1. Author Response:

    Reviewer #1 (Public Review):

    This manuscript describes single molecule measurements of rotation of the C10 ring of E. coli ATP synthase in intact complexes embedded in lipid nanodiscs. The major point of the work is to identify the mechanisms by which protonation/deprotonation steps produce torque between the a-subunit and the C10 ring, which is subsequently conveyed to F1 to couple to ATP synthesis. The work explores the pH-dependent of the "transient dwell" (TD) phenomenon of rotation motion to identify likely intermediates, showing a likely step of 11o in the "clockwise" (ATP synthase-related) direction. The results are then interpreted in the context of detailed structural information from previous cryo-EM and X-ray crystallographic reports, to arrive at a more detailed model for the partial steps for coupling of proton translocation to motion. The effects of site-specific mutations in the c-subunits appears to support the overall model.

    While the detailed structural arguments seem, at least to this reader, to be plausible, the text is not structured for any hypothesis testing, and one might imagine that alternative models are possible. No alternative models were presented, so it is not clear to what extent the 11o rotation step rules out such possibilities. This leaves the reader with the feeling that a lot of speculation occurs in the Discussion, but it is very difficult to figure out which parts are solid and which parts are speculation.

    We have now presented the results in terms of hypothesis testing specifying alternative hypotheses that exist in the literature. We then specify results presented in the manuscript that discriminate between alternate hypotheses.

    The Discussion also tries to pack in too many concepts, going well beyond the advances enabled by the TD results themselves. For example, the proton "funnel" concept is quite interesting, but it is not easy to see how the TD leads up to it. This overpacking makes it difficult to pinpoint the real advances, and dilutes the message sets the reader up to ask for more support for such extensive modeling. Do the mechanistic details set up good testable hypothesis for future experimental tests?

    It is clear that the pKa values we determined are the result of multiple residues involved in the proton transfer process. It is currently not possible to determine where the input channel starts. The recent structures now show that the residues that were thought to define the input channel are far from the surface and must communicate with the periplasm via the funnel. Residues in the funnel likely impact the pKa values that we have measured. The proton transfer-dependent 11 degree step that we measured must also depend upon the funnel. Our results clearly show that this 11 degree step depends upon the correct protonation states of both the input and output channels, and that this depends on the differences in the high and low pKa values. The possibility also exists that this funnel that is absent in the output channel may provide a proton reservoir to supply the input channel, which promotes the ability of input and output channels to drive the 11 degree synthase steps. We have now included this information in the manuscript, and for these reasons, we decided that discussion of the funnel must remain.

    Overall, the text would be far more impactful if it focused more tightly on the implications of the TD results themselves, testing specific sets of models, and taking more care to guide the readers through the interpretation.

    We have extensively rewritten the entire manuscript to address these issues. To help guide the readers, we added more background information to the introduction and pose alternate hypotheses. In the results, we now guide the readers by restating how the experiments can test a given hypothesis, and include brief conclusions that explain why a hypothesis is eliminated or favored based on the results. We shortened the Discussion to make it more focused, with the exception that we provided additional information that has been requested by the reviewers. We also tie each point in the discussion to the results presented. Of course, a good discussion is meant to put the results and conclusions of the manuscript into the context of results and conclusions from other laboratories, which we have done.

    Reviewer #2 (Public Review):

    This brilliant, beautiful and important study provides the essential kinetic framework for the recent, static high-resolution cryo-EM structures of F1FO ATPases from bacteria, chloroplasts and mitochondria. The elegantly conducted single-molecule work is necessarily complex, and its analysis is difficult to follow, even for someone who is intimately familiar with F1FO ATPases. Some more background and better explanations would help.

    We added additional background information to the Introduction, and we now periodically explain the reasoning and conclusions in the Results to help guide the readers.

    For F1FO ATPases, CCW rotation has little if any biological relevance, whereas CW rotation is centrally important. Evidently, the CW ATP synthesis mode is not accessible to the approach taken in this manuscript, since the ATP synthase is reconstituted into lipid nanodiscs rather than liposomes. This critical fact should be stated more clearly in the introduction.

    We now state explicitly that net rotation was observed as the result of F1-ATPase activity as requested. We also note that E. coli does sometimes use F1Fo as an ATPase-dependent proton pump to maintain a pmf across the membrane depending upon metabolic conditions.

    The central concepts of "transient dwells", "dwell times" and "power strokes" need to be introduced more fully for a general, non-expert audience.

    We added this information to the Introduction as requested.

    The manuscript describes the power stroke and dwell times in CCW ATP hydrolysis mode in unprecedented detail. Presumably the dwell times and power strokes apply equally to the physiologically relevant CW ATP synthesis mode, but are they actually the exact reverse? Is there evidence for transient dwells and 36{degree sign} power strokes divided into 11{degree sign}+25{degree sign} substeps during ATP synthesis?

    The 36° subunit-c stepping that contain 11° synthase-direction steps is a novel observation first reported in this study. To date, single-molecule studies of rotation during net ATP synthesis have been carried out using single-molecule FRET that have been able to observe only 3 or 4 consecutive synthase steps for a given F1Fo molecule (Dietz et al. ((2004) Proton-powered subunit rotation in single membrane-bound FoF1-ATP synthase. Nature Struct & Mol Bio). The FRET measurements do not have the time resolution to resolve sub-steps. Whether or not continuous rotation in the synthesis direction is the exact reverse of ATPase-dependent rotation is an important question that remains to be answered.

    The meaning of low, medium and high efficiency of transient dwell formation (Figure legend 2; lines 189/190; Figure 3; line 365) is not obvious and not well explained. How are these efficiencies defined? Why are they important? What would be 100% efficiency? And what would be 0%?

    Background information concerning the three efficiencies of transient dwell formation has been added to the Introduction, and we now explain their importance. We also now explain what 100% and 0% efficiency is in the Results.

    Why is it important whether transition dwells do or do not contain a synthase step? Is this purely stochastic? If not, what does it depend on?

    We added a paragraph to the discussion to explain that their formation depends on the kinetics of the rate of formation of the interaction between subunit-a and the c-ring versus the velocity of ATPase-depending rotation in the opposite direction, and that it depends on the energy that can drive the synthase-direction step relative to the energy that drives the ATPase direction power stroke. More work is required to define the energetic parameters of these opposing rotations that is beyond the scope of the work presented here.

    The formation of a salt bridge between aR210 of subunit-a and cD61 of the c-ring rotor would seem to be counter-productive for unhindered rotary catalysis. What is the evidence for such a salt bridge from the cryo-EM structures or molecular dynamics simulations?

    This is an excellent question, especially since the distances between aR210 and cD61 are more consistent with intervening water molecules. We revised the paragraph in the Discussion describing this point and have been more explicit about the importance of the aqueous vestibule between the output channel and aR210 must play during rotation, which includes the impact of the dielectric constant inside the vestibule. As a direct answer to the reviewer’s question, in the absence of water, a salt bridge between aR210 and cD61 in such a hydrophobic environment would be so strong that the energy of a proton from the input channel would never be able to dislodge them.

    Reviewer #3 (Public Review):

    Yanagisawa and Frasch utilise a gold nanorod single molecule method to probe the pH dependency of F1FO rotation. The experimental setup has been previously used to investigate both F1-ATPase and FO function in multiple studies. In this study, clockwise rotations are observed in transient dwells which may correlate to synthesis sub-steps. Mutations along the proposed proton path modify the pH dependency of the transient dwells.

    The strength of this manuscript can be seen in the rigorous way in which the problem has been explored. Testing the pH dependence of mutants along the proposed proton path and linking this to potential sub-steps using the known atomic structure.

    In my view, the main weakness of this study is the experimental design (shown in Fig. 1C). Strictly, the measurements show rotation of the c-ring relative to subunit-Beta rather than relative to subunit-a. Recent structures of E. coli F1FO ATP synthase inhibited by ADP (doi: 10.1038/s41467-020-16387-2) have shown that the peripheral stalk is flexible and can accommodate movements of the c-ring relative to the F1 (AlphaBeta)3-subunit ring. For example, comparison of PDB entries 6PQV and 6OQS shows that FO (the c-ring and subunit-a) can rotate 10 degrees as a rigid body relative to the F1 (AlphaBeta)3-subunit ring - with no relative rotation between the c-ring and subunit-a, or rotation of subunit-gamma. The authors discuss structures from this study related by a 25 degree rotation of the c-ring relative to subunit-a, but I do not believe they have ruled out the possibility that their observations show rotation of the FO as a rigid body. A preprint investigating E. coli F1FO ATP synthase in the presence of ATP has proposed that the complex becomes more flexible during ATP hydrolysis (doi: 10.1101/2020.09.30.320408), with the central stalk twisting by up to 65 degrees. The small CW movements seen in the transient dwells in this study could be attributed to 36 degree FO sub steps, facilitated by central stalk flexibility, with counter rotation facilitated by peripheral stalk flexibility.

    The data clearly show that the mutations of subunit-a residues in the input or output channels significantly change the pKa values of TD formation (Figs 2B and 2C), and can dramatically change the occurrence of the synthase-direction steps (see Figs 4D and 4E). These results clearly indicate that the rotational events observed in this study do not result from rotation of subunit-a and the c-ring as a unit.

    With regard to the recent structures that the reviewer refers to, we report differences in the efficiency of TD formation that are consistent with torsion induced by rotation of the c-ring relative to the beta subunit, which we reported previously (Yanagisawa and Frasch, JBC 2017), and which has been confirmed by independent single-single molecule studies by the Junge lab and by the Boersch lab using different approaches to our own (Sielaff et al., Molecules 2019). Both papers are cited in the manuscript. We have now expanded the introduction to include these results describing the impact of central stalk flexibility on the ability to form synthase-direction steps, and how these results are consistent with E. coli cryo-EM structures similar to those referred to (27).

    It is also unclear what causes the stochastic nature of transient dwells. Are these related to inhibition of F1-ATPase? Could increased drag in FO increase the likelihood of F1-ATPase inhibition?

    We now include background information from our prior publications that characterizes the kinetic component that affects the ability to form a transient dwell. Ishmukhametov et al. EMBO J (2010), reported an increase in TDs upon increasing the drag on the nanorod that slowed the power stroke angular velocity. We decribed the kinetics of TD formation in that paper. In the Discussion, we also now provide information concerning how the bioenergetics can impact the probability of TD formation.

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  2. Evaluation Summary:

    This paper is of outstanding interest to the broad community of scientists interested in biological energy conversion in general and rotary ATPases in particular. The authors show that the 36{degree sign} power stroke in ATP synthesis is subdivided into two steps of 11{degree sign} and 25{degree sign} in the E. coli enzyme, which serves as a comparatively simple model of the fundamental and universally important process of ATP production in mitochondria and chloroplasts. By combining precise and sophisticated single-molecule studies with directed mutagenesis, this work provides the much-needed functional context for recent high-resolution cryo-EM structures of rotary ATPases.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    This manuscript describes single molecule measurements of rotation of the C10 ring of E. coli ATP synthase in intact complexes embedded in lipid nanodiscs. The major point of the work is to identify the mechanisms by which protonation/deprotonation steps produce torque between the a-subunit and the C10 ring, which is subsequently conveyed to F1 to couple to ATP synthesis. The work explores the pH-dependent of the "transient dwell" (TD) phenomenon of rotation motion to identify likely intermediates, showing a likely step of 11o in the "clockwise" (ATP synthase-related) direction. The results are then interpreted in the context of detailed structural information from previous cryo-EM and X-ray crystallographic reports, to arrive at a more detailed model for the partial steps for coupling of proton translocation to motion. The effects of site-specific mutations in the c-subunits appears to support the overall model.

    While the detailed structural arguments seem, at least to this reader, to be plausible, the text is not structured for any hypothesis testing, and one might imagine that alternative models are possible. No alternative models were presented, so it is not clear to what extent the 11o rotation step rules out such possibilities. This leaves the reader with the feeling that a lot of speculation occurs in the Discussion, but it is very difficult to figure out which parts are solid and which parts are speculation.

    The Discussion also tries to pack in too many concepts, going well beyond the advances enabled by the TD results themselves. For example, the proton "funnel" concept is quite interesting, but it is not easy to see how the TD leads up to it. This overpacking makes it difficult to pinpoint the real advances, and dilutes the message sets the reader up to ask for more support for such extensive modeling. Do the mechanistic details set up good testable hypothesis for future experimental tests?

    Overall, the text would be far more impactful if it focused more tightly on the implications of the TD results themselves, testing specific sets of models, and taking more care to guide the readers through the interpretation.

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  4. Reviewer #2 (Public Review):

    This brilliant, beautiful and important study provides the essential kinetic framework for the recent, static high-resolution cryo-EM structures of F1FO ATPases from bacteria, chloroplasts and mitochondria. The elegantly conducted single-molecule work is necessarily complex, and its analysis is difficult to follow, even for someone who is intimately familiar with F1FO ATPases. Some more background and better explanations would help.

    For F1FO ATPases, CCW rotation has little if any biological relevance, whereas CW rotation is centrally important. Evidently, the CW ATP synthesis mode is not accessible to the approach taken in this manuscript, since the ATP synthase is reconstituted into lipid nanodiscs rather than liposomes. This critical fact should be stated more clearly in the introduction.

    The central concepts of "transient dwells", "dwell times" and "power strokes" need to be introduced more fully for a general, non-expert audience.

    The manuscript describes the power stroke and dwell times in CCW ATP hydrolysis mode in unprecedented detail. Presumably the dwell times and power strokes apply equally to the physiologically relevant CW ATP synthesis mode, but are they actually the exact reverse? Is there evidence for transient dwells and 36{degree sign} power strokes divided into 11{degree sign}+25{degree sign} substeps during ATP synthesis?

    The meaning of low, medium and high efficiency of transient dwell formation (Figure legend 2; lines 189/190; Figure 3; line 365) is not obvious and not well explained. How are these efficiencies defined? Why are they important? What would be 100% efficiency? And what would be 0%?

    Why is it important whether transition dwells do or do not contain a synthase step? Is this purely stochastic? If not, what does it depend on?

    The formation of a salt bridge between aR210 of subunit-a and cD61 of the c-ring rotor would seem to be counter-productive for unhindered rotary catalysis. What is the evidence for such a salt bridge from the cryo-EM structures or molecular dynamics simulations?

    Read the original source
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  5. Reviewer #3 (Public Review):

    Yanagisawa and Frasch utilise a gold nanorod single molecule method to probe the pH dependency of F1FO rotation. The experimental setup has been previously used to investigate both F1-ATPase and FO function in multiple studies. In this study, clockwise rotations are observed in transient dwells which may correlate to synthesis sub-steps. Mutations along the proposed proton path modify the pH dependency of the transient dwells.

    The strength of this manuscript can be seen in the rigorous way in which the problem has been explored. Testing the pH dependence of mutants along the proposed proton path and linking this to potential sub-steps using the known atomic structure.

    In my view, the main weakness of this study is the experimental design (shown in Fig. 1C). Strictly, the measurements show rotation of the c-ring relative to subunit-Beta rather than relative to subunit-a. Recent structures of E. coli F1FO ATP synthase inhibited by ADP (doi: 10.1038/s41467-020-16387-2) have shown that the peripheral stalk is flexible and can accommodate movements of the c-ring relative to the F1 (AlphaBeta)3-subunit ring. For example, comparison of PDB entries 6PQV and 6OQS shows that FO (the c-ring and subunit-a) can rotate 10 degrees as a rigid body relative to the F1 (AlphaBeta)3-subunit ring - with no relative rotation between the c-ring and subunit-a, or rotation of subunit-gamma. The authors discuss structures from this study related by a 25 degree rotation of the c-ring relative to subunit-a, but I do not believe they have ruled out the possibility that their observations show rotation of the FO as a rigid body. A preprint investigating E. coli F1FO ATP synthase in the presence of ATP has proposed that the complex becomes more flexible during ATP hydrolysis (doi: 10.1101/2020.09.30.320408), with the central stalk twisting by up to 65 degrees. The small CW movements seen in the transient dwells in this study could be attributed to 36 degree FO sub steps, facilitated by central stalk flexibility, with counter rotation facilitated by peripheral stalk flexibility.

    It is also unclear what causes the stochastic nature of transient dwells. Are these related to inhibition of F1-ATPase? Could increased drag in FO increase the likelihood of F1-ATPase inhibition?

    Read the original source
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