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  1. Author Response

    Reviewer #1 (Public Review):

    Rasicci et al. have developed a FRET biosensor that is designed to light up when cardiac myosin folds. This structure is extremely important to understand, and its link to the super-relaxed (SRX) state has not been fully shown. Their study provides a comprehensive review of the literature and provides compelling data that the 15 heptad+leucine zipper+GFP construct does function well and that the DCM mutant E525K has a similar IVM velocity despite a reduced ATPase compared with HMM. They rely on the ionic strength-dependent changes in the rate of MantATP release to argue that the E525K mutation stabilizes the 'interacting heads motif' (IHM) state, which makes logical sense.

    Strengths:

    Well written and comprehensive.

    Utilizes the appropriate fluorescence-based sensor for measuring the folding of the myosin structure. Provides a detailed range of techniques to support the premise of the study

    Weaknesses:

    Over-interpretation of the outcomes from this study means that the IHM and SRX are the same. Similar studies, e.g. Anderson 2018 and Chu 2021 support the opposite view that IHM and SRX are not necessarily the same, Anderson (and Rohde 2018) point out that S1 has some element of a reduced ATPase, this clearly cannot be due to folding of the molecule. Also, mavacamten was used in these studies to show that even S1 is inhibited suggesting that SRX and IHM are not connected. This is not to say that with enough supporting evidence that these observations cannot be over-ridden, it is just not clear that there is enough in this study to support this conclusion.

    We have revised our discussion to emphasize that our results support a model in which the SRX state is enhanced by formation of the IHM, but given the S1 and 2HP data the IHM may not be required for populating the SRX biochemical state (see page 8).

    I felt that the authors passed over the recent Chu 2021 paper too quickly, the Thomas group used a FRET sensor as well and provides a direct comparison as a technique, but with opposite conclusions. They also have supporting data in Rohde 2018 that their constructs were less ionic strength sensitive. It would be useful to understand what the authors think about this.

    We have discussed the Rohde and Chu papers in more detail in the discussion (see page 8). In the Rhode paper they used proteolytically prepared HMM and S1. Rohde found 20% SRX at all KCl concentrations in S1, while HMM shifted from 50% to 20% SRX in low and high salt conditions, respectively. Our results are different in terms of the absolute fraction of the SRX state but the trend is similar in terms of S1 being salt-insensitive and HMM being salt-sensitive. The difference could be proteolytic HMM, which is a longer construct, and proteolytic S1, which is prone to internal cleavage that can impact ATPase activity. Another difference could be the mixed isoform of mantATP used in previous studies and the single isoform of mantATP used on our study (see page 5)

    Reviewer #2 (Public Review):

    The paper by Rasicci et al. examines the impact of the DCM mutation E525K in beta-cardiac myosin on its function and regulation by autoinhibition. The role of the auto-inhibited state of beta-cardiac myosin in fine-tuning cardiac contractility is an active and exciting area of current research related to muscle biology and cardiomyopathies. Several studies in the past have linked the destabilization of the autoinhibited, super-relaxed (SRX) state of myosin to the pathogenesis of hypertrophic cardiomyopathy. This timely study provides one of the first few examples where the hypocontractile phenotype of a DCM mutation has been linked to the stabilization of the SRX state.

    One of the strengths here is the utilization of a wide variety of both pre-existing and novel biochemical and biophysical assays for the study. The authors have characterized a new two-headed long-tailed myosin construct containing 15-heptad repeats of the proximal S2 (15HPZ), which they show allows myosin to form the SRX state in vitro using single ATP turnover assays. The authors go on to compare the E525K and WT proteins using the 15HPZ myosin construct in terms of their steady-state actin-activated ATPase activity, in-vitro actin-sliding velocity and single ATP turnover measurements. These assays reveal that the predominant effect of this mutation is the stabilization of the SRX state which is maintained even at 150 mM salt concentration where the WT SRX is largely disrupted. This is an important observation because DCM mutations so far have been believed to only affect the force-generating capacity of myosin.

    One of the biggest strengths of this study is the attempt to develop a FRET-based approach to directly ask if the biochemical SRX state here correlates well with the structural IHM state, which is an important unresolved question in the field. The authors have designed a FRET pair (C-terminal GFP and Cy3ATP bound to the active site) that is sensitive to the relative position of the heads and the tail, allowing them to distinguish between the low-FRET closed IHM conformation and the no-FRET open conformation. Remarkably, the authors show that the salt dependence of the FRET efficiency values closely follows their results from the salt dependence of the percent SRX for both WT and E525K proteins. The authors then attempt to substantiate their FRET results by a direct visual analysis of the conformational states populated by both WT and E525K proteins at low salt using negative staining EM analysis. The authors have optimized conditions to allow the deposition of the IHM state on grids without adding the small molecule mavacamten, which was found to be necessary in an earlier study to visualize the closed state using EM. The authors conclude that the SRX state correlates well with the IHM state and that the E525K mutation indeed stabilizes the folded-back conformation of myosin.

    This study significantly strengthens the previously illustrated correlation between the SRX and IHM states and provides methodological advances (especially visualization of the IHM state by negative EM in the absence of cross-linking agents) that will be very useful to the field going forward. The observation that a DCM mutation can lead to stabilization of the folded back state is a novel insight that should spark interest in the field to test how broadly this applies to other DCM mutations. The conclusions of the paper are mostly supported by the data; however, some clarifications and qualifications are needed.

    Weaknesses:

    The extremely low enzymatic activity of the M2β 15HPZ myosins as compared to the WT S1 control (which is a historical control not assayed in parallel with the 15HPZ proteins), is concerning for the low protein quality of the 15HPZ myosins. The authors attribute the low kcat to the high proportion of SRX population in their ensembles. However, the DRX rates reported for the WT and E525K 15HPZ proteins in the single ATP turnover assay are ~3-4 fold lower than those of their S1 counterparts. These rates reflect basal turnover of ATP in the open state and thus should not be affected by the presence of the S2 tail, which leads to concerns about the 15HPZ protein activity. In addition, the very high percentage of stuck filaments in the in vitro motility assay for the 15HPZ constructs (despite the use of dark actin) is also concerning for significant amounts of enzymatically inactive protein.

    We thank the reviewer for pointing out the differences in the S1 and HMM DRX rates. We performed additional single turnover measurements with S1, adding two sets of measurements from one additional preparation (N=3), and we demonstrate that there is a significant increase in the DRX rates of WT S1 compared to WT HMM (see pages 4-5, Table 3, Figure 3- figure supplement 3). A faster rate in S1 was also reported in Rohde et al. 2018. Indeed, the DRX rates of E525K S1 are significantly higher than WT in S1, which we also now report in the results (see page 5, Figure 3 – figure supplement 3). We addressed the concerns about 15HPZ activity by performing NH4+ ATPase assays to demonstrate that the number of active heads was similar in S1 and 15HPZ HMM (see page 4). It is possible that the higher percentage of stuck filaments in the HMM motility is due to myosin heads in the IHM state on the motility surface, which generate a drag force by non-specifically interacting with actin, but further study is necessary to examine this question.

    The authors assert that the E525K mutation represents a new mechanism by which DCM-causing mutations lead to decreased contractility - by stabilizing the sequestered state rather than affecting motor function. However, there is no evaluation of the motor function (actin-activated ATPase activity or in vitro motility) of the E525K S1, which would reveal the effects of the mutation without confounding effects due to the sequestering of heads. Interestingly, in the single ATP turnover assay, the DRX rate of the E525K S1 is >2-fold higher than the WT control, suggesting that the mutation may have effects beyond stabilization of the SRX state. The conclusion that the E525K mutation's effect on myosin function is mediated via stabilization of the SRX state would be strengthened if the effects of the mutation on the motor domain alone were also known.

    We thank the reviewer for this suggestion. We performed actin-activated ATPase assays with WT and E525K S1 and found that E525K increases kcat and lowers KATPase, demonstrating enhanced intrinsic motor activity in the mutant S1 construct (see page 4, Figure 2B). This adds an interesting dimension to the manuscript because we report a mutant that enhances the intrinsic motor activity but stabilizes the SRX/IHM (see Discussion page 10). We did not perform in vitro motility, because this assay depends on the surface attachment strategy, and we would like to compare all constructs with the same attachment strategy using a C-terminal GFP tag (mutant and WT S1 and 15HPZ HMM). Therefore, we are making the S1 construct with a C-terminal GFP tag for this purpose, to be examined in a future study.

    While the authors show strong qualitative correlations between the SRX and IHM states using single ATP turnover, FRET, and EM experiments, attempts to quantitatively compare the fraction of heads in the IHM state using the various experimental approaches is problematic. For example, the R0 value of the FRET pair used here doesn't allow precise measurement of the distances being probed here to be made, but the distances are reported and compared to predicted distances. The authors report that the R0 for their FRET pair is 63 Å. Surprisingly the authors go on to use the steady-state FRET efficiency values to determine the average D-A distance (Fig 5B) which is 100 Å when all heads are in the IHM configuration and becomes larger than that when heads open. R0 of 63 Å allows a precise distance measurement to be made in the 31.5-94.5 Å range which corresponds to 0.5-1.5 R0. It is therefore technically incorrect to use the steady-state FRET efficiency values to determine the D-A distance here. Besides, there are several unknown factors here like orientation factor (κ2) which further complicate these calculations. Similarly, the quantification of IHM state molecules from the negative stain EM experiments is significantly hampered by the disruptive effect of the grid surface on the structure of the IHM state. The authors find that limiting the contact time with the grid to ~ 5s is necessary to preserve the IHM state.

    Despite that, only ~15% WT molecules were seen in the IHM state at low salt (Fig. 6B). In contrast, ~56% E525K molecules were in the IHM state. Both these proteins have similar SRX proportions (Fig. 3C) and similar FRET efficiency values (Fig. 5A) at this salt concentration. This mismatch highlights the problem arising due to not having a measure of the populations from the FRET data. It is not clear if the hugely different proportions of the IHM state in EM experiments are indicative of the relative stability of this state in the two proteins or a random difference in the electrostatic interactions of WT vs mutant with the grid. These experiments do not provide a correct idea of the %IHM in the two proteins. In the absence of any IHM population measurement, it is important to proceed with caution when quantitatively correlating the SRX and IHM.

    We thank the reviewer for pointing out that measuring precise distances by FRET can be difficult. We agree that the low FRET efficiency makes precise distance determination even more challenging. However, FRET is quite good at measuring a change in distance given a specific donor-acceptor pair. We feel our FRET biosensor clearly demonstrates FRET efficiencies that are salt-insensitive in E525K but a clear decrease in FRET at higher salt concentrations in WT. In order to compare the trend in the predicted FRET, based on the single turnover measurements, and the actual FRET we thought it was important to plot the two together on the same graph. We understand that this could have been misleading that we were reporting actual distances. We have now plotted the FRET efficiency instead of distance as a function of KCl concentration (Figure 5B), to prevent any confusion with reporting distances. In addition, we have emphasized that the data are plotted to allow for a comparison of the trend in the single turnover and FRET data (see page 6, 10, Figure 5B).

    We agree that it is important to proceed with caution when comparing the EM to the FRET and single turnover data. The EM does not give a quantitative estimate of the fraction of IHM molecules, due to the disruptive effect of the grid surface on protein conformation. However, it does provide direct (though qualitative) evidence that the conformation underlying SRX and enhanced FRET is the IHM, and it is consistent with our interpretation that the E525K mutation enhances FRET and SRX by stabilizing the IHM. To consolidate this result, we have performed EM experiments now with a total of 3 preparations of WT and mutant (see page 6-7 and Figure 6D). We find that while there is variability from experiment to experiment, likely because the grid surface is slightly different each time the experiment is performed, in all cases there was a ~4-fold higher fraction of folded molecules in the mutant. Since each WT/mutant experimental pair was studied in parallel, using identically prepared grids, the results provide further evidence that the mutant stabilizes the IHM. However, we agree that a quantitative, direct visual correlation of the SRX and IHM is not possible based on the current EM data.

    Finally, the utility of the methods described in the paper to the field would be greatly enhanced if they were described in more detail. As currently written, it would be difficult for others to replicate these experiments.

    Thank you for the comment. We have made significant changes in the methods to clarify the details of the experiments (see pages 11-14). In addition, we have added details to the results and figure legends.

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  2. Evaluation Summary:

    This study seeks to develop the use of a FRET-based sensor for the formation of the folded 'interacting heads motif' structure for cardiac myosin, which is thought by some to represent a super-relaxed state with lower basal ATPase activity. This study offers some evidence that there is a relationship between the super-relaxed state and the 'interacting heads motif' structure, and that a specific dilated cardiomyopathy mutant in this myosin stabilizes the 'interacting heads motif' conformation. This paper will be of interest to muscle and cardiovascular biologists as it provides important insights into the correlation of structural and functional states of motor proteins in the context of cardiac muscle. The data qualitatively support this correlation and suggest a new mode of action of disease-causing mutations that lead to impaired contractile function.

    (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):

    Rasicci et al. have developed a FRET biosensor that is designed to light up when cardiac myosin folds. This structure is extremely important to understand, and its link to the super-relaxed (SRX) state has not been fully shown. Their study provides a comprehensive review of the literature and provides compelling data that the 15 heptad+leucine zipper+GFP construct does function well and that the DCM mutant E525K has a similar IVM velocity despite a reduced ATPase compared with HMM. They rely on the ionic strength-dependent changes in the rate of MantATP release to argue that the E525K mutation stabilizes the 'interacting heads motif' (IHM) state, which makes logical sense.

    Strengths:

    Well written and comprehensive.
    Utilizes the appropriate fluorescence-based sensor for measuring the folding of the myosin structure.
    Provides a detailed range of techniques to support the premise of the study

    Weaknesses:

    Over-interpretation of the outcomes from this study means that the IHM and SRX are the same. Similar studies, e.g. Anderson 2018 and Chu 2021 support the opposite view that IHM and SRX are not necessarily the same, Anderson (and Rohde 2018) point out that S1 has some element of a reduced ATPase, this clearly cannot be due to folding of the molecule. Also, mavacamten was used in these studies to show that even S1 is inhibited suggesting that SRX and IHM are not connected. This is not to say that with enough supporting evidence that these observations cannot be over-ridden, it is just not clear that there is enough in this study to support this conclusion.

    I felt that the authors passed over the recent Chu 2021 paper too quickly, the Thomas group used a FRET sensor as well and provides a direct comparison as a technique, but with opposite conclusions. They also have supporting data in Rohde 2018 that their constructs were less ionic strength sensitive. It would be useful to understand what the authors think about this.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    The paper by Rasicci et al. examines the impact of the DCM mutation E525K in beta-cardiac myosin on its function and regulation by autoinhibition. The role of the auto-inhibited state of beta-cardiac myosin in fine-tuning cardiac contractility is an active and exciting area of current research related to muscle biology and cardiomyopathies. Several studies in the past have linked the destabilization of the autoinhibited, super-relaxed (SRX) state of myosin to the pathogenesis of hypertrophic cardiomyopathy. This timely study provides one of the first few examples where the hypocontractile phenotype of a DCM mutation has been linked to the stabilization of the SRX state.
    One of the strengths here is the utilization of a wide variety of both pre-existing and novel biochemical and biophysical assays for the study. The authors have characterized a new two-headed long-tailed myosin construct containing 15-heptad repeats of the proximal S2 (15HPZ), which they show allows myosin to form the SRX state in vitro using single ATP turnover assays. The authors go on to compare the E525K and WT proteins using the 15HPZ myosin construct in terms of their steady-state actin-activated ATPase activity, in-vitro actin-sliding velocity and single ATP turnover measurements. These assays reveal that the predominant effect of this mutation is the stabilization of the SRX state which is maintained even at 150 mM salt concentration where the WT SRX is largely disrupted. This is an important observation because DCM mutations so far have been believed to only affect the force-generating capacity of myosin.

    One of the biggest strengths of this study is the attempt to develop a FRET-based approach to directly ask if the biochemical SRX state here correlates well with the structural IHM state, which is an important unresolved question in the field. The authors have designed a FRET pair (C-terminal GFP and Cy3ATP bound to the active site) that is sensitive to the relative position of the heads and the tail, allowing them to distinguish between the low-FRET closed IHM conformation and the no-FRET open conformation. Remarkably, the authors show that the salt dependence of the FRET efficiency values closely follows their results from the salt dependence of the percent SRX for both WT and E525K proteins. The authors then attempt to substantiate their FRET results by a direct visual analysis of the conformational states populated by both WT and E525K proteins at low salt using negative staining EM analysis. The authors have optimized conditions to allow the deposition of the IHM state on grids without adding the small molecule mavacamten, which was found to be necessary in an earlier study to visualize the closed state using EM. The authors conclude that the SRX state correlates well with the IHM state and that the E525K mutation indeed stabilizes the folded-back conformation of myosin.

    This study significantly strengthens the previously illustrated correlation between the SRX and IHM states and provides methodological advances (especially visualization of the IHM state by negative EM in the absence of cross-linking agents) that will be very useful to the field going forward. The observation that a DCM mutation can lead to stabilization of the folded back state is a novel insight that should spark interest in the field to test how broadly this applies to other DCM mutations. The conclusions of the paper are mostly supported by the data; however, some clarifications and qualifications are needed.

    Weaknesses:

    The extremely low enzymatic activity of the M2β 15HPZ myosins as compared to the WT S1 control (which is a historical control not assayed in parallel with the 15HPZ proteins), is concerning for the low protein quality of the 15HPZ myosins. The authors attribute the low kcat to the high proportion of SRX population in their ensembles. However, the DRX rates reported for the WT and E525K 15HPZ proteins in the single ATP turnover assay are ~3-4 fold lower than those of their S1 counterparts. These rates reflect basal turnover of ATP in the open state and thus should not be affected by the presence of the S2 tail, which leads to concerns about the 15HPZ protein activity. In addition, the very high percentage of stuck filaments in the in vitro motility assay for the 15HPZ constructs (despite the use of dark actin) is also concerning for significant amounts of enzymatically inactive protein.

    The authors assert that the E525K mutation represents a new mechanism by which DCM-causing mutations lead to decreased contractility - by stabilizing the sequestered state rather than affecting motor function. However, there is no evaluation of the motor function (actin-activated ATPase activity or in vitro motility) of the E525K S1, which would reveal the effects of the mutation without confounding effects due to the sequestering of heads. Interestingly, in the single ATP turnover assay, the DRX rate of the E525K S1 is >2-fold higher than the WT control, suggesting that the mutation may have effects beyond stabilization of the SRX state. The conclusion that the E525K mutation's effect on myosin function is mediated via stabilization of the SRX state would be strengthened if the effects of the mutation on the motor domain alone were also known.

    While the authors show strong qualitative correlations between the SRX and IHM states using single ATP turnover, FRET, and EM experiments, attempts to quantitatively compare the fraction of heads in the IHM state using the various experimental approaches is problematic. For example, the R0 value of the FRET pair used here doesn't allow precise measurement of the distances being probed here to be made, but the distances are reported and compared to predicted distances. The authors report that the R0 for their FRET pair is 63 Å. Surprisingly the authors go on to use the steady-state FRET efficiency values to determine the average D-A distance (Fig 5B) which is 100 Å when all heads are in the IHM configuration and becomes larger than that when heads open. R0 of 63 Å allows a precise distance measurement to be made in the 31.5-94.5 Å range which corresponds to 0.5-1.5 R0. It is therefore technically incorrect to use the steady-state FRET efficiency values to determine the D-A distance here. Besides, there are several unknown factors here like orientation factor (κ2) which further complicate these calculations. Similarly, the quantification of IHM state molecules from the negative stain EM experiments is significantly hampered by the disruptive effect of the grid surface on the structure of the IHM state. The authors find that limiting the contact time with the grid to ~ 5s is necessary to preserve the IHM state. Despite that, only ~15% WT molecules were seen in the IHM state at low salt (Fig. 6B). In contrast, ~56% E525K molecules were in the IHM state. Both these proteins have similar SRX proportions (Fig. 3C) and similar FRET efficiency values (Fig. 5A) at this salt concentration. This mismatch highlights the problem arising due to not having a measure of the populations from the FRET data. It is not clear if the hugely different proportions of the IHM state in EM experiments are indicative of the relative stability of this state in the two proteins or a random difference in the electrostatic interactions of WT vs mutant with the grid. These experiments do not provide a correct idea of the %IHM in the two proteins. In the absence of any IHM population measurement, it is important to proceed with caution when quantitatively correlating the SRX and IHM.

    Finally, the utility of the methods described in the paper to the field would be greatly enhanced if they were described in more detail. As currently written, it would be difficult for others to replicate these experiments.

    Was this evaluation helpful?