Cystic Fibrosis (CF) is an incurable, devastating inherited disease caused by mutations in the CF Transmembrane Conductance regulator (CFTR) chloride ion channel (Riordan et al., 1989). A large number of mutations identified in CF patients scatter throughout the entire protein sequence and cause defects in protein synthesis, maturation, stability, channel gating, and/or anion permeation through the open channel pore (O'Sullivan and Freedman, 2009). In recent clinical trials small molecule drugs that target the CFTR protein and rectify either processing (‘correctors’) or gating defects (‘potentiators’) have proven beneficial for some CF patients carrying specific mutations (Ramsey et al., 2011), underscoring the importance of understanding the structure and mechanism of this disease-associated protein at a molecular level.

CFTR belongs to the large family of ATP-Binding Cassette (ABC) proteins (Riordan et al., 1989), most of which function as active transporters (Dean and Annilo, 2005). A conserved molecular mechanism that involves ATP binding and hydrolysis drives opening and closing (gating) of the CFTR pore and substrate translocation through other ABC proteins (Li et al., 1996). The exceptional resolution of single-channel patch-clamp recordings makes CFTR a model ABC protein eminently suitable for molecular-level biophysical studies.

ABC proteins are built from two homologous halves each containing a transmembrane domain (TMD) and a cytosolic nucleotide binding domain (NBD). In CFTR these two canonical halves are linked by the unique regulatory (R) domain which must be either phosphorylated by cyclic AMP-dependent protein kinase (PKA) (Anderson et al., 1991), or deleted (Figure 1A) (Csanády et al., 2000) to allow channel gating. The highly conserved NBD fold comprises an alpha/beta ‘head’ subdomain harboring the Walker A and B motifs, and an alpha-helical ‘tail’ subdomain which contains the ABC-specific ‘signature’ sequence (Hung et al., 1998). Binding of ATP to the Walker motifs in each of the two NBDs promotes their association into a tight head-to-tail dimer that occludes the two bound ATP molecules at interfacial binding sites formed by the Walker motifs of one NBD and the signature motif of the other (Smith et al., 2002). Such NBD dimers are extremely stable but dissociate following ATP hydrolysis (Moody et al., 2002; Smith et al., 2002). Formation/disruption of the NBD dimer, communicated to the TMDs through an interface formed by four short coupling helices (CH1-4, Figure 1A, violet loops), drives the major conformational rearrangements of the substrate translocation pathway (Locher, 2009). Upon formation of the NBD dimer, the CFTR anion pore opens to a ‘burst’ state interrupted by brief (~10 ms) ‘flickery’ closures, and upon dimer disruption following ATP hydrolysis at the consensus site (‘catalytic site 2’, formed by NBD2 Walker motifs and NBD1 signature sequence) the channel returns to a long-lived (~1 s) ‘interburst’ closed state (Figure 1B; [Vergani et al., 2005]). The closed-pore CFTR structure is inward facing (Zhang and Chen, 2016; Liu et al., 2017), whereas the open-pore structure, with dimerized NBDs, likely resembles the outward-facing TMD conformation of ABC proteins. In a subset of ABC transporters, including CFTR, only one of the two composite sites is catalytically active, whereas the other contains non-canonical substitutions at several key residues that impair ATPase activity. In CFTR that degenerate ATP site (‘non-catalytic site 1’, formed by NBD1 Walker motifs and NBD2 signature sequence), keeps ATP bound but unhydrolyzed throughout several gating cycles (Aleksandrov et al., 2002; Basso et al., 2003). The precise range of gating-related movements in non-catalytic site 1 is unclear (Tsai et al., 2010b; Szollosi et al., 2011; Chaves and Gadsby, 2015), but mutations here impair ATP hydrolysis at the catalytic site 2 (Ramjeesingh et al., 1999), affect channel gating (Tsai et al., 2010a; Csanády et al., 2013), and may cause CF (e.g., G1349D).

Figure 1

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Resolving the relative timing of motions in various protein regions allows the molecular forces that drive protein conformational rearrangements to be understood. In particular, the structural organization of the transition (T) state for channel opening may shed light on the sources of molecular strain that underly the large Gibbs energy of this highest-energy intermediate conformation, which rate limits the overall transition (Csanády et al., 2006). Such T-state structures cannot be studied by standard structural approaches, as their life times fall into the sub-microsecond range (Chakrapani and Auerbach, 2005). However, relative timing of motions of a given channel position is reported by its Φ value, the slope of a log-log plot of the opening rate constant (k_CO) as a function of the closed-open equilibrium constant (K_eq = k_CO/k_OC) for a series of point substitutions (Brønsted plot). For the opening step of a simple idealized two-state channel with a single intervening T state, Φ is a measure of how far the conformation of the position has progressed along the reaction coordinate in the T state (0≤Φ ≤1). For a position that moves very early, and has therefore reached its open-like conformation in the T state (Φ = 1), perturbations affect T-state and open-state stabilities to similar extents, and thus impact only opening but not closing rate. At the other extreme, for a position that moves very late, and is still in its closed-like conformation in the T-state (Φ = 0), perturbations affect closing but not opening rate (Auerbach, 2007). In reality, the overall closed-to-open conformational change of large ion-channel proteins is best described by a sequence of conformational steps across a chain of high-energy intermediary states (the ‘transition-state ensemble’), and the Φ value of a position reports whether its perturbations affect early (large Φ) or late (small Φ) steps within that chain of events. However, on the plausible assumption that perturbation of a position affects the microscopic step during which the position undergoes a rearrangement, but not steps during which it remains static, the Φ value is reasonably interpreted to reflect relative timing of movements even in such cases (Zhou et al., 2005). The terms ‘early’ and ‘late’ will be used here to reflect sequentiality of movements based on this interpretation.

Because Φ value analysis requires equilibrium (Csanády, 2009), it can be applied to CFTR pore opening only in the presence of a background mutation (Figure 1A–B, red star) that disrupts ATP hydrolysis at catalytic site 2 (Figure 1B, red cross), thus reducing gating in saturating ATP to a simple closed-open equilibrium (C₁↔O₁; Figure 1B, red box). These conditions are satisfied in a background construct (Sorum et al., 2015) in which mutation of the NBD2 Walker B aspartate disrupts ATP hydrolysis and removal of the R domain obviates the need for prior phosphorylation (cut-ΔR(D1370N); Figure 1A). In this background, Φ value analysis detected a clear temporal gradient in opening-related movements which spreads along the longitudinal, cytoplasmic to extracellular, protein axis from catalytic site 2 towards the pore (Figure 1C): in the T state catalytic site 2 is already dimerized (Φ ~ 1 for position 1246), the NBD-TMD interface is on the move (Φ ~ 0.5 for position 275), whereas the pore is still shut (Φ ~ 0.2 for position 348), suggesting strain in the region of the main CF locus (Sorum et al., 2015).

Here we exploit the same background construct to compare the roles of the two ATP binding sites in pore opening, and find markedly asynchronous movements. An outline of the transition-state structure based on Φ values of eleven positions at four different levels along the longitudinal protein axis, from the two ATP binding sites to the extracellular surface, suggests distinctly different energetic roles for the two sites in supporting channel gating. This provides the first mechanistic clues for understanding the division of tasks and functional cross-talk between the catalytic and the non-catalytic ATP binding site in asymmetric ABC proteins. The findings also clarify the molecular mechanisms of the CFTR channel gating defects caused by two common CF causing mutations.

The closed-to-open transition of the CFTR channel is a major structural rearrangement which involves dimerization of the NBDs and flipping of the TMDs from an inward- to an outward-facing conformation ([Vergani et al., 2005], cf., homology models for phosphorylated closed and open CFTR channels in Figure 6A, left-to-right). Whereas X-ray or cryo-EM structures may capture stable closed (Zhang and Chen, 2016; Liu et al., 2017) and open ground states, the structure of the transition state traversed during opening, and the relative timing of motions in various protein regions may presently be inferred only from Φ value analysis (Auerbach, 2007). Previous work revealed a Φ value gradient along the longitudinal protein axis, suggesting that the opening conformational change spreads from catalytic site 2 towards the pore (Sorum et al., 2015). By expanding the axial distance spanned by our target positions here we show a gradient of Φ values from ~1 for both sides of catalytic ATP site 2, all the way to ~0 for the extracellular protein surface (Figure 6A). These findings corroborate the earlier proposal that in the T state catalytic site 2 is already dimerized but the pore is still in its closed-like (inward-facing) conformation.

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Phenylalanine 508, the locus of the most common CF mutation, is located at the NBD-TMD interface, that is, in a region expected to experience strain in the T state. Here we show that position 508 is indeed just on the move in the T state, together with spatially adjacent CH4 (Zhang and Chen, 2016; Liu et al., 2017), as reported by their intermediate, essentially indistinguishable Φ values of ~0.5 (Figures 4O and 5E). Because all tested perturbations of the F508 side chain reduce opening and accelerate non-hydrolytic closing rate (Figure 5B–C), the simplest interpretation is that the native phenylalanine side chain is involved in stabilizing interactions that become stronger as the closed channel reaches the T state, and even stronger when it relaxes to the open state. Loss (or weakening) of these interactions by 508-position perturbations destabilizes the T state relative to the closed state (reducing opening rate, Figure 5C; [Miki et al., 2010]), and further destabilizes the open state relative to the T state (enhancing non-hydrolytic closing rate, Figure 5B; [Jih et al., 2011]). The alternative explanation that the native phenylalanine side chain forms destabilizing interaction(s) in the closed state seems unlikely, as mutations here dramatically reduce protein stability, suggesting an overall stabilizing role for residue F508 in its native environment. Of note, because in wild-type (WT) CFTR the hydrolytic pathway for channel closure is much faster than the non-hydrolytic pathway, the strong acceleration of non-hydrolytic closing rate by deletion or substitutions of residue F508 is not apparent when studied in a WT (hydrolytic) background (Miki et al., 2010; Kopeikin et al., 2014), a fact that has led to previous misassignment of a Φ value close to 1 for position 508 (Aleksandrov et al., 2009).

Mutation R117H is associated with a mild form of CF and causes, beside a moderate reduction in single channel chloride conductance, a strong channel gating defect (Sheppard et al., 1993; Yu et al., 2016), suggesting that this position is involved in interactions that change in a gating-state dependent manner. Indeed, all substitutions at position 117 dramatically reduced open probability (Figure 2I) by accelerating closure (Figure 2G) while leaving opening unaffected (Figure 2H). Because in closed-state structures of CFTR the native R117 side chain is not seen to form strong interactions with other residues (Zhang and Chen, 2016; Liu et al., 2017), the implication is that it forms a stabilizing interaction (or interactions) in the open state, that is (are) lost when the arginine at position 117 is replaced. Furthermore, the stabilizing interaction is not yet formed in the T state: its appearance is among the last movements associated with pore opening (Φ ~ 0). Thus, R117 substitutions selectively destabilize the open state, but not the transition state, relative to the closed state, as indicated by the unaltered opening rate.

In striking contrast to the large Φ values of positions on both sides of the dimer interface in catalytic site 2, positions flanking non-catalytic site 1 display low-to-intermediate Φ values (Figure 6A, orange vs. red residues (left) and numbers (right)). Thus, unlike catalytic site 2 which has already adopted its open-like conformation (Figure 6A, right, red residues) in the T state, non-catalytic site 1 is still on the move between its closed- and open-like conformations (Figure 6A, left and right, orange residues). Interestingly, such pronounced asymmetry in the timing of motions at one end of the dimer interface compared to the other cannot be detected at the level of the four coupling helices, which are all characterized by Φ values of 0.5–0.6 (Figure 6A, violet residues (left) and numbers (right)). Thus, the movements that take place in non-catalytic site 1 between the T state and the open state likely remain confined to the site-1 interface.

ATP bound at the two composite sites acts as molecular glue that stabilizes the open-pore conformation by bonding the NBD interfaces together (Moody et al., 2002; Smith et al., 2002). Our data suggest a division of labor in the bonding activities of the two sites. Phosphorylated WT CFTR channels open infrequently even in the absence of ATP (Figure 6B, top current trace), and such ‘spontaneous’ openings reflect occasional dimerization of empty, unliganded NBDs (Mihályi et al., 2016) (Figure 6B, top cartoon). Comparison of such spontaneous gating of WT channels with the equilibrium gating of a hydrolysis-deficient mutant, such as the NBD2 Walker A lysine mutant K1250R, in saturating ATP (Figure 6B, bottom current trace) reveals two robust effects of bound ATP: a shortening of closed interburst durations (Figure 6B, compare green bars above current traces) and a lengthening of open burst durations (Figure 6B, compare red bars below current traces), both by >100 fold (Mihályi et al., 2016). Thus, bound ATP reduces the energetic barrier for opening, but increases the barrier for non-hydrolytic closure (Figure 6C, black vs. orange standard Gibbs energy profiles).

Which of the two bound ATP molecules affects opening rate? Our Φ value analysis indicates that in the T state catalytic site 2 has already finished moving, that is, the site-2 glue is already bonded (Figure 6B, bottom cartoon, lower site; red semi-circles in states T and O₁): that bonding readily explains the reduction in the energetic barrier for opening, that is, stimulation of opening rate, by ATP. Whether stably bound ATP in non-catalytic site 1 might also contribute to speeding opening, by maintaining some contact across the site-1 interface even in the closed state and hence preventing complete NBD disengagement, is still unsettled (Tsai et al., 2010b; Szollosi et al., 2011), but, cf. Chaves and Gadsby, 2015).

Which of the two bound ATP molecules affects non-hydrolytic closing rate? Because it is bonded both in the T and O₁ states, the site-2 glue cannot affect the height of the barrier for non-hydrolytic closure. On the other hand, the low Φ value of non-catalytic site 1 indicates localized movements between the T state and the open state, which we propose reflects tight bonding of the site-1 glue (i.e., tightening of the site-1 interface; Figure 6B, bottom cartoon, upper site; red semi-circle only in state O₁). By selectively stabilizing the open state relative to the T state, the site-1 glue increases the height of the barrier for non-hydrolytic closure, explaining the observed prolongation of non-hydrolytic bursts by ATP.

In the context of the functional cycle of WT CFTR (Figure 1B) ATP bound at catalytic site 2 promotes efficient opening (speeds step C₁→O₁) whereas ATP bound at non-catalytic site 1, by slowing rate O₁→C₁, ensures progress through ATP hydrolysis: such unidirectional cycling is essential for ABC proteins to mediate uphill transport. In asymmetric ABC proteins flipping to the outward-facing, NBD-dimerized conformation is followed by a step in which the consensus site becomes committed to ATP hydrolysis (Timachi et al., 2017): that step might involve bonding of the glue in the non-catalytic site. This interpretation is consistent with the functional cross-talk between the two sites observed in CFTR or related MRP1 (Ramjeesingh et al., 1999; Gao et al., 2000; Hou et al., 2002): perturbations in the non-catalytic site of CFTR reduce the ATPase turnover rate of the catalytic site (Ramjeesingh et al., 1999) by lowering the fraction of open bursts that are terminated by ATP hydrolysis as opposed to non-hydrolytic dissociation of the NBD dimer (Csanády et al., 2010).

The hydrolysis-disrupting D1370N mutation, an essential component of our background construct, precludes targeting position 1370 for Φ value analysis. However, the non-hydrolytic closing rate of D1370N CFTR channels is substantially faster than that of other non-hydrolytic catalytic site-2 mutants, suggesting that this mutation affects not only opening, but also non-hydrolytic closing rate (Gunderson and Kopito, 1995; Vergani et al., 2003): the implication is that the Φ value of position 1370 might be lower than those of the catalytic site-2 positions studied here. Although functionally involved in Mg²⁺ coordination in catalytic site 2 (cf., Zaitseva et al., 2005), aspartate 1370 is physically located in between the two ATP sites, in the center of the NBD interface, at the boundary between core and α-helical subdomains of NBD2 (Figure 6—figure supplement 1, salmon). Moreover, it is in contact with the conserved Q-loop glutamine Q1291 (Figure 6—figure supplement 1, yellow) (Liu et al., 2017) which acts as a γ-phosphate sensor to induce an ~15^o rotation of the α-helical subdomain towards the core subdomain upon ATP binding (Karpowich et al., 2001). Thus, residue D1370 might also be involved in subdomain closure, essential for stable NBD dimerization (Smith et al., 2002). Whether in CFTR that subdomain closure already happens in closed channels upon ATP binding (state C₁, Figure 1B), or is completed only in the tight-dimer open-channel state as in the maltose transporter (Orelle et al., 2010), might be clarified by establishing a different non-hydrolytic background construct which allows targeting positions at the intra-NBD subdomain interface for Φ value analysis.

In conclusion, the Φ value map obtained here supports distinct roles for the two composite ATP sites in CFTR channel gating and reveal the molecular mechanism of the gating defects caused by two common CF mutations. Whereas this work reports relative timing of movements in the two ATP sites during pore opening, clarifying the extent of these movements during each step in the channel opening/closing cycle will require comparisons among high-resolution structures of CFTR trapped in phosphorylated closed- and open-channel states. The recent structures of unphosphorylated closed (Liu et al., 2017) and phosphorylated occluded (Zhang et al., 2017) CFTR have just started to set the stage for such comparisons.

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Author details

1. Ben Sorum

   Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6742-1094

2. Beáta Töröcsik

   1. Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
   2. MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

3. László Csanády

   1. Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
   2. MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary

   Contribution

   Conceptualization, Software, Supervision, Funding acquisition, Validation, Methodology, Writing—original draft, Project administration

   For correspondence

   csanady.laszlo@med.semmelweis-univ.hu

   Competing interests

   Reviewing Editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-6547-5889

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