Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation

  1. Noriyo Mitome
  2. Shintaroh Kubo
  3. Sumie Ohta
  4. Hikaru Takashima
  5. Yuto Shigefuji
  6. Toru Niina
  7. Shoji Takada

  • Curated by eLife

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    Evaluation Summary:

    This is an interesting manuscript describing for the first time experimentally the cooperative effects of mutations to individual key Glu residues in the c-ring of ATP synthase. The main result is that mutations in nearby c subunits are less inhibitory than those in subunits further apart in the ring. This is explained on the basis of MD/MC simulations as a shared waiting time for delayed proton uptake in case of neighboring subunits, which appears logical. Overall the manuscript is well presented, but with some caveats. The works will be of interest to specialists in bioenergetics, and to a wider biochemical, biophysical and structural biology audience.

    (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 #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

In F o F 1 -ATP synthase, proton translocation through F o drives rotation of the c -subunit oligomeric ring relative to the a -subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c -subunits contribute to proton release to and proton uptake from the a -subunit. However, no studies have demonstrated cooperativity among c -subunits toward F o F 1 -ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring a c -ring with various combinations of wild-type and c E56D, enabled by genetically fused single-chain c -ring. ATP synthesis and proton pump activities were decreased by a single c E56D mutation and further decreased by double c E56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c -subunits. Similar results were obtained for proton transfer-coupled molecular simulations. The simulations revealed that prolonged proton uptake in mutated c -subunits is shared between two c -subunits, explaining the cooperation observed in biochemical assays.

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  1. Version published to 10.7554/elife.69096 on eLife
  2. eLife

    Reviewer #2 (Public Review):

    Mitome et al. investigated the possible cooperativity among the proton-carrying subunits (c-subunit) of Fo motor in ATP synthase by biochemically analyzing the ATP synthesis/ATP-driven proton-pump activities of mutant ATP synthases. The c-subunits form the rotor oligomer ring (c10 oligomer in the Fo system that they investigated) that rotates against the stator complex of Fo composed of a-subunit and b2 dimer complex, upon transmembrane proton translocation in Fo motor. It is widely thought that each of c-subunit executes proton-transfer between c-subunit an a-subunit, coupled with 36-degree rotation of c-oligomer ring. For the investigation of the possible cooperativity among c-subunits, they prepared mutants of Fo in which 10 c-subunits were genetically fused into a single polypeptide, and they introduced a mutation at the proton-carrying residue of c-subunit, cE56 to produce the mutated c-subunit (cE56D) at particular positions in the c10 repeat. The main observation is that when cE56D mutation was introduced into two c-subunit repeats that were separated to each other, the impact of the cE56D mutation on catalysis were almost additive. On the other hand, when the mutation was introduced into two neighboring c-subunit repeats, the double mutation effect was weakened, and very close to that of single mutation. These findings suggest some cooperativity exists in c10 oligomer ring. In order to investigate the molecular mechanism of the observed cooperativity, they conducted in silico simulation where Monte Carlo simulation for the proton transfer was integrated into coarse grained molecular dynamics simulation. They observed the dwelling states for the rate-determining step on neighboring two c-subunits were often overlapped to diminish the mutation effect of the second c-subunit, while the impact of the mutation was additive when mutated c-subunit were separated in c10 ring.

    This study uncovers the cooperativity among c-subunits and provides a possible molecular mechanism for that. This work gives new and important insights on molecular mechanism of Fo motor of ATP synthase. Therefore, this paper would be suitable for the publication in eLIFE when the following concerns are addressed.

    One of the main concerns is the accuracy of biochemical assays, ATP synthesis activity measurement and ATP-driven proton pumping activity measurement. To my knowledge, it is not easy to achieve highly accurate and precise biochemical assays such as error within a few % even if we use highly purified enzymes. In this paper, the authors reported very small experimental error: around 1 % or even less. However, I have not found the description on how the author did determine the experimental errors. They used not purified enzymes but inverted vesicles of E. coli expressing the mutated enzymes. One of critical parameters for the accuracy is the quantification of the enzymes in the vesicles that were estimated from the decoupled ATP hydrolysis activity measurement. The error in this quantification should be substantially smaller than 1 % to achieve such a high accuracy. In addition, the ATP synthesis activity and ATP-driven proton pumping activity were measured from the time courses of the assays that should also include some experimental errors as found in the noise and drift in the time courses of proton pumping measurement (Fig. 2c). Because the activity difference among the double mutants were subtle, the accuracy and precision of the biochemistry part are the critical points to prove the validity of their arguments. The detailed explanation son the estimation of experimental error as well as reproducibility are required.

    The second concern is the validity of the simulation. The authors conduced Monte Carlo simulation for proton transfer step between c-subunit and a-subunit. The rate constant was represented in a simple exponential factor: exp(-A(r-r0)) where 'A' represents the decay rate, 'r' is physical distance between c-subunit and a-subunit and 'r0' is the offset value that represents the sum of sidechain length of the proton transferring residues on c-subunit and a-subunit. They assumed smaller 'r0' and larger 'A' for cE59D mutant. Although the smaller 'r0' would be reasonable considering the shorter side change of aspartic acid, the reason for higher 'A' for the mutant is not clear. In addition, different values for pKa were given to the glutamic acid in the wild type c subunit (cE59) and to the aspartic acid in the mutant (cE59D), without rationalization. These parameters should be critical for the simulation results. The validity of the different 'A' and pKa in the mutant should be explained.

  3. eLife

    Evaluation Summary:

    This is an interesting manuscript describing for the first time experimentally the cooperative effects of mutations to individual key Glu residues in the c-ring of ATP synthase. The main result is that mutations in nearby c subunits are less inhibitory than those in subunits further apart in the ring. This is explained on the basis of MD/MC simulations as a shared waiting time for delayed proton uptake in case of neighboring subunits, which appears logical. Overall the manuscript is well presented, but with some caveats. The works will be of interest to specialists in bioenergetics, and to a wider biochemical, biophysical and structural biology audience.

    (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 #1 and Reviewer #2 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    FoF1-ATP synthase couples proton translocation across the membrane to ATP synthesis/hydrolysis. When the proton motive force dominates, the rotor within this complex moves in one direction, promoting ATP synthesis. On the other hand, when in ATP-driven mode, the rotor moves in the opposite direction and ATP hydrolysis is used to translocate protons against their concentration gradient. The internal symmetry of the homo-oligameric rotor (8-17 units, depending on the variant) suggests cooperative mode of action. Here an engineered variant with 10 concatenated copies of the unit is used to examine cooperativity. Single and double mutants of the key glutamic acid that shuttles the protons from the rotor to the stator are used and the results support cooperativity because of the dependence of activity on the distance between the mutated amino acids. Simulations, using a recently introduced coarse grained model, support this suggestion and provide molecular interpretation of the results. A simple kinetic model that accounts for the observed cooperativity is derived.

    Cleverly integrated experimental and computational approaches support the conclusion, and the results are honestly and modestly presented, admitting to imperfections. The one thing that bothers me some is whether the relatively minor activity differences between the double mutants with close vs more remote positions are significant enough. The statistical analysis suggests that they are, but sill... I also added a few minor suggestions to improve the manuscript further.

  5. eLife

    Reviewer #3 (Public Review):

    This is an interesting manuscript describing for a first time experimentally the cooperative effects of mutations to individual key Glu residues in the c-ring of ATP synthase. The main result is that mutations in nearby c subunits are less inhibitory than those in subunits further apart in the ring. This is explained on the basis of MD/MC simulations as a shared waiting time for delayed proton uptake in case of neighboring subunits, which appears logical. Overall the manuscript is well presented, but with some caveats described below, which should be addressed. It will be of interest to the specialists in bioenergetics, and to a wider audience working in biochemistry.

    General comment: the cooperativity is shown here in case of mutants, but it is not so obvious how it relates to WT enzyme. One clue, to which authors only briefly relate, is that according to their earlier simulations in WT the preferred pathway is when 2 or 3 Glu are unprotonated at any time rather than just one Glu being protonated/unprotonated. This kind of "cooperativity" in WT enzyme and its relation to presented here data should be discussed in more detail here.

    Also, parts of text, such as the introduction, as not very clearly written and can be improved.

  6. Version published to 10.1101/2021.03.25.436961v2 on bioRxiv
  7. Version published to 10.1101/2021.03.25.436961v1 on bioRxiv