Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein

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    This study uncovers a unique feature of the nucleotide binding domain interface in human CFTR, offering valuable insights into the effects of different non-hydrolytic mutations on CFTR gating. While the evidence presented is solid, a more thorough examination of the non-hydrolytic mutants of zebrafish CFTR for comparison would strengthen the authors' claims. In the current form, more cautious interpretations of some of the data are needed. This study will be of interest to researchers in the fields of cystic fibrosis and proteins in the ATP Binding Cassette (ABC) transporter family.

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CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure – which determines how tightly bursts and ATP hydrolysis are coupled – is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.

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  1. eLife assessment

    This study uncovers a unique feature of the nucleotide binding domain interface in human CFTR, offering valuable insights into the effects of different non-hydrolytic mutations on CFTR gating. While the evidence presented is solid, a more thorough examination of the non-hydrolytic mutants of zebrafish CFTR for comparison would strengthen the authors' claims. In the current form, more cautious interpretations of some of the data are needed. This study will be of interest to researchers in the fields of cystic fibrosis and proteins in the ATP Binding Cassette (ABC) transporter family.

  2. Reviewer #1 (Public Review):

    The CFTR ion channel belongs to the family of ABC transporters, alternating between inward-facing (IF) and outward-facing (OF) conformations driven by binding and hydrolysis of ATP. ABC transporters are involved in a wide variety of physiologically essential transport processes. In contrast to all other ABC transporters, the OF conformation of CFTR includes an anion-conducting transmembrane pore, which has enabled investigators to use single-channel patch clamp electrophysiology to characterize the energetics of most of the relevant transitions that can be observed during a gating cycle. A transition that had remained elusive to quantify is the non-hydrolytic closing rate in which the channel switches back to an IF conformation even though both nucleotide-binding domain sites are occupied by non-hydrolyzed ATP molecules. The reason for this is that the rate of closure due to ATP hydrolysis occurs much faster, such that the non-hydrolytic closing rate cannot be quantified in WT channel recordings. Further, channels with mutations that are expected to disrupt ATP hydrolysis exhibit high variability between mutants in the non-hydrolytic closing rates, precluding an extrapolation for this rate onto the WT channel. It is presently unclear whether the large spread in the rates is caused by distinct degrees of remnant hydrolytic activity in each of the mutant channels. Regardless of this uncertainty, several of these mutations have been successfully employed in structural studies to stabilize the channel in an OF conformation with two ATP molecules bound. In the present manuscript, Márton A. Simon and collaborators use patch-clamp electrophysiology to measure the rates of non-hydrolytic channel closure in human CFTR channels containing single and double mutations expected to disrupt ATP hydrolysis. First, they find that the E1371Q but not the E1371S mutation significantly stabilizes the OF state and slows channel closure in the human but not the zebrafish channel. Looking at the structures of both human and zebrafish E1371Q mutant channels, a non-native hydrogen bond is identified between the side-chain of E1371Q and the main chain at position G576 that is observed only in the human structure and would be expected to stabilize the OF state. Double mutant cycle analysis is then utilized to compare the effect of removal of either of the hydrogen-bonding partners in the human CFTR, and found to be consistent with a large energy of interaction between the two sites that is interpreted to occur selectively in the OF state. Notably, none of these perturbations altered the intra-burst activity of the CFTR, indicating that those closures do not involve major changes in the NBD. The rates of closure for other mutants are then investigated, and it is found that combining two hydrolysis-disrupting mutations that retain relatively fast closure rates does not slow closure any further, suggesting that their fast closure rates are not due to residual ATP hydrolytic activity. Further, this observation also suggests that these mutations do not affect the intrinsic non-hydrolytic closing rate, allowing their rates to be used as a measure of what occurs in WT channels. The experiments presented are high-quality, the conclusions are well supported by the data and the findings clarify a series of questions that had remained in the field, in addition to finding and precisely quantitating the role of a hydrogen bond in stabilizing a conformation state of this channel. The results could have implications for other members of the ABC family that are harder to study because they do not produce ionic currents. Some methodological details could be explained better, such as the voltage at which each of the recordings was performed, and how data was normalized, which is presently unclear. Additional testing of the hypothesis could have been carried out through double mutant cycle analysis with E1371Q + G576Δ in the zebrafish receptor or the other non-hydrolytic mutants.

  3. Reviewer #2 (Public Review):

    Gating of the CFTR chloride channel is controlled by its nucleotide binding domains (NBDs) where ATP binding-induced dimerization leads to channel opening and ATP hydrolysis in the catalytic ATP binding site terminates CFTR's opening burst. Mutations that diminish ATP hydrolysis, including Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371Q and E1371S, have been used extensively to trap the channel in the open state by researchers studying CFTR function. The E1371Q human CFTR (hCFTR) has an extremely longer burst duration than all the other hydrolysis-deficient mutants, including E1371S hCFTR. An unexpected finding that the E-to-Q and E-to-S mutants of zebrafish CFTR (zCFTR) have similar non-hydrolytic closing rates inspired Simon et al to investigate the underlying mechanism for this discrepancy between the human and zebrafish CFTR orthologs, and examine how hydrolysis deficient mutations have differential effects on the CFTR's burst duration. Their data support the idea that all the above mutations completely abolish ATP hydrolysis. The closing rate of K1250A and E1371S CFTR represents the true non-hydrolytic closing rate of wildtype CFTR, while the closing rate of D1370N is accelerated presumably due to the lack of interaction between the negatively charged aspartate and magnesium ion in the ATP binding site. On the other hand, an artificial H-bond between the G576-Q1371 of hCFTR, which is absent in zCFTR, stabilizes the NBD dimer and slowers non-hydrolytic closure.

    The conclusions of this paper are mostly well supported by the data, but some additional experiments will strengthen the claim on the role of the artificial inter-NBD hydrogen bond (point 1 below). Some aspects of data interpretation need to be further clarified (point 2-5 below).

    1. The author hypothesized that in hCFTR an artificial H-bond between the side-chain of glutamine at position 1371 (i.e., in E1371Q mutant) and the backbone carbonyl at G576 of the D-loop stabilizes the NBD dimer. Such H-bond is absent in E1372Q zCFTR. The authors employed mutant cycle analysis on the G576Δ-E1371S mutation pair to demonstrate an energetic coupling between the hG576 and hE1371Q. However, how the deletion of G576 might alter the local structure is unpredictable. The result does not directly address the discrepancy between zCFTR and hCFTR, either. The D-loop is highly conserved across species with a consensus sequence PFGYLD (residue 574-579 in hCFTR), but in zCFTR the analogous sequence is PFTHLD. The backbone carbonyl oxygen could therefore be harder to access in zCFTR. A simple yet critical experiment would have strengthened the authors' claim that the interaction between Q1371 and G576 stabilizes the dimer: introducing mutation in the D-loop of zCFTR to match the sequence of hCFTR (and vice versa). The authors' hypothesis would predict that zCFTR with hCFTR's D-loop sequence should recapitulate hCFTR's phenotype: the E-to-Q mutation on the catalytic glutamate would further lengthen the burst duration compared to the E-to-S mutation.

    2. The authors speculated that the reason for D1370N's relatively fast closing rate compared to other non-hydrolytic mutants is the loss of interaction between Mg2+ and the negatively charged aspartate. However, this reasoning fails to explain why non-hydrolytic closure of wildtype CFTR in the absence of Mg2+ (e.g., Levring et al. 2023 Extended Data Fig. 7g) is even slower than the non-hydrolytic closure of D1370N CFTR opened by MgATP, where at least the Mg2+ is present. The authors should caution the readers that so far no definitive experimental evidence can explain the destabilizing effect of D1370N.

    3. Based on the results that the double mutant E1371S/K1250A hCFTR has similar burst duration as single mutant E1371S and K1250A, the authors made a strong claim that both mutations completely abolish ATP hydrolysis. Similar reasoning was applied to D1370N. The limitations in such interpretations should be discussed. The authors made the assumption that the termination of a burst is solely controlled by site 2 (Figure 1C). However, when hydrolysis is significantly diminished, binding of ATP in site 2 is very stable, and thus dissociation of ATP from site 2 versus site 1 becomes hard to distinguish. Whether all hydrolysis-deficient mutants share the same open-to-close transition by releasing ATP from site 2 but retaining ATP in site 1 is still a question. As the authors have elaborated in the text, it is known that mutations in the degenerate site 1 can affect non-hydrolytic closing. When mutations are introduced to site 2, they might as well result in allosteric effects on the stability of ATP binding in site 1, which could subsequently alter the channel's closing rate. The authors might want to make the readers aware of the complicated relationship between channel closure and CFTR's two ATP binding sites, and that the estimation of the "true non-hydrolytic closing rate" is based on an oversimplified gating scheme shown in Figure 1C.

    4. It is known that non-hydrolytic closing rate of CFTR is phosphorylation dependent, which the authors briefly mentioned in the Discussion. Vergani et al. (2003) documented that τburst of K1250A and D1370N in PKA is ~80 s and ~4 s respectively, but both are reduced by roughly twofold when PKA was removed. In this study the burst durations of K1250A (~30 s, Figure 4C) and D1370N (~2 s, Figure 4E) indicate that these channels are not strongly phosphorylated. Similarly, the τburst of E1371S in PKA is over 100 s (Bompadre et al. 2005), significantly longer than that in the current study. Although it is unclear how a different degree of R domain phosphorylation affects non-hydrolytic closing, the fact that it does again suggests that the simplified scheme used as the base for data interpretation may have its limitation. The Discussion would benefit from a more cautionary note on the oversimplification of the IB1↔B1 transition, and clarify that channels are not strongly phosphorylated in the current experimental condition.

    5. The τburst of E1371Q CFTR is over 400 second while the τburst of K1250A-E1371Q double mutant is shortened to ~200 second (Figure 3B, black vs Figure 4C, black). The K1250A-E1371S CFTR also seems to have a shorter τburst than E1371S CFTR (Figure 4C, blue vs Figure 3B, blue). Although the effect of the K1250A mutation on shortening τburst of E1371Q and E1371S CFTR is not as dramatic as the D1370N mutation, the authors might want to clearly state if there is indeed a significant difference and address how K1250A mutation has such destabilizing effect.

    Bompadre, S. G., Cho, J. H., Wang, X., Zou, X., Sohma, Y., Li, M., and Hwang, T. C. (2005) CFTRgating II: Effects of nucleotide binding on the stability of open states. J Gen Physiol 125, 377-394

    Levring,J., Terry,D.S., Kilic,Z., Fitzgerald,G., Blanchard,S.C., and Chen,J. (2023). CFTR function,
    pathology and pharmacology at single-molecule resolution. Nature 616, 606-614.

    Vergani,P., Nairn,A.C., and Gadsby,D.C. (2003). On the mechanism of MgATP-dependent gating of CFTR Cl- channels. J. Gen. Physiol 121, 17-36.

  4. Reviewer #3 (Public Review):

    CFTR is an anion-selective channel that plays important roles in epithelial physiology. In this paper, Simon and colleagues focus on the step of the CFTR gating cycle that opens the pore. But the authors are particularly interested in the reversal of this opening step. Wild-type (WT) CFTR channels do not usually close by reversal of the opening step, as closure via this "non-hydrolytic" pathway is slow. Instead, hydrolysis of the ATP molecule bound at site 2 destabilizes the open (or bursting) channel and triggers rapid "hydrolytic" channel closure - before the open channel has time to overcome the energetic barrier on the non-hydrolytic pathway. While it is generally (but not universally) accepted that such a non-equilibrium kinetic scheme underlies CFTR gating, how tightly gating and ATPase cycles are coupled is still quite controversial.

    Here, combining simple electrophysiology measurements on mutant channels with solid arguments, the authors provide an improved estimate for the backward rate on the opening transition (rate k-1) in WT-CFTR channels. It turns out that this rate is indeed slow, compared to the rate of the hydrolytic step (k1) allowing authors to conclude that WT CFTR channels close via reversal of the opening step only less than once every 100 gating cycles. In addition, results of thermodynamic mutant cycles and careful analysis of cryo-EM structures are used to support plausible molecular mechanisms that explain why different mutations in CFTR's catalytic site slow down, speed up or barely affect non-hydrolytic closure.

    The strength of this study is twofold. First, the methods are sound, and the effects seen are clear-cut. Records are competently acquired, with a high number of repeats, are well analysed and very clearly presented. Second, the authors interpret their results with interdisciplinary competence, drawing on structural knowledge of ABC transporter catalytic mechanism, as well as on an in-depth understanding of studies investigating kinetics and thermodynamics of CFTR gating. This study, bringing together conclusions obtained in many previous studies, is a useful step forward towards a comprehensive description of the energetic landscape CFTR channel proteins wander through when gating. The Csanády lab has greatly contributed to developing this over the past years, and this paper reads as a "capstone".

    However the reliance on previous conclusions is, in some ways, also a weakness. Many of the inferences made in interpreting the data depend on assumptions being met. There is evidence supporting the validity of these, but more clarity in stating implicit assumptions, and why the authors believe them to be valid, could improve the manuscript. The results fit well within the conceptual framework of CFTR's non-equilibrium gating. But some scientists, still sceptical of its basic premises, will not be convinced by these new results.

    Within this context, the authors achieve their aim of estimating the microscopic rate constant for non-hydrolytic closure. The study will be of interest not only to scientists studying CFTR gating, but also to those wishing to understand how small-molecule drugs affect such gating. The mechanism of action of ivacaftor (currently taken by thousands of people for treatment of cystic fibrosis) is still not completely clear, and some evidence suggests that it stabilizes the pre-hydrolytic bursting state investigated here. Aspects of CFTR's conformational dynamics will probably also be true for some of its phylogenetic relatives. Thus, those studying other ABC transporters, many of which have medical relevance, will find it interesting to learn how CFTR couples its gating and hydrolytic cycles. This is especially true now, when cryogenic electron microscopy and other methods allow detailed structural comparisons between related ABC transporters, which can be correlated with differences in their function. Now more than ever CFTR could be a "model ABC protein".