1. Structural Biology and Molecular Biophysics
Download icon

Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond

  1. Márton A Simon
  2. László Csanády  Is a corresponding author
  1. Department of Biochemistry, Semmelweis University, Hungary
  2. HCEMM-SE Molecular Channelopathies Research Group, Hungary
  3. MTA-SE Ion Channel Research Group, Semmelweis University, Hungary
Research Article
  • Cited 0
  • Views 320
  • Annotations
Cite this article as: eLife 2021;10:e74693 doi: 10.7554/eLife.74693

Abstract

The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here, we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state-dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived ‘flickery’ nor in the long-lived ‘interburst’ closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

Editor's evaluation

Multiple inherited mutations in the epithelial CFTR anion-permeable channel cause cystic fibrosis through different molecular mechanisms, and some of these mechanisms can be specifically targeted by drugs to treat the disease. Drawing from available structural information and double-mutant cycle analysis applied to patch-clamp recordings, Simon and Csanády find that one of the most common CFTR disease-causing mutations, R117H, disrupts an interaction between the R117 side-chain and a main-chain carbonyl that selectively stabilizes the open state of the channel. These findings may open new paths of exploration for treating patients carrying this mutation, and provide important mechanistic constraints towards understanding the gating mechanism of CFTR channels.

https://doi.org/10.7554/eLife.74693.sa0

Introduction

Loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel disrupt transepithelial salt-water transport in the lung, intestine, pancreatic duct, and sweat duct, and cause cystic fibrosis (CF), the most common inherited lethal disease among caucasians (O’Sullivan and Freedman, 2009). Based on their molecular consequences the several hundred identified CF mutations have been categorized into classes, such as those that impair synthesis of the full-length CFTR polypeptide (Class I), processing and trafficking of the CFTR protein (Class II), channel gating (Class III), or anion permeation through the open pore (Class IV) (De Boeck and Amaral, 2016). Until recently symptomatic therapy remained the only available treatment option for CF patients, but this has been profoundly changed by a major breakthrough in the development of small-molecule drugs that target the CFTR protein itself. Treatments by ‘corrector’ drugs that improve processing and maturation of Class II mutant CFTR protein, and by the ‘potentiator’ compound Vx-770 (ivacaftor) which increases open probability (Po) of Class III mutant channels, have led to significant symptomatic improvement for patients with Class II/III mutations (Ramsey et al., 2011; Davies et al., 2018), and have been approved by the FDA. Because the responsiveness to potentiators of different Class III mutants is greatly variable (Van Goor et al., 2014), understanding the molecular pathologies of such mutations bears strong clinical relevance.

CFTR is an ATP-binding cassette (ABC) protein which contains two transmembrane domains (TMD1, -2; Figure 1A, gray) and two cytosolic nucleotide-binding domains (NBD1, -2; Figure 1A, blue and green). These two ABC-typical TMD-NBD halves are linked by a cytosolic regulatory (R) domain (Figure 1A, magenta) which is unique to CFTR and contains multiple serines that must be phosphorylated by cAMP-dependent protein kinase (PKA) to allow channel activity (Riordan et al., 1989). CFTR pore opening/closure (gating) is linked to an ATP hydrolysis cycle at the NBDs and resembles the active transport cycle of ABC exporters (reviewed, e.g., in Csanády et al., 2019). In the presence of ATP single phosphorylated CFTR channels open into ‘bursts’: groups of openings separated by brief (~10 ms) ‘flickery’ closures and flanked by long (~1 s) ‘interburst (IB)’ closures. Upon ATP binding CFTR’s NBDs associate (Figure 1B–C) into a tight head-to-tail dimer which is accompanied by a large rearrangement of its TMDs from an inward-facing (Figure 1B) to an outward-facing (Figure 1C) orientation. The stable IB closed state corresponds to an inward-facing TMD conformation with separated NBDs in which the channel gate, near the extracellular membrane surface, is closed (Liu et al., 2017). The bursting (B) state features a tight NBD dimer that occludes two ATP molecules in interfacial binding sites (sites 1 and 2), and an outward-facing TMD conformation in which a large lateral gap between TM helices 4 and 6 continues to connect the pore to the cytosol, bypassing the constricted ABC-transporter inner gate (El Hiani and Linsdell, 2015; El Hiani et al., 2016; Zhang et al., 2018a). The B state is a compound state that includes the fully open (O) and the flickery closed (Cf) states. For wild-type (WT) CFTR the majority of sojourns in the highly stable B state are terminated by hydrolysis of the ATP at site 2, which prompts NBD dimer dissociation and resets the channel into the inward-facing IB state (Csanády et al., 2010).

Figure 1 with 1 supplement see all
An H-bond between the R117 side chain and the E1124 backbone carbonyl group is apparent in the quasi-open CFTR structure.

(A) Cartoon topology of CFTR and location of target positions (blue and red dot). TMDs, gray; NBD1, blue; NBD2, green; R domain, magenta; membrane, yellow. Orange star denotes catalytic site mutation in the NBD2 Walker B motif. (B–C) Target residues R117 (blue) and E1124 (red) shown in space fill on the cryo-EM structures of (B) inward-facing dephospho-CFTR (pdbid: 5uak) and (C) outward-facing phosphorylated ATP-bound CFTR (pdbid: 6msm). Domain color coding as in A, the R domain is not depicted. (D–E) Close-up views of the extracellular region highlighted in B–C by dotted purple boxes. Target residues are shown as sticks, target distances are illustrated by orange dotted lines.

The cryo-electron microscopic (cryo-EM) studies have provided unprecedented structural insight, but have left several questions open. First, in the outward-facing CFTR structure (pdbid: 6msm) the external end of the pore is not quite as wide as would be required for passage of chloride ions. Nevertheless, at present that structure is the best available model for the O state (Zhang et al., 2018a). Second, the structure of the Cf state is unknown although, based on its fast kinetics, the O↔Cf transition is likely a localized movement. Third, the inward-facing structure (pdbid: 5uak) does not represent the closed state of an active channel gating in the presence of ATP, as it was obtained from unphosphorylated CFTR in the absence of ATP, and correspondingly contains density for the unphosphorylated R domain wedged in between the two unliganded NBDs and the cytosolic ends of the TMD helices. Nevertheless, functional studies suggest that the overall organization of the TMDs in the above two structures resembles those of the open and of the ‘active’ IB state, respectively (Bai et al., 2011; Cui et al., 2014; Wang et al., 2014). Thus, the former structure (6msm) will be referred to as ‘outward-facing’ or ‘quasi-open’, and the latter (5uak) as ‘inward-facing’ or ‘closed’ throughout this study.

Substitution of the conserved arginine at position 117, and in particular mutation R117H, is among the most common CF mutations (Dean et al., 1990; Wilschanski et al., 1995; https://cftr2.org/). In heterologous expression systems the R117H mutation does not impair surface expression (Sheppard et al., 1993; Hämmerle et al., 2001), but severely reduces whole-cell CFTR currents (Sheppard et al., 1993). Consistent with its location in the first extracellular loop (ECL1), replacement of the R117 side chain (Figure 1A, blue dot) causes a mild conductance defect suggesting that its positive charge is normally involved in recruiting extracellular chloride ions to the outer mouth of the pore (Sheppard et al., 1993; Zhou et al., 2008). However, the robust reduction of whole-cell currents is not explained by that small conductance defect, but rather by a strong gating defect (Sheppard et al., 1993) which places the R117H mutation into Class III. The reduced Po of the mutant is primarily caused by a large reduction in the mean burst duration (τburst) (Sheppard et al., 1993; Yu et al., 2016). In addition, the kinetics of intraburst gating is also altered in the mutant: shortened open times are paired with prolonged flickery closed times (Yu et al., 2016). Because R117H-CFTR currents are only modestly stimulated by Vx-770 (Van Goor et al., 2014; Yu et al., 2016), understanding the molecular pathology of this mutation will be important for developing better drugs optimized for personalized treatment.

How a mutation at the outer mouth of the pore may affect channel gating, thought to be controlled by NBD dimer formation/disruption, seemed puzzling at first. CFTR’s cyclic gating mechanism precludes equilibrium thermodynamic analyses (Csanády, 2009), and makes it difficult to discern whether a mutational effect on burst durations reflects an alteration in the rate of ATP hydrolysis or in the stability of the prehydrolytic open state. Using a non-hydrolytic site 2 mutation which reduces CFTR gating in saturating ATP to reversible IB↔B transitions, Φ-value analysis (Auerbach, 2007) revealed that the pore opening (IB→B) step is a spreading conformational wave initiated at site 2 of the NBD dimer interface and propagated along the longitudinal protein axis toward the extracellular surface. In the high-energy transition state (T) site 2 has already adopted its B-state conformation (i.e., it is already tightly dimerized) whereas the extracellular portion of the TMDs, which includes the gate, is still in its IB-state (closed) conformation (Sorum et al., 2015). Thus, mutations at extracellular positions, like 117, are expected to similarly perturb the stabilities of the T state and the IB closed state, and thus to selectively impact closing rate but not opening rate. That expectation was later confirmed for a series of perturbations at position 117: because Q, A, H, and C substitutions here all selectively accelerated closure (shortened τburst), it was concluded that in WT CFTR the R117 side chain is involved in a stabilizing interaction that is formed only in the B state but neither in the IB state nor in the T state for opening (Sorum et al., 2017). However, in the absence of high-resolution structures the interaction partner of the R117 side chain could not be identified.

Here, we exploit the recent cryo-EM structures of human CFTR obtained in inward- and outward-facing conformations (Liu et al., 2017; Zhang et al., 2018a), to identify the functionally important molecular interaction partner of the R117 side chain. We then test the functional role of this putative interaction using detailed single-channel kinetic analysis, and employ thermodynamic double-mutant cycles to rigorously quantitate how its strength changes between specific gating states. Our analysis provides a mechanistic explanation for the strong energetic role that the conserved arginine at position 117 plays in CFTR channel gating, as well as for the molecular pathology caused by its mutations. It also provides new structural inference for the organization of the Cf state.

Results

Cryo-EM structures suggest a strong H-bond between the R117 side chain and the E1124 peptide carbonyl group in the open-channel state

In cryo-EM structures of both inward-facing (Figure 1B; Liu et al., 2017) and outward-facing (Figure 1C; Zhang et al., 2018a) CFTR, the R117 side chain is resolved (Figure 1B–C, blue space fill). In the quasi-open structure it approaches, and forms a strong H-bond with, the backbone carbonyl oxygen of residue E1124 (Figure 1E, Figure 1—figure supplement 1), located in ECL6 (Figure 1A, red dot). In the closed-pore structure it is pointing out toward the solvent, and is not in contact with any other residue of the protein (Figure 1D). Thus, based on the structures, the identified 117–1124 H-bond might represent an interaction that changes dynamically during the gating cycle. However, as these structures need to be interpreted with caution, we set out to study the functional relevance of this putative interaction for CFTR gating energetics. To study gating under equilibrium conditions, we employed two different background mutations which disrupt ATP hydrolysis at site 2 (Figure 1A, orange star) and reduce CFTR gating in saturating ATP to a reversible IB↔B mechanism.

Deletion of residue 1124 reproduces the R117 phenotype

The NBD2 Walker B glutamate (E1371) side chain forms the catalytic base in site 2, and its mutations disrupt ATP hydrolysis (Zhang et al., 2018a). Correspondingly, the E1371S mutation prolongs τburst by >100-fold (Vergani et al., 2003; Csanády et al., 2013), revealing slow reversibility of the IB→B gating step. That slow rate of the (non-hydrolytic) B→IB transition is conveniently measured in macroscopic inside-out patch recordings by activating pre-phosphorylated E1371S CFTR channels using a brief (~1 min) exposure to 5 mM ATP, and then observing the slow rate of macroscopic current decay upon sudden ATP removal (Figure 2A, black trace); the time constant of a fitted exponential reports τburst (Figure 2B, black bar), that is, the average life time of the B state. Introduction of the R117H mutation into this background robustly accelerated the current decay (Figure 2A, blue trace), yielding ~6-fold shortening of τburst (Figure 2B, blue bar), consistent with an earlier report using the E1371Q background (Yu et al., 2016).

Figure 2 with 2 supplements see all
Energetic coupling between positions 117 and 1124 changes during non-hydrolytic closure.

(A, C) Macroscopic current relaxations following ATP removal for indicated CFTR mutants (color coded) harboring the E1371S background mutation. Inside-out patch currents were activated by exposure of pre-phosphorylated channels to 5 mM ATP. Current amplitudes are shown rescaled by their steady-state values. The gray trace in C illustrates the speed of solution exchange, estimated as the time constant of deactivation (~100 ms) upon Ca2+ removal of the endogenous Ca2+-activated chloride current, evoked by brief exposure to Ca2+. Membrane potential (Vm) was –20 mV. (B, D) Relaxation time constants of the currents in A and C, respectively, obtained by fits to single exponentials. Data are shown as mean ± SEM from 6 to 10 experiments. Note logarithmic ordinate in D. (E) Thermodynamic mutant cycle showing mutation-induced changes in the height of the free enthalpy barrier for the B→IB transition (ΔΔGT++-B0, numbers on arrows; k, Boltzmann’s constant; T, absolute temperature). Each corner is represented by the mutations introduced into positions 117 and 1124 of E1371S-CFTR. ΔΔGint(BT++)(purple number) is obtained as the difference between ΔΔGT++-B0 values along two parallel sides of the cycle.

If the phenotype caused by the R117H mutation is indeed due to loss of the R117-E1124 interaction, then a similar phenotype should be brought about by perturbations of position 1124. However, designing a suitable perturbation here is complicated by the fact that E1124 interacts through its peptide carbonyl oxygen. Notably, positioning of a backbone carbonyl group is unaffected by side chain substitutions, and its targeted elimination using nonsense suppression-based unnatural amino acid incorporation (Pless and Ahern, 2013) has not yet been developed. As expected, truncation of the E1124 side chain using the E1124A mutation did not affect closing rate (Figure 2A, violet trace; Figure 2B, violet bar), confirming that the E1124 side chain is not required for a normal burst duration. A slight shortening of τburst by the E1124G mutation suggested that increasing flexibility of ECL6 only marginally accelerates closing rate (Figure 2A, green trace; Figure 2B, green bar). We next examined whether insertion of a proline might distort ECL6 sufficiently to move the E1124 backbone carbonyl group out of reach of the R117 side chain. However, neither the E1124P (Figure 2A, orange trace; Figure 2B, orange bar) nor the E1126P (Figure 2—figure supplement 1) mutation caused a shortening of τburst. Thus, shortening of the ECL6 loop using a single-residue deletion appeared to be the only viable strategy to increase the distance between the R117 side-chain guanidino group and the nearest ECL6 backbone carbonyl (in this case that of G1123). Indeed, in our E1371S background construct introduction of the E1124Δ mutation produced a phenotype very similar to that of R117H, accelerating the macroscopic current decay (Figure 2A, red trace), and shortening τburst by ~6-fold (Figure 2B, red bar).

Energetic coupling between positions 117 and 1124 changes during non-hydrolytic closure

Functional effects of mutations of any individual amino acid residue in a protein provide little information regarding the role of a specific hypothesized residue-residue interaction, because in the WT protein the targeted residue might be involved in multiple interactions, all of which are altered by its mutations. To dissect and quantify the energetic effect of perturbing a specific residue-residue interaction, thermodynamic mutant cycle analysis must be employed (Vergani et al., 2005). The concept behind that approach is that the background construct, two single mutants that each perturb one or the other target residue, and the corresponding double-mutant form a thermodynamic cycle. If the two target residues do not interact, or if their interaction remains static (does not change between various gating states), then the energetic effects of mutating either target residue will be additive in the double mutant. Conversely, if the strength of the interaction between the target residues changes between gating states S1 and S2, then the effect on the relative stability of state S2 caused by mutating one target residue (ΔΔGS2-S10) will depend on the nature of the residue present at the other target position – thus, the energetic effects of the two target-site mutations will not be additive in the double mutant. The change in strength of the target interaction during step S1→S2 (ΔΔGint(S1S2)) is obtained as the difference between ΔΔGS2-S10 values along two parallel sides of the mutant cycle (Figure 2E).

Introducing the R117H mutation into an E1124Δ background caused only marginal further acceleration of E1371S non-hydrolytic closing rate (Figure 2C, green trace), shortening τburst ~1.47-fold (Figure 2D, green vs. red symbol). In energetic terms, the ~6-fold shortening of τburst brought about by the R117H mutation alone (Figure 2D, blue vs. black symbol) signals an ~1.8 kT decrease in the height of the free enthalpy barrier for the B→IB transition (ΔΔGT++-B0; Figure 2E, number on top horizontal arrow). In contrast, the ~1.47-fold acceleration caused by the same mutation in the E1124Δ background reports a decrease in the same barrier height by only ~0.39 kT (Figure 2E, number on bottom horizontal arrow). The difference between those two numbers quantifies the change in the strength of the R117-E1124 interaction in an E1371S channel while it progresses from the B state to the T state, on its way toward the IB closed state (Figure 2E, purple number). That value of ΔΔGint(BT++) = 1.4 ± 0.23 kT is significantly different from zero (p = 0.00043), and its positive signature reports that a stabilizing interaction present in the B state is lost in the T state. (The theoretical alternative of a lack of interaction in the B state but a destabilizing interaction in the T state is inconsistent with structural evidence.)

The stabilizing interaction between the target positions is present only in the bursting state

To address how the 117–1124 interaction changes between the IB state and the T state, mutational effects on opening rate need to be quantified in steady-state single-channel recordings. Because gating of the E1371S background construct is prohibitively slow for steady-state single-channel analysis, we employed a different non-hydrolytic background mutation, D1370N, which perturbs the Walker B aspartate side chain that coordinates catalytic Mg2+ in site 2 (Hung et al., 1998; Rai et al., 2006; Gunderson and Kopito, 1995). This mutation disrupts ATP hydrolysis but results in shorter burst durations, allowing steady-state kinetic analysis of the IB↔B gating process (Csanády et al., 2010; Sorum et al., 2015). The R117H, E1124Δ, and R117H/E1124Δ target-site mutations were introduced into this background, and gating of single prephosphorylated channels was studied in the presence of 5 mM ATP (Figure 3A), a saturating concentration for all four constructs (Figure 3—figure supplement 1). In such steady-state recordings flickery and IB closures can be readily discriminated by dwell-time analysis (Materials and methods; Figure 3—figure supplement 2), allowing calculation of not only τburst, but also of mean IB durations (τinterburst) (Table 1).

Figure 3 with 3 supplements see all
The H-bond between the target positions is formed only in the bursting state.

(A) Single-channel currents in 5 mM ATP of indicated CFTR mutants (color coded) harboring the D1370N background (BG) mutation. Channels were pre-phosphorylated by ~1 min exposure to 300 nM PKA +5 mM ATP. Vm was –80 mV. (B, D) Mean burst (B) and interburst (IB) (D) durations obtained by steady-state dwell-time analysis (see Materials and methods). Data are shown as mean ± SEM from five to eight patches; note logarithmic ordinates. (C, E) Thermodynamic mutant cycles showing mutation-induced changes in the height of the free enthalpy barrier for the (C) B→IB and (E) IB→B transition (ΔΔGT++-B0 and ΔΔGT++-IB0, respectively, numbers on arrows; k, Boltzmann’s constant; T, absolute temperature). Each corner is represented by the mutations introduced into positions 117 and 1124 of D1370N-CFTR. ΔΔGint (purple number) is obtained as the difference between ΔΔG0 values along two parallel sides of the cycle. (F) (Top) Free enthalpy profile of the entire channel protein (black curve) and of the 117–1124 interaction (purple curve) along the states of the IB↔B gating process (see Materials and methods). (Bottom) Cartoon representation of channel conformations in the IB, T, and B states. Color coding as in Figure 1A; ATP, yellow circles. Blue and red moieties represent the R117 side chain and the E1124 backbone carbonyl group, respectively. Yellow star in state B represents formation of the H-bond.

Table 1
Model-independent descriptive gating parameters for the indicated mutant CFTR constructs in the D1370N background.

Gating parameters were obtained from steady-state recordings as described in Materials and methods. Data are displayed as mean ± SEM from five to eight patches.

τburst (ms)τinterburst (ms)τflicker (ms)nflickerPo
Background (D1370N)2000 ± 2505100 ± 110010 ± 1.64.8 ± 0.820.35 ± 0.056
R117H67 ± 8.82800 ± 80033 ± 5.01.2 ± 0.120.017 ± 0.0055
E1124Δ100 ± 164800 ± 81023 ± 4.02.1 ± 0.410.013 ± 0.0033
R117H E1124Δ58 ± 4.43200 ± 88050 ± 7.91.1 ± 0.0990.0054 ± 0.0011

Mutational effects on closing rate resembled those observed in the E1371S background: both the R117H and the E1124Δ mutation robustly shortened τburst (by ~30-fold and ~20-fold, respectively, Figure 3B, blue and red symbols, respectively, vs. black symbol), whereas little further shortening was observed in the double mutant (Figure 3B, green symbol). Thus, whereas the ~30-fold shortening of τburst by the R117H mutation alone (Figure 3B, blue vs. black symbol) reports a decrease in ΔΔGT++-B0 by ~3.4 kT (Figure 3C, top horizontal arrow), the ~1.76-fold shortening of τburst by the same mutation in the E1124Δ background (Figure 3B, green vs. red symbol) reports a decrease in the height of the same barrier by only ~0.57 kT (Figure 3C, bottom horizontal arrow). The disparity between those two values, ΔΔGint(BT++) = 2.8 ± 0.23 kT (Figure 3C, purple number), is significantly different from zero (p = 0.000034) confirming disruption of a strong stabilizing interaction between the target positions during the B→T transition.

In contrast, channel opening rates were little affected by the target-site mutations, with τinterburst values remaining within 2-fold for all four constructs (Figure 3D, Table 1). Correspondingly, the calculated change in the strength of the 117–1124 interaction during step IB→T, ΔΔGint(IBT++) (Figure 3E, purple number), was not significantly different from zero. Of note, correct estimation of τinterburst strongly depends on correct estimation of the number of channels in the patch, which becomes increasingly difficult when the Po is very low. Thus, for our target-site mutants and the double mutant the presence of more than one channel in the patch was excluded by strongly boosting channel Po at the end of each recording using N6-(2-phenylethyl)-dATP (P-dATP) and/or the potentiator drug Vx-770 (Figure 3—figure supplement 3, also see Materials and methods). Nevertheless, compared to τburst, estimates of τinterburst necessarily retain some degree of uncertainty, as the number of active channels can never be determined with 100% confidence (cf., Yu et al., 2016).

Taken together, these steady-state data reveal that in D1370N channels a strong stabilizing interaction between positions 117 and 1124 is formed in state B, but is absent in states T and IB (Figure 3F, purple bond energy profile; Figure 3F bottom, cartoon).

The H-bond between the target positions is broken in the flickery closed state

The bursting state comprises the open (O) and flickery closed (Cf) states. If the strength of an interaction changes between states O and Cf then perturbing that interaction should affect intraburst Po (PO|B), that is, the fraction of time the pore is open within a burst. To address whether the R117-E1124 H-bond is disrupted in the flickery closed state, we studied additivity of effects of the R117H and E1124Δ mutations on the intraburst closed-open equilibrium constant Keq|B, obtained as Keq|B = PO|B/(1−PO|B). Effects on intraburst gating kinetics are most conveniently studied in the E1371S background, by briefly applying ATP to activate the channels and then focusing on the last bursting channel following ATP removal (Figure 4A, Csanády et al., 2010; Yu et al., 2016). Because in the absence of bath ATP reopening from the IB state is no longer possible, in such ‘last-channel’ segments of record all closures (except for the final) are necessarily flickery closures, and the long life time of the B state in E1371S allows sampling of a large number of O↔Cf transitions.

The H-bond between the target positions is broken in the flickery closed state.

(A) Currents of last open channels surviving ATP removal, for indicated CFTR mutants (color coded) harboring the E1371S background mutation. Inside-out patch currents were activated by exposure of pre-phosphorylated channels to 5 mM ATP. Insets (right) show 5 s segments corresponding to gray boxes at an expanded time scale. Vm was –80 mV. (B) Closed-open equilibrium constants within a burst (see Materials and methods). Data are shown as mean ± SEM from five to six patches; note logarithmic ordinate. SEM was calculated by error propagation (see Materials and methods). (C) Thermodynamic mutant cycle showing mutation-induced changes in the stability of the O state relative to the Cf state (ΔΔGO-Cf0, numbers on arrows; k, Boltzmann’s constant; T, absolute temperature). Each corner is represented by the mutations introduced into positions 117 and 1124 of E1371S-CFTR. ΔΔGint (purple number) is obtained as the difference between ΔΔG0 values along two parallel sides of the cycle. (D) (Top) Free enthalpy profile of the entire channel protein (black curve) and of the 117–1124 interaction (purple curve) along the states of the intraburst (O↔Cf) gating process (see Materials and methods). (Bottom) Cartoon representation of channel conformations in the Cf and O states. Color coding as in Figure 3F.

As reported previously (Yu et al., 2016), the R117H mutation caused a strong reduction in PO|B, due to an ~20-fold shortening of mean open times (τopen) and a 5-fold prolongation of mean flickery closed times (τflicker) (Figure 4A, insets, blue vs. black trace; Table 2). Interestingly, this aspect of the R117H phenotype was also reproduced by the E1124Δ mutant, which demonstrated a similarly lowered PO|B (Figure 4A, red trace, Table 2). Although PO|B was even further reduced for R117H-E1124Δ (Figure 4A, green trace, Table 2), the effects of the two single mutations on Keq|B were not additive in the double mutant. Whereas the ~90-fold reduction in Keq|B caused by the R117H mutation alone (Figure 4B, blue vs. black symbol) reported an increase in the Cf-O free enthalpy difference (ΔΔGO-Cf0) by ~4.5 kT (Figure 4C, top vertical arrow), the same mutation reduced Keq|B only ~10-fold (Figure 4B, green vs. red symbol), amounting to ΔΔGO-Cf0 = ~ 2.3 kT (Figure 4C, bottom vertical arrow), when introduced into an E1124Δ background. The obtained interaction energy, ΔΔGint(CfO) = 2.2 ± 0.34 kT (Figure 4C, purple number), is significantly different from zero (p = 0.0017) indicating that, even within the bursting state, the R117-E1124 H-bond is formed only in the open state, not in the flickery closed state (Figure 4D, purple bond energy profile; Figure 4D bottom, cartoon).

Table 2
Model-independent descriptive parameters of intraburst gating for the indicated mutant CFTR constructs in the E1371S background.

Gating parameters were obtained from last-open-channel recordings as described in Materials and methods. Data are displayed as mean ± SEM from five to six patches.

τflicker (ms)τopen (ms)Keq|B
Background (E1371S)11 ± 2.3200 ± 2717 ± 3.9
R117H58 ± 7.311 ± 1.10.20 ± 0.031
E1124Δ42 ± 6.018 ± 1.70.42 ± 0.070
R117H E1124Δ150 ± 186.2 ± 0.500.042 ± 0.0060

Both kinetic schemes used to describe CFTR bursting behavior adequately explain observed effects

CFTR’s bursting behavior may be explained by two alternative linear three-state models, Cs(low)↔Cf(ast)↔O or Cs↔O↔Cf. Because those two models cannot be differentiated in any individual steady-state recording, the true model is still unknown. All the analysis presented so far was therefore performed in a model-independent manner. Nevertheless, we also sought to address whether the mutational effects observed here might provide any support for one or the other kinetic mechanism.

Maximum likelihood fits (Csanády, 2000b) by the Cs↔Cf↔O and Cs↔O↔Cf model of steady-state single-channel recordings, obtained for the four constructs in the D1370N background (Figure 3A), yielded rate constants for all four microscopic transition rates of the respective scheme (Figure 5C and D, center, average rates (in s–1) are color coded by mutation). Thus, for both models, a mutant cycle could be built on each of the four transition rates, to quantitate ΔΔGint of the target H-bond for the respective gating step (Figure 5—figure supplement 1). Finally, those four ΔΔGint values allowed reconstruction of the entire 117–1124 interaction free enthalpy profile assuming one or the other model (Figure 5A–B, purple plots). Assuming either model, these profiles predict formation of the target H-bond selectively in state O. Correspondingly, a comparison of the standard free enthalpy profiles of gating for each of the four constructs (Figure 5C–D, top), calculated for both models from the fitted microscopic transition rate constants (see Materials and methods), unanimously predict a predominant destabilization of the O state by the target-site mutations (Figure 5C–D, top, colored profiles vs. black profile). Moreover, using the Cs↔O↔Cf model the change in target bond strength between states O and Cs (~2.6 kT) and between states O and Cf (~2.5 kT) turned out to be nearly identical (Figure 5A). All in all, the data are equally consistent with either model.

Figure 5 with 1 supplement see all
Both alternative linear three-state models adequately explain the effects of the mutations.

(A–D) Free enthalpy profiles (A–B) of the 117–1124 interaction in D1370N-CFTR, and (C–D) of the entire channel protein for various mutants (color coded) in the D1370N background, along all states of the gating process assuming either the Cs↔O↔Cf (A, C) or the Cs↔Cf↔O (B, D) model. The profiles in (A–B) were obtained from the ΔΔGint values for the four gating steps, those in (C–D) from the microscopic transition rate constants (numbers in s–1, color coded by mutants) obtained by fitting either model to the steady-state recordings in Figure 3A (see Materials and methods). Cartoons in C–D (bottom) represent the channel conformations in the Cs, O, and Cf states. Color coding as in Figure 3F.

Discussion

Here, we have shown that a strong H-bond between the side chain of R117 and the backbone carbonyl group of E1124 is formed in the open state but neither in the IB nor in the flickery closed state of the CFTR channel. We have further shown that loss of that hydrogen bond is responsible for the strong gating phenotype caused by CF mutation R117H, including a dramatic shortening of burst durations and a strong reduction of intraburst Po. These findings have clarified the molecular pathology of a relatively common CF mutation (and of the less frequent variants R117C/G/L/P) and might prove useful for targeted design of better potentiator drugs for this mutant (see Ideas and speculation).

The fact that it is the backbone carbonyl group, not the side chain, of E1124 that participates in this H-bond is consistent with several observations. First, mutations of position 1124 have not been identified in CF patients, consistent with our conclusion that the E1124 side chain does not play an important role. Second, whereas the arginine at the position corresponding to 117 in hsCFTR is absolutely conserved across species, the residue at the position equivalent to 1124 is not (Figure 2—figure supplement 2). On the other hand, the lengths of both ECL6 and ECL1 are absolutely conserved (Figure 2—figure supplement 2), consistent with 3D spacing between those two loops playing an important role.

Based on homology modeling the R117 side chain was earlier proposed to interact with the E1126 side chain (Cui et al., 2014). Our data suggest that is not the case, as mutation E1126P which truncates the E1126 side chain did not result in an R117H-like phenotype, but rather prolonged τburst (Figure 2—figure supplement 1). The reason for the latter effect is unclear, but might be due to changes in flexibility of the ECL6 loop which natively contains three glycines (sequence: GEGEG; Figure 2—figure supplement 2). Mutation E1126P (GEGEG → GEGPG) is expected to reduce, whereas mutation E1124G (GEGEG → GGGEG) is expected to increase ECL6 flexibility: correspondingly, τburst was prolonged by the former, but slightly shortened by the latter (Figure 2A–B, green) mutation. Of note, although the cryo-EM structures of quasi-open and closed CFTR do not accurately represent the fully open and the active closed state, respectively, in both structures, the E1126 side chain points away from R117.

Based on the structures the IB→B transition involves a large conformational rearrangement during which dozens of intramolecular interactions are broken and dozens of others are formed (Liu et al., 2017; Zhang et al., 2018a). How can disruption of a single H-bond result in such a pronounced gating phenotype as that caused by the R117H mutation? Although both conformations of the protein are stabilized by large numbers of local interactions, evolution has carefully balanced the energetic contributions of these, such that net ΔG0 for the overall IB→B protein conformational change is not far from zero. Indeed, for the D1370N background construct that net ΔG0 for the entire protein (~1 kT; Figure 3F, black free enthalpy profile) is smaller than the bond energy of our single target H-bond (~2.6 kT; Figure 3F, purple free enthalpy profile). Thus, elimination of that single H-bond, selectively present in the B state, can profoundly shift the gating equilibrium toward the IB conformation.

In contrast, based on its fast kinetics, the O↔Cf transition may involve a smaller conformational change with only a few local interactions broken/formed. Because the overall ΔG0 for that transition is again comparable to that of formation/disruption of the 117–1124 H-bond (~2.9 kT vs. ~2.2 kT in the E1371S background construct; Figure 4D, black vs. purple free enthalpy profile), that bond might represent a key interaction that governs intraburst gating in CFTR. In the cryo-EM structure of phosphorylated ATP-bound zebrafish CFTR (Zhang et al., 2017; pdbid: 5w81), although the NBDs are dimerized and the TMDs outward-facing, the external extremity of the pore is tightly sealed. The latter feature is due to a localized displacement of the external portions of TM helices 1 and 12, as compared to the quasi-open structure of human CFTR. Interestingly, that displacement increases the ECL1-ECL6 distance in the zebrafish structure, moving the residues equivalent to our target positions (R118 and D1132) too far away to form an H-bond. Thus, out of two suggested possible alternative explanations (Zhang et al., 2017), our findings support the interpretation (Zhang et al., 2018b) that the zebrafish outward-facing structure might represent the flickery closed state (cf., cartooned Cf state in Figures 45).

The magnitude of the effect on B-state stability of disrupting the 117–1124 H-bond depended on the choice of the non-hydrolytic model: τburst was shortened by ~6-fold in the E1371S (Figure 2C–D), but by ~30-fold in the D1370N (Figure 3A–B) background. That discrepancy suggests that the precise arrangement of the ECLs in the B state is not exactly identical in those two constructs, so that the distance between the two target positions, and thus the strength of the H-bond between them, is slightly different (1.4 kT [Figure 2E] vs. 2.8 kT [Figure 3C]). Which of those two values is more representative of a WT channel? Considering the effect of the R117H mutation on τburst of WT CFTR, a semi-quantitative lower estimate can be made. In an earlier study (Yu et al., 2016) the rate of the B→IB transition was increased from ~3.5 s–1 in WT CFTR to ~11 s–1 in R117H CFTR. Since the life time of the posthydrolytic B state is very short (Csanády et al., 2010), that rate essentially reflects the life time of the prehydrolytic B state, and is approximated by the sum of the rates for ATP hydrolysis (kh) and for non-hydrolytic closure (knh). In WT channels hydrolytic closure dominates (knh<< kh; Csanády et al., 2010), that is, kh >3 s–1, knh <0.5 s–1 (cf., knh~0.5 s–1 for the fastest-closing among several known non-hydrolytic models, D1370N [Figure 3B, black symbol]). Based on the plausible assumption that the ECL1 mutation R117H does not alter the catalytic rate in site 2 of the NBD dimer, in R117H CFTR kh remains ~3 s–1, and thus knh is ~8 s–1. These arguments provide a lowest possible estimate of ~16-fold for the increase in non-hydrolytic closing rate caused by the R117H mutation in WT CFTR, more in line with our findings in the D1370N background.

CFTR bursting may be accounted for by two possible linear three state models, Cs↔O↔Cf and Cs↔Cf↔O. In the past, multiple studies have addressed whether ATP dependence (Winter et al., 1994), voltage dependence (Cai et al., 2003), or pH dependence (Chen et al., 2017) of CFTR gating might allow differentiation between those two mechanisms, but in each case both models proved equally adequate to explain the data. Because the 117–1124 H-bond is formed only in state O but not in state Cs or Cf (Figures 34), we reasoned that mutant cycles built on microscopic transition rates obtained by fits to one or the other model might provide some support for a choice. In particular, if analysis based on the Cs↔O↔Cf model had returned significantly different values of ΔΔGint for the Cs↔O and Cf↔O steps, that would have rendered this model highly unlikely, given that in both of those steps ΔΔGint should reflect the strength of the same H-bond. However, no such discrepancy emerged from our analysis (Figure 5A), thus, our data are adequately explained by either model. Moreover, there is currently no structural evidence to support the existence of a fixed order of transitions among the three channel conformations seen in the cryo-EM structures of dephosphorylated human, phosphorylated ATP-bound zebrafish, and phosphorylated ATP-bound human CFTR, which are thought to resemble states Cs, Cf, and O, respectively (cartooned in Figure 5C, bottom). Thus, more structural information will be required to finally settle this question.

Ideas and speculation

The findings presented here define ECL1-ECL6 as a candidate drug target region on the CFTR protein. Pharmacological stabilization of the open-state interaction between those two loops might efficiently stimulate R117H (and R117C/G/L/P) CFTR channels present in CF patients. Because the target region is extracellular, potentiator compounds acting by such a mechanism would not have to be membrane permeable, that is, could be administered in inhalation form to stimulate CFTR channels in the lung.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
biological sample (Xenopus laevis)Xenopus laevis oocytesEuropean Xenopus Resource CentreRRID: NXR_0.0080
Commercial assay or kitHiSpeed Plasmid Midi KitQiagen12643
Commercial assay or kitQuickChange II Mutagenesis KitAgilent Technologies200524
Commercial assay or kitmMESSAGE mMACHINE T7 Transcription KitThermoFisher ScientificAM1344
Chemical compound, drugCollagenase type IIThermoFisher Scientific17101–015
Chemical compound, drugAdenosine 5′-triphosphoribose magnesium (ATP)Sigma-AldrichA9187
Chemical compound, drugProtein kinase A catalytic subunit, bovineSigma-AldrichP2645
Chemical compound, drugVx-770 (solid)Selleck ChemicalsS1144
Chemical compound, drugN6-(2-phenylethyl)-dATPBiolog LSID 104
Software, algorithmPclamp9Molecular DevicesRRID: SCR_011323

Molecular biology

Request a detailed protocol

Mutations were introduced into the human CFTR(E1371S)/pGEMHE and CFTR(D1370N)/pGEMHE sequences using the QuikChange II XL Kit (Agilent Technologies) and confirmed by automated sequencing (LGC Genomics GmbH). To generate cRNA, the cDNA was linearized (Nhe I HF, New England Biolabs) and transcribed in vitro using T7 polymerase (mMessage mMachine T7 Kit, Thermo Fisher), cRNA was stored at –80°C.

Functional expression of human CFTR constructs in Xenopus laevis oocytes

Request a detailed protocol

Oocytes were removed from anesthetized Xenopus laevis following Institutional Animal Care Committee guidelines, and separated by collagenase treatment (Collagenase type II, Gibco). Isolated oocytes were kept at 18°C, in a modified frog Ringer’s solution (in mM: 82 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.5 with NaOH) supplemented with 1.8 mM CaCl2 and 50 μg/ml gentamycin. Injections by 0.1–10 ng of cRNA, to obtain microscopic or macroscopic currents, was done in a fixed 50 nl volume (Nanoject II, Drummond). Recordings were performed 1–3 days after injection.

Excised inside-out patch recording

Request a detailed protocol

The patch pipette solution contained (in mM): 138 NMDG, 2 MgCl2, 5 HEPES, pH = 7.4 with HCl. The bath solution contained (in mM): 138 NMDG, 2 MgCl2, 5 HEPES, 0.5 EGTA, pH = 7.1 with HCl. Following excision into the inside-out configuration patches were moved into a flow chamber in which the composition of the continuously flowing bath solution could be exchanged with a time constant of <100 ms using electronic valves (ALA-VM8, Ala Scientific Instruments). CFTR channel gating was studied at 25°C, in the presence of 5 mM MgATP (Sigma), following activation by ~1 min exposure to 300 nM bovine PKA catalytic subunit (Sigma P2645). Macroscopic currents were recorded at –20 mV, microscopic currents at –80 mV membrane potential. For the low-Po mutants R117H, E1124Δ, and R117H-E1124Δ, evaluation of the number of active channels in the patch was facilitated by stimulating the channels with 50 μM N6-(2-phenylethyl)-dATP (P-dATP; Biolog LSI), with or without 10 nM Vx-770 (Selleck Chemicals), at the end of each experiment. Currents were amplified and low-pass filtered at 1 kHz (Axopatch 200B, Molecular Devices), digitized at a sampling rate of 10 kHz (Digidata 1550B, Molecular Devices) and recorded to disk (Pclamp 11, Molecular Devices).

Kinetic analysis of electrophysiological data

Request a detailed protocol

To obtain relaxation time constants (Figure 2B and D), macroscopic current relaxations were fitted to single exponentials by non-linear least squares (Clampfit 11).

For kinetic analysis of steady-state single-channel recordings in the D1370N background, baseline-subtracted unitary currents, from recordings with no superimposed channel openings, were Gaussian-filtered at 100 Hz and idealized by half-amplitude threshold crossing. Recordings were further analyzed only if the presence of a second active channel could be excluded with >90% confidence using statistical tests described earlier (Csanády et al., 2000a). All events lists were fitted with both the C1(s)↔O3↔C2(f) and the C1(s)↔C2(f)↔O3 model using a maximum likelihood approach which accounted for the presence of an imposed fixed dead time of 4 ms, and returned a set of microscopic transition rate constants k12, k21, k23, and k32 for both models (Csanády, 2000b). As verified on simulated data sets analyzed at the same bandwidth, these rate estimates remained unbiased even for the low-Po constructs. The descriptive kinetic parameters τburst, τinterburst, τflicker, and nflicker (average number of flickery closures per burst) were calculated from the rate constants as follows Colquhoun and Sigworth, 1995. For the C1↔O3↔C2 model τburst=(1/k31)(1+k32/k23), τinterburst=1/k13, τflicker=1/k23, nflicker=k32/k31. For the C1↔C2↔O3 model τburst=(1/k21)((k21+k23)/k32+k23/(k21+k23)), τinterburst=((k12+k21+k23)/(k12k23)) + 1/(k21+k23), τflicker=1/(k21+k23), nflicker=k23/k21. Importantly, fits using either model necessarily yield identical sets of calculated parameters τburst, τinterburst, τflicker, and nflicker, thus, the latter descriptive parameters (Figure 3B–D, Table 1) are model-independent. The rate of the IB→B step was taken as 1/τinterburst, and that of the B→IB step as 1/τburst.

For intraburst kinetic analysis of the last open channel in the E1371S background, mean open times (τopen) and mean flickery closed times (τflicker) were obtained as the simple arithmetic averages of the mean open and closed dwell-time durations, respectively, and Keq|B was calculated as Keq|B=τopen/τflicker (Figure 4B, Table 2).

Mutant cycle Analysis

Request a detailed protocol

Changes in the strength of the R117-E1124 interaction between various gating states were evaluated using thermodynamic mutant cycle analysis as described (Mihályi et al., 2016). For an S1↔T↔S2 gating step mutation-induced changes in the relative stabilities of those three states were calculated as follows. The change in the height of a transition-state barrier, for example, for step S1→T, was calculated as ΔΔGT++-S10=kTln(r`12/r12), where k is Boltzmann’s constant, T is absolute temperature, and r12 and r`12 are the rates for the S1→S2 transition in the background construct and in the mutant, respectively. The change in the stability of the two ground states relative to each other was calculated as ΔΔGS2S10=kTln(Keq/Keq), where Keq and K`eq are the equilibrium constants for the S1↔S2 transition in the background construct and in the mutant, respectively. Interaction free energy (ΔΔGint) was defined as the difference between ΔΔG0 values along two parallel sides of a mutant cycle. All ΔΔG values are given as mean ± SEM; SEM values were estimated assuming that rij and Keq are normally distributed random variables, using second-order approximations of the exact integrals (Mihályi et al., 2016).

Calculation of free enthalpy profiles

Request a detailed protocol

To construct standard free enthalpy profiles for channel gating (Figures 3F and 4D, black profiles; Figure 5C–D) ΔΔG0 between two ground states was calculated as ΔΔG0 = −kT lnKeq. For example, for Figure 3F ΔΔG0(IB→B) = −kT ln( τburst/(τinterburst)), values for τburst and τinterburst are given in Table 1; for Figure 4D, ΔΔG0(Cf→O) = −kT ln(τopen/(τflicker)); values for τopen and τflicker are given in Table 2. The SEM for Keq|B in Figure 4B was calculated by error propagation from the variances of τopen and τflicker. The absolute values of the barrier heights were not calculated (see breaks), but the relative heights of the barriers were appropriately scaled as follows. The difference between the heights of two alternative barriers for exiting a single source state was calculated as ΔΔGT1++-T2++0=kTln(r1/r2), where r1 and r2 are the rates of exit along those two pathways. Similarly, the difference between the heights of a given barrier for two different constructs was calculated using the same equation, but with r1 and r2 reflecting the corresponding transition rates for constructs 1 and 2, respectively.

Statistics

All values are given as mean ± SEM, with the numbers of independent samples provided in each figure legend. Statistical significances of interaction free energies were calculated using Student’s t test, ΔΔGint is reported significantly different from zero for p < 0.05.

Data availability

All data generated or analysed during this study are included in the main text figures, tables, and supporting figures.

References

  1. Book
    1. Colquhoun D
    2. Sigworth FJ
    (1995) Fitting and statistical analysis of single-channel records
    In: Sakmann B, Neher E, editors. In Single Channel Recording. New York: Plenum Press. pp. 483–587.
    https://doi.org/10.1007/978-1-4419-1229-9

Decision letter

  1. Andrés Jara-Oseguera
    Reviewing Editor; The University of Texas at Austin, United States
  2. Richard W Aldrich
    Senior Editor; The University of Texas at Austin, United States
  3. Andrés Jara-Oseguera
    Reviewer; The University of Texas at Austin, United States
  4. Tzyh-Chang Hwang
    Reviewer

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Molecular pathology of a cystic fibrosis mutation is explained by loss of a hydrogen bond" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Andrés Jara-Oseguera as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Tzyh-Chang Hwang (Reviewer #2).

The reviewers have discussed their reviews with one another, and are all in agreement that the study is interesting and that the data and analysis are of high quality. However, the reviewers also identified a series of weaknesses in the study that would need to be addressed, the main one being the lack of direct structural evidence in support of the open-state dependence of the interaction between R117 and the main-chain carbonyl of residue E1224. Based on the discussion between reviewers, the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

Title:

1. The title should be improved. As the authors stated in the abstract and introduction, CFTR has a wide variety of mutations and a broad range of pathologies. This paper focuses on the R117-E1124 interaction and therefore does not reflect all molecular pathologies of CFTR. A title that reflects the focus of this paper is considered more appropriate.

Introduction:

1. P3 first sentence: Gut and intestine are the same thing.

2. P3 second sentence: The actual CF-causing mutations are less than 1700. Many of the mutations in the data bank are polymorphisms.

3. P3: The sentence "Treatment by.…" may need revision.

4. P3 second sentence of the second paragraph: Please spell out the R domain.

5. P3: I would caution the authors not to use "inward-facing" and "outward-facing" to describe CFTR gating conformational changes since there is really no typical outward-facing configuration in CFTR.

6. P4: The major reason for a drastic reduction of the Po by the R117H mutation is actually not a shortening of the burst duration, but a > 3-fold prolongation of the interburst duration (see Yu et al., 2016). The authors seem to discount this observation throughout the manuscript. It is therefore not appropriate to claim that "loss of this hydrogen bond explains the entire gating phenotype of the R117H mutant" in the Abstract. This should be addressed in the manuscript text.

Results:

1. P6: Please specify the duration of exposure to 5 mM ATP.

2. P7, line 8: Without extensive studies of E1124A, it is probably premature to claim "the E1124 side chain is not required for normal gating."

3. P8 last sentence: The D1370N mutation does not "remove" the Walker B glutamate side chain.

4. P9: It seems that the τinterburst for D1370N in the current study is much larger than one reported previously (5 s versus 2 s in Sorum et al., 2017). Previous work was done in a background that is independent of phosphorylation. Doesn't this discrepancy implicate the importance of phosphorylation on the estimated τinterburst? Indeed, the model proposed in Mihalyi et al. (2020) also suggests that the level of phosphorylation in the R domain can affect τinterburst. In light of a lack of stringent control of phosphorylation in the oocyte expression system (Csanady et al., 2013) and the effect of R domain phosphorylation on the tinterburst, some discussion over this caveat is needed. This, plus the fact that extreme long interburst times pose a severe limit on the number of events collected (Figure 3-Figure Suppl 2), makes it very difficult to accurately estimate the τinterburst. This uncertainty out of technical and conceptual considerations should be borne in mind while discussing the opening rate.

5. Figure 3F: Please explain how 0.96 kT was obtained.

6. P11, line 8-10: Please revise.

7. It would strengthen the conclusions by the authors if they included in Figure 1B the experimental densities associated with the structures, to provide further support for the interaction on which the manuscript is based.

8. The authors should provide more information regarding the computational work. From the main text, it appears that the authors carried out molecular dynamics simulations, but the information in the methods suggests that the authors carried out molecular modeling. If MD simulations were carried out, it would strengthen the manuscript if the authors provided additional data with the MD trajectories showing that the structures on which the calculations were carried out are stable, and that the minimal distance shown in Figure 2 Suppl. 2 has the highest occupancy. If multiple models were compared, it would be also beneficial if the authors provided a distribution of the calculated distances in the top-scoring models, rather than simply showing the mean value.

9. The authors should comment on the potential uncertainty in estimating the intraburst gating parameters from the data in Figure 3A for the constructs with low open probability.

10. The authors should comment on the effects of R117H or E1124Δ on the intraburst kinetic parameters (Figure 4), which appear to suggest that the introduced structural perturbations are not entirely limited to the open state of the channel.

11. For all group data presented in figures (especially bar graphs), the authors should not only show the mean+/-S.E.M but also the individual data points.

Discussion:

1. It is clear that recent structure biological studies of CFTR have provided exquisite mechanistic insights into CFTR function as a phosphorylation-activated, ATP-gated chloride channel. However, the structures solved so far also raise a plethora of questions. Moreover, equating the unphosphorylated closed conformation to phosphorylated closed state seems problematic as previous studies suggest that the two NBDs in phosphorylated CFTR may not assume a conformation that is widely separated. While I applaud the authors' attempt to connect the dots between CFTR functional states and the available structures, I also believe that a more conservative approach is more appropriate at this stage. I therefore suggest that authors tone down their speculations in the Discussion.

2. P13, lines 353-356: A two-armed scale and a protein are energetically different systems. The authors are clearly stating their points in the paragraph already even without the metaphor. The metaphor is not necessary.

3. Since authors are mentioning the implications of the study for CF treatment with targeted potentiator drugs in the Abstract and Introduction, it would be interesting for readers if authors included their opinion on how the findings presented here could help to develop a potentiator drug. eLife now offers authors the opportunity to include an "Ideas and Speculation" subsection within their Discussion, which would enable the authors to offer some amount of speculation in this regard.

https://doi.org/10.7554/eLife.74693.sa1

Author response

Essential revisions:

Title:

1. The title should be improved. As the authors stated in the abstract and introduction, CFTR has a wide variety of mutations and a broad range of pathologies. This paper focuses on the R117-E1124 interaction and therefore does not reflect all molecular pathologies of CFTR. A title that reflects the focus of this paper is considered more appropriate.

We have made the title more focused by specifying the CF mutation in focus:

"Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond".

Introduction:

1. P3 first sentence: Gut and intestine are the same thing.

Amended, thank you.

2. P3 second sentence: The actual CF-causing mutations are less than 1700. Many of the mutations in the data bank are polymorphisms.

Thank you, corrected for "several hundred".

3. P3: The sentence "Treatment by.…" may need revision.

Revised, thank you.

4. P3 second sentence of the second paragraph: Please spell out the R domain.

Done, thank you.

5. P3: I would caution the authors not to use "inward-facing" and "outward-facing" to describe CFTR gating conformational changes since there is really no typical outward-facing configuration in CFTR.

We agree, and have actually discussed this issue in detail (original lines 97-100). Nevertheless, the structures do fall into two distinct groups: structures with separated NBDs and an inward-facing arrangement of TMDs, and structures with dimerized NBDs and an overall outward-facing arrangement of the TMDs. We now provide a more in-depth coverage of the limitations associated with the structures, and provide a clear definition for the nomenclature adopted in this paper:

"… the former structure (6msm) will be referred to as "outward-facing" or "quasi-open", and the latter (5uak) as "inward-facing" or "closed" throughout this study". (lines 103-115).

6. P4: The major reason for a drastic reduction of the Po by the R117H mutation is actually not a shortening of the burst duration, but a > 3-fold prolongation of the interburst duration (see Yu et al., 2016). The authors seem to discount this observation throughout the manuscript. It is therefore not appropriate to claim that "loss of this hydrogen bond explains the entire gating phenotype of the R117H mutant" in the Abstract. This should be addressed in the manuscript text.

In the Yu et al., 2016 study a direct comparison of WT and R117H opening rates (Figure 1B) revealed a ~25% decrease in opening rate for R117H relative to WT (interburst duration was ~300 ms for WT but ~400 ms for R117H), but the difference was claimed insignificant ("the R117H mutation probably also decreases the opening rate, although the difference in the closed time constant between WT- and R117H-CFTR is not yet statistically significant (Figure 1B)"). In contrast, in the same figure the mean burst duration of R117H was shown to be ~3-fold shorter than that of WT CFTR (~300 ms for WT but ~100 ms for R117H). These findings are in good agreement with those described in our study.

Although in later figures of the Yu et al. 2016 study the authors obtained somewhat smaller opening rates for R117H when accurate channel counting was facilitated by stimulating the channels with Vx770, no direct comparison was performed to evaluate how estimation of opening rate for WT CFTR is affected when a similar approach for channel counting is employed. Therefore, although the data in Yu et al. suggested that opening rate might perhaps be slightly affected by the R117H mutation, no direct proof for that hypothesis was provided. The Reviewer's claim that the "major reason for a drastic reduction of the Po by the R117H mutation.. is… a prolongation of the interburst duration" is therefore inconsistent both with the data in the Yu et al. 2016 study and those presented here. (Moreover, the ~3fold reduction in τb together with the drastic reduction in intraburst Po reported by Yu et al. adequately accounts for the ~6-fold reduction of CFTR whole-cell conductance by the R117H mutation observed earlier (Sheppard et al., 1993), without the postulation of a slowed opening rate.)

Of note, in the present study, and also in Sorum et al. 2017, estimation of opening rates was based exclusively on patches in which channel number could be estimated with high confidence following strong stimulation by Vx-770 and/or P-dATP (Figure 3—figure supplement 3) – these studies did not reveal any significant reduction of opening rate by the R117H mutation.

Nevertheless, we have changed the word "entire" to "strong" in the Abstract, and also acknowledge limitations of accurately estimating channel opening rate (lines 270-272).

Results:

1. P6: Please specify the duration of exposure to 5 mM ATP.

The duration of exposure to 5 mM ATP was ~ 1 minute, this has been added to the text (line 180).

2. P7, line 8: Without extensive studies of E1124A, it is probably premature to claim "the E1124 side chain is not required for normal gating."

We agree. We have toned down that claim:

"the E1124 side chain is not required for a normal burst duration" (line 193).

3. P8 last sentence: The D1370N mutation does not "remove" the Walker B glutamate side chain.

Thank you, moreover, D1370 is an aspartate not a glutamate. We now say "which perturbs the Walker B aspartate side chain" (line 242).

4. P9: It seems that the τinterburst for D1370N in the current study is much larger than one reported previously (5 s versus 2 s in Sorum et al., 2017). Previous work was done in a background that is independent of phosphorylation. Doesn't this discrepancy implicate the importance of phosphorylation on the estimated τinterburst? Indeed, the model proposed in Mihalyi et al. (2020) also suggests that the level of phosphorylation in the R domain can affect τinterburst. In light of a lack of stringent control of phosphorylation in the oocyte expression system (Csanady et al., 2013) and the effect of R domain phosphorylation on the tinterburst, some discussion over this caveat is needed.

The τib values reported for the D1370N mutant in various studies are indeed somewhat variable. The estimate in the present study is ~2x longer than that reported in Sorum et al. (2017) or Csanády et al. (2010), but comparable to that reported in Vergani et al. (2003) (all of those studies were done in the Xenopus oocyte expression system). Although phosphorylation does affect opening rate, we do not think that the above scatter is attributable to differential degrees of phosphorylation. The "lack of stringent control of phosphorylation in the oocyte expression system (Csanady et al., 2013)" mentioned by the Reviewer refers to the rapid partial current decline which is observed immediately upon PKA removal in inside-out patches excised from Xenopus oocytes and was earlier interpreted to reflect rapid dephosphorylation of a subset of phosphoserines in the R domain. However, the recent study by Mihalyi et al. (2020) has demonstrated that the rapid partial current decline reflects immediate loss of a direct stimulation provided by PKA binding, whereas endogenous phosphatase activity in the patches is minimal. Thus, in retrospect, in all of the above studies D1370N channels were likely fully phosphorylated. The differences are more likely to reflect biological variability due to unknown factors. Of note, such degree of variability is typical for biological systems, and is not inherent to the Xenopus oocyte expression system or the D1370N construct. (E.g., τib values ranging from 300 ms (Yu et al., 2016) to 500 ms (Tsai et al., 2010) have been reported for WT CFTR expressed in CHO cells.)

This, plus the fact that extreme long interburst times pose a severe limit on the number of events collected (Figure 3-Figure Suppl 2), makes it very difficult to accurately estimate the τinterburst. This uncertainty out of technical and conceptual considerations should be borne in mind while discussing the opening rate.

In each individual patch the numbers of interburst events analyzed were sufficient to estimate τib to within <20% of the mean value (for an exponential distribution S.E.M. = mean/sqrt(n); i.e., already for 25 events S.E.M. = 0.2*mean), and 5-8 patches were analyzed for each construct. Nevertheless, we agree that estimation of opening rates is more challenging than estimation of closing rates due to the uncertainty in the number of active channels, which can never be completely eliminated. This caveat was already discussed in the Results section (original lines 263-268), but we have now added the following sentence:

"Nevertheless, compared to τburst, estimates of τinterburst necessarily retain some degree of uncertainty, as the number of active channels can never be determined with 100% confidence (cf., (Yu et al., 2016))" (lines 270-272).

5. Figure 3F: Please explain how 0.96 kT was obtained.

This is described in Materials and methods (line 497, now expanded on lines 497-500):

∆∆G0(IB→B) = -kT ln(Keq) = -kT ln(Pburst/(1-Pburst)) = -kT ln(τburst/(τinterburst)) Values for τburst and τinterburst are given in Table 1.

6. P11, line 8 -10: Please revise.

Based on the explanation provided in our response to comment (10.), below, we believe that this sentence is adequately worded as written.

7. It would strengthen the conclusions by the authors if they included in Figure 1B the experimental densities associated with the structures, to provide further support for the interaction on which the manuscript is based.

Done. A close-up view of the R117-E1124 interaction in the quasi-open human CFTR structure is shown with the electron density in Fig. 1—figure supplement 1. Both loops are well represented, and density for the R117 side chain is visible down to the delta carbon.

8. The authors should provide more information regarding the computational work. From the main text, it appears that the authors carried out molecular dynamics simulations, but the information in the methods suggests that the authors carried out molecular modeling. If MD simulations were carried out, it would strengthen the manuscript if the authors provided additional data with the MD trajectories showing that the structures on which the calculations were carried out are stable, and that the minimal distance shown in Figure 2 Suppl. 2 has the highest occupancy. If multiple models were compared, it would be also beneficial if the authors provided a distribution of the calculated distances in the top-scoring models, rather than simply showing the mean value.

We acknowledge that the way we used the term "molecular dynamics simulations" was misleading. As described in Methods, our approach should be better referred to as modeling, although the program Modeler implements a molecular dynamics simulation run in the final optimization of loops. Although the results of these calculations rationalized our experimental approach, we have decided to omit this supplementary figure for two reasons. First, the strategy of using a deletion to try to shorten a loop is a trivial idea which does not require in silico modeling. Second, our use of Modeler for this purpose is unconventional, and would require extensive validation and testing to establish its reliability.

9. The authors should comment on the potential uncertainty in estimating the intraburst gating parameters from the data in Figure 3A for the constructs with low open probability.

The dead time of our low-pass (Gaussian) filter was 1.8 ms, but a 4-ms artifical dead time was imposed on the events lists to eliminate dwell-times with durations between td and ~2td that are distorted by filtering (Colquhoun and Sigworth, 1995). This bandwidth limitation indeed truncates the dwell-time distributions of the short-burst constructs (see Figure 3—figure supplement 2.). However, the ML fitting procedure employed for the extraction of gating parameters from the dwell-time distributions includes a robust correction for missed events, which was extensively verified on simulated data (Csanády, 2000). Thus, the obtained steady-state kinetic parameters (Figure 5) are reasonably reliable and allowed us to build mutant cycles even on the intraburst gating parameters (Figure 5—figure supplement 1). Of note, whereas interpretation of IB↔B gating rates (Figure 3) is model-independent, interpretation of the intraburst gating rates is model-dependent – as evident from Figures 5C-D. We have included a comment on the reliability of our kinetic analysis in methods (lines 463-465).

(In contrast, the dwell-time analysis of last open channels in the E1371S background (Figure 4A-B and Table 2) is based on simple mean intraburst open and closed times, which might be indeed slightly distorted by the 1.8-ms filter dead time. That is why we refrained from analyzing the intraburst gating rates in Figure 4, and provide only an analysis off Keq|B values which are (i) less sensitive to filtering, and (ii) also model-independent.)

10. The authors should comment on the effects of R117H or E1124Δ on the intraburst kinetic parameters (Figure 4), which appear to suggest that the introduced structural perturbations are not entirely limited to the open state of the channel.

The Reviewers point out that "if the interaction occurs only in the open state, then the transition from the Cf state to the open state should not be affected by any of the perturbations, but this rate seems to also become altered". The Reviewers are correct that the mutations affect not only the mean open time (τopen) but also the mean flickery closed time duration (τflicker) (Table 2); a similar pattern is observed also in the D1370N background (Table 1). However, the inference of an underlying stabilization of the flickery closed state by the H-bond is incorrect. Two points must be considered here.

i) The rate of opening from the flickery closed state reflects the height of the energy barrier (∆∆GTf-Cf). The mutation-induced prolongations of τflicker suggest that the mutations destabilize not only ground state O, but to some extent also the transition state Tf, relative to ground state Cf.

ii) Prolongation of τflicker by a mutation at either position 117 or 1124 provides little information regarding the role of the R117-E1124 H-bond, because in the WT protein the targeted residue might be involved in multiple interactions, all of which are altered by its mutation. To dissect and quantify the effect on τflicker of perturbing the target H-bond, thermodynamic mutant cycle analysis must be employed (see Page 7, bottom). From our analysis the energetic effects of the R117H and E1124∆ mutations on rate Cf→O appeared largely additive in the double mutant, i.e., ∆∆Gint(Cf→Tf) was not significantly different from zero, regardless of the chosen gating model (Figure 5—figure supplement 1D, G). This suggests that the H-bond is formed neither in state Cf nor in state Tf, but exclusively in state O, as depicted in the free energy profiles in Figure 5A-B.

11. For all group data presented in figures (especially bar graphs), the authors should not only show the mean+/-S.E.M but also the individual data points.

Done. (Except for Figure 4B, in which S.E.M. values were calculated by error propagation; see Figure 4 legend and Materials and methods.)

Discussion:

1. It is clear that recent structure biological studies of CFTR have provided exquisite mechanistic insights into CFTR function as a phosphorylation-activated, ATP-gated chloride channel. However, the structures solved so far also raise a plethora of questions. Moreover, equating the unphosphorylated closed conformation to phosphorylated closed state seems problematic as previous studies suggest that the two NBDs in phosphorylated CFTR may not assume a conformation that is widely separated.

Both the discrepancy between the outward-facing CFTR structure and the CFTR open state and that between the inward-facing structure and the CFTR closed state pointed out by the Reviewer have been discussed in our manuscript (original lines 97-100 and 155-158). In fact, it is exactly these shortcomings of the available structures that lend significance to our functional studies. Thus, it was not our intention to "equate" CFTR gating states with the existing structures, but rather to perform functional studies to test the validity and functional relevance of a putative interaction suggested by those structures. "The latter structure does not represent the closed state of an active channel gating in the presence of ATP… the identified 117-1124 H-bond might represent an interaction that changes dynamically… To study the functional relevance of this putative interaction…" (original lines 155-162). We acknowledge that all this information appeared somewhat scattered in the original text, which must have been confusing to the reader. To clarify these issues we have moved this entire information to the Introduction, and now devote a separate paragraph to the exposure of limitations associated with the available structures, as well as the nomenclature adopted in our study (lines 103115):

"The cryo-EM studies have provided unprecedented structural insight, but have left several questions open. […] Thus, the former structure (6msm) will be referred to as "outward-facing" or "quasi-open", and the latter (5uak) as "inward-facing" or "closed" throughout this study."

While I applaud the authors' attempt to connect the dots between CFTR functional states and the available structures, I also believe that a more conservative approach is more appropriate at this stage. I therefore suggest that authors tone down their speculations in the Discussion.

Discussion is focused on the functional aspects of our findings, cryo-EM structures are discussed in only two instances. One of these regarded the positioning of side chain E1126 in the open and closed state. The reference to the cryo-EM structures is actually not needed here, as our functional data suffice to rule out the involvement of the E1126 side chain in forming an H-bond with R117. The structures are now mentioned on lines 349-351, in a cautiously reworded note. The other instance in which a cryo-EM structure is referred to, that of zebrafish CFTR (lines 366-375), is adequately worded, as we do not make any firm statement here. We simply note that our functional data support one of two earlier interpretations of the structure. We believe that this level of "speculation" is adequate for a Discussion section.

2. P13, lines 353-356: A two-armed scale and a protein are energetically different systems. The authors are clearly stating their points in the paragraph already even without the metaphor. The metaphor is not necessary.

We have deleted the metaphor.

3. Since authors are mentioning the implications of the study for CF treatment with targeted potentiator drugs in the Abstract and Introduction, it would be interesting for readers if authors included their opinion on how the findings presented here could help to develop a potentiator drug. eLife now offers authors the opportunity to include an "Ideas and Speculation" subsection within their Discussion, which would enable the authors to offer some amount of speculation in this regard.

Done. ("Ideas and Speculation", lines 411-417.)

https://doi.org/10.7554/eLife.74693.sa2

Article and author information

Author details

  1. Márton A Simon

    1. Department of Biochemistry, Semmelweis University, Budapest, Hungary
    2. HCEMM-SE Molecular Channelopathies Research Group, Budapest, Hungary
    Contribution
    Formal analysis, Funding acquisition, Investigation, Software, Visualization, Writing – original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5411-9408
  2. László Csanády

    1. Department of Biochemistry, Semmelweis University, Budapest, Hungary
    2. HCEMM-SE Molecular Channelopathies Research Group, Budapest, Hungary
    3. MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary
    Contribution
    Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Software, Supervision, Validation, Writing – original draft
    For correspondence
    csanady.laszlo@med.semmelweis-univ.hu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6547-5889

Funding

Magyar Tudományos Akadémia (LP2017-14/2017)

  • László Csanády

Cystic Fibrosis Foundation (CSANAD21G0)

  • László Csanády

Ministry for Innovation and Technology (ÚNKP-20-3-I-SE-34)

  • Márton A Simon

European Union (739593)

  • László Csanády

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

Supported by EU Horizon 2020 Research and Innovation Program grant 739593, MTA Lendület grant LP2017-14/2017, and Cystic Fibrosis Foundation Research Grant CSANAD21G0 to LC. MAS received support from the ÚNKP-20–3-I-SE-34 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.

Ethics

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of Semmelweis University (last approved 06-03-2021, expiration 06-03-2026).

Senior Editor

  1. Richard W Aldrich, The University of Texas at Austin, United States

Reviewing Editor

  1. Andrés Jara-Oseguera, The University of Texas at Austin, United States

Reviewers

  1. Andrés Jara-Oseguera, The University of Texas at Austin, United States
  2. Tzyh-Chang Hwang

Publication history

  1. Received: October 13, 2021
  2. Preprint posted: October 21, 2021 (view preprint)
  3. Accepted: December 3, 2021
  4. Accepted Manuscript published: December 6, 2021 (version 1)
  5. Version of Record published: December 15, 2021 (version 2)

Copyright

© 2021, Simon and Csanády

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 320
    Page views
  • 65
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Physics of Living Systems
    2. Structural Biology and Molecular Biophysics
    Rui Fang et al.
    Research Article

    The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits1,2. How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.

    1. Neuroscience
    2. Structural Biology and Molecular Biophysics
    Johannes Elferich et al.
    Research Article Updated

    Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the ‘tip link.’ Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location, and conformational states of intact tip links, on stereocilia, remains unknown. Here, we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.