Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization

Issues to address:

1) Figure 2A. It appears that there is some significant rundown in the 'background' recording. The effect is subtle, but looks real. (But I can't tell because to the inherently anecdotal character of the displayed record.) Could this possibly undermine the quantification of energies being used here?

There are two features in the recordings that the Reviewers might have perceived as "rundown".

First, the current decline upon ATP removal is typically biexponential, which is better appreciated by looking at the macroscopic current trace in Figure 1. The rapid decline with a large fractional amplitude likely reflects immediate loss of ATP from low-affinity site 2, whereas the subsequent slower phase is due to slower loss of ATP from high-affinity site 1 (see Tsai et al., 2010; J Gen Physiol 135:399-414). The current typically settles to a quasi-steady level within ~1 min following ATP removal. To extract gating parameters of unliganded channels we therefore analyzed only the last ~4 min of the 5 min ATP-free segments, as indicated in Methods. Of course in single-channel recordings loss of ATP from site 1 is a discrete event and so the actual time required to reach this "unliganded" steady state is variable. Thus, in individual recordings the precise starting time point for the "steady-state" segment to be analyzed was chosen by eye. E.g., for the trace in Figure 2A only the last ~3 min were analyzed. We have added a corresponding statement to Materials and methods (subsection “Excised inside-out patch recording”, last paragraph).

Second, the currents in ATP appear slightly smaller in the rear bracketing segments, as apparent in the macroscopic recording in Figure 1C. One reason for this is that following a prolonged exposure to an ATP-free solution not all channels recover instantaneously, giving rise to the slowly creeping phase of the current in the second bracket in Figure 1C. This phenomenon is also well known, and much more pronounced for WT CFTR (see Figure 1A), but the underlying mechanism is unclear (see Tsai et al., 2010; J Gen Physiol 135:399-414). However, both in our background construct, and in the single- and double-mutants studied in this background, the open probability in the rear bracket, following full "reactivation", was only slightly smaller than in the front brackets (see Figure 1—figure supplement 2). We do not think that small decline – probably due to dephosphorylation, and observed similarly for all four constructs in the mutant cycle – would have substantially distorted the extracted values of interaction energies.

2) Figure 3C is somewhat confusing. At the bottom (just above -0.4 kT) there are to arrow pointing towards each other: why?

The three double-headed arrows in Figure 3C are used in the manner of dimension lines in technical drawings, to gauge distances between open-state and closed-state values for the three free energy profiles. The 0.4 kT distance for the gray profile is too small to fit both arrowheads into it. We have therefore used two arrowheads pointing in opposite directions, consistent with technical drawing guide lines. It is true that these two arrowheads should not have been connected to each other – we have removed the connecting line.

The y axis contains a break, which makes it difficult to find all the numbers back that are calculated in the manuscript. For example, the 3.3 kT (Discussion, fourth paragraph).

Unfortunately, a plot in which all the cited numbers are readily visible cannot be constructed. This is not due to the y-axis break (which is necessary in order to be able to draw at least all the changes in free energies to scale, given that the absolute heights of the barriers are disproportionately large (and also uncertain)), but to the fact that energy levels for three states (closed, transition, open) are compared to each other under three conditions (WT unliganded, BG unliganded, WT ATP-bound). For such comparisons to be made, one of the states must be arbitrarily chosen as a reference, and we have chosen state C as our reference state. This choice allows changes in △G^o_(T-C) and △G^o_(O-C) – caused by the BG mutations (downward black arrows) or by ATP binding (downward gray arrows) – to be directly visualized, whereas changes in △G^o_(T-O) can be inferred as the difference between the former two numbers but cannot be simply illustrated. A similar limitation arises regardless of the choice of the reference state.

3) In the subsection “Mutant cycle analysis” it is stated that SEMs were calculated using the mean value for n. It is not clear why this was done. Also the justification is weird: there were "similar numbers of observations". Looking through the manuscript, the number of observation is not explicitly presented for all experiments; in addition, it varies from 5-16.

The numbers of observations were included in parentheses for all experiments, in the form "0.11±0.04 (6)". We realize that these numbers might have been mistaken as citations to references, so we have now explicitly added "n=" throughout the text (e.g., "0.11±0.04 (n=6)" (subsection “Gain-of-function mutations allow quantitative characterization of spontaneous CFTR gating”).

The statement (subsection “Mutant cycle analysis”) on using the mean value of n for SEM calculations applies only for the △△G values calculated from the mutant cycles (but, cf. Statistics section for all other data). In fact, estimation of △△G standard errors is not trivial, so we have added a more detailed description of this mathematical procedure (in the aforementioned subsection). Of note, n values for the four corners of each mutant cycle were quite similar to each other: n=19-21 for Figure 2H, and n=15-19 for Figures 2F and G (a few patches that were not sufficiently "clean" were analyzed for P_o, but not for gating kinetics).

4) In the subsection “The site-2 NBD interface is already tightened in the transition state for spontaneous pore opening”: 0.62 +/- 0.34 kT is not significantly different from zero. Why not?

As stated in the Statistics section, differences were considered significant for p<0.05. For the above parameter p=0.09 (see Figure 2J).

5) Figure 2H, I, J would benefit from labels indicating what exactly was analyzed.

Done.

6) In the first sentence of the subsection “Gain-of-function mutations allow quantitative characterization of spontaneous CFTR gating”: Replace 'instantaneously' by 'rapidly' or some such adverb.

Done.

7) The manuscript is also generally well written, with the exception of the first sentence of the Abstract, which is full of commas. Please rewrite.

Done: "In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it."