2 mM ATP (blue bars) are replotted from Csanády et al. (2010). Error bars represent 0.5-unit log-likelihood intervals. Note that in the presence of the site-1 perturbations, rate k2 could be estimated only to a limited precision because of the increased discrepancy between the values of k1 and k2 (compare Csanády, 2006; k2/k1 is 11, 18, and 29, respectively, for WT+ATP, WT+P-ATP, and H1348A+ATP).
Figure 8. Energetic interpretation of the gating effects of P-ATP and the H1348A mutation. Free energy profiles (top; not drawn to scale) of channels moving sequentially through the gating states depicted below (from Fig. 7 C), in the absence (blue energy profile) or presence (red energy profile) of a perturbation (red star in cartooned states) in composite site 1. A selective stabilization of the O1 ground state (by δÎGO1, relative to both C1 and O2) is predicted to increase the heights of the free energy barriers for exiting O1 in both directions (ÎGâ¡-1 and ÎGâ¡1; compare red and blue vertical double arrows), thereby slowing both rate k-1 and k1 (cartoon).
Figure 9. Partial separation of degenerate site in an asymmetrical bacterial ABC exporter. Ribbon diagram of NBD dimer from the crystal structure of TM287/288 (Protein Data Bank accession no. 3QF4), with AMPPNP (yellow) bound at the degenerate site. Conserved Walker A threonines (T368 in TM287 and T390 in TM288; red), as well as D-loop residue N521 of TM288 and corresponding TM287 residue S499 (violet), are highlighted in space fill. The empty active site is wide open, whereas the nucleotide-bound degenerate site retains some contact between the two NBD surfaces through opposing Walker A and D-loop motifs. NBD color coding follows that used in Fig. 7 C: green, TM287; blue, TM288.
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