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  1. Author Response

    Reviewer #1 (Public Review):

    The authors have employed a variety of techniques (single-molecule fluorescence kinetic and steady state measurements, cryo-EM structure determination, and in vivo measurements of protein synthesis and cell proliferation) to investigate the mechanism of action of two molecule products: Didemnin B and Ternatin-4. Both molecules have previously shown to target eEF1A and have potential as cancer therapeutics. In addition, the structure of Didemnin B, bound to eEIF1A and to an elongation complex, have previously been solved.

    The authors here show that both compounds disrupt the dynamic accommodation of tRNA driven by eEF1A and its activation by the GTPase activation center of the ribosomal large subunit, relying on previous assignment of the FRET intensities observed in pre-steady state single-molecule fluorescence experiments in which peptide-tRNA and incoming aminoacylated tRNA are labeled with donor and acceptor dyes, respectively. They further show that this inhibition is dose dependent for both compounds and sensitive to the A399V eEF1A mutant, which creates a steric clash with didemnin B in its usual binding site. Subsequent analysis of steady-state single-molecule FRET experiments shows that didemnin B more strongly inhibits transitions between the intermediate (0.45) FRET state and the high (0.8) FRET states (though the authors choose to focus only on the effect of transitions from 0.45 to 0.8) previously assigned to the GTPase activated and fully accommodated conformations of the ternary complex, respectively. Further single-molecule experiments provide initial evidence that Didemnin B remains more stably bound to elongation complexes than does Ternatin-4.

    The authors then turn to cryo-EM structures of each compound bound to elongation complexes purified either from lysate or assembled from purified components. The structure of the Ternatin-4 complex shows additional density in the same binding cleft observed for Didemnin B in a prior structure reported elsewhere, with which the Didemnin B structures reported here also agree. This binding location provides structural evidence for both compounds effects on ternary complex dynamics, as well as their previously described effects on tRNA accommodation and elongation. Further comparison of the Didemnin B and Ternatin-4 structures reveals decreased electron density in the Ternatin-4 structure for elements of eEF1A (switch loops 1 and 2 and helix alpha2), as compared to the Didemnin B structures. The authors interpret this as evidence for greater mobility of these elements, which might explain the more modest restriction of A-site tRNA dynamics they observe in the presence of Ternatin-4 (as opposed to Didemnin B). Certainly this decreased density (which might be more convincingly demonstrated using difference maps of the two structures) is consistent with that interpretation. That said, it is certainly not a smoking gun.

    We have worked to soften the language pertaining to this point and have updated Fig. 4 to more accurately highlight the observed differences between the didemnin and ternatin-4 structures.

    Finally, the authors turn to in vivo measurements of protein synthesis and effects on cellular proliferation or survival in the presence of both compounds. Consistent with their single-molecule experiments, they observe more severe and durable inhibition of protein synthesis in the presence of Didemnin B, whereas Ternatin-4 exhibits more modest effects that are more rapidly restored upon removal of the drug in solution. Interestingly, Ternatin-4 appears to elicit similar, and perhaps more rapid, effects on cellular survival, increasing apoptosis more rapidly than Didemnin B, though these effects (like those on protein synthesis rates) are once again more sensitive to removal of the drug. The authors describe these results as evidence that Didemnin-B "irreversibly inhibits" protein synthesis in cells. I find this assertion strange, given that the authors have previously measured a dissociation rate for this molecule from elongation complexes and they have not performed measurements to ensure that activity is not simply restored at timescales longer than their initial measurements. That said, I concede that this might be a semantic distinction if the vast majority of cells perish prior to dissociation of the drug. In either case, I would suggest the authors apply a somewhat more nuanced interpretation of these results lest they be misunderstood.

    We thank Reviewer 1 for bringing this point to our attention. We have changed the title of this section to “Protein synthesis inhibition by ternatin-4, but not didemnin, can be reversed in cells,” and have softened the interpretation.

    Overall, this is a rigorous and well reasoned study that employs multiple complementary techniques to investigate the mechanism of action of compounds of potential therapeutic interest. In places, the higher order interpretation of the experimental data leaks into the results section (as opposed to being fully explored in the discussion) and is at times somewhat aggressive. Nonetheless, the results presented here illuminate important questions at the intersection of translational mechanism, cell proliferation, and cancer.

    We are grateful to Reviewer 1 for their assessment of this work as rigorous and well-reasoned. We have made significant updates to the text and figures, and we hope they find that we have addressed all concerns.

    Reviewer #2 (Public Review):

    The manuscript of Juette et al presents a combined structural and dynamic view of how a class of inhibitors (Didemins) block human ribosomal elongation. Prior work had shown that these cyclic peptide drugs bind to eEF1A in the ternary complex on the ribosome, between the Domains I and III of the factor, blocking the dissociation of the elongation factor from elongator tRNA and ribosome during decoding. Here the authors use beautiful single-molecule and structural approaches to probe the mechanisms of two related drugs-Didemnin B and Ternatin-4. Their results expand on prior observations of drug mechanism, and provide clarity for the similarities and differences on how the two drugs work both in vitro and in vivo. Using single-molecule tRNA-tRNA FRET, the authors show that the drugs (at saturating concentrations) block progression of the tRNA from a mid-FRET (GTPase activating) state to the fully accommodated (high FRET) state; they observe slightly more transitions to high FRET in the presence Ternatin-4 than Didemnin B (more below on this). These results are consistent with the idea that the drugs trap the ternary complex on the ribosome after GTP hydrolysis. Using the fraction of ribosomes that lead to accommodation, the authors performed a titration to determine the apparent Ki for the drugs (which were similar in the range of 5-10nM). They also performed clever washout experiments (always in presence of cycloheximide to block further conformational dynamics once a tRNA accommodates). These experiments probed the drug dissociation rate and showed marked differences between Didemnin-B (slow rate) and Ternatin-4 (faster rate). The authors then recapitulate the prior structural work (at lower resolution in RRL) using a reconstituted system. Their results show a similar structure as that solved previously, but with more disordered loops in the presence of ternatin-4, although the resolution here is moderate (3.2 and 3.8Å for the two drug complexes). Finally, the authors perform in vivo analyses of drug action on protein synthesis using clickable amino acid incorporation. They show that the two drugs block protein synthesis in a dose dependent manner, and that the effect of ternatin-4 can be reversed by washout of the drug, whereas that of didemnin-2 is poorly reversed, explaining differences in drug action despite the similar binding site.

    Overall, this is a rigorous and well performed study probing the mechanisms of drug action in human translation elongation. The combination of dynamics measurements and structure are particularly novel, and will complement ongoing investigations (and publications) by the Blanchard lab on human elongation in general.

    We thank Reviewer 2 for their assessment of this work as rigorous and novel.

    Reviewer #3 (Public Review):

    In this article, Juette et al employed single-molecule FRET, cryo-EM, and Hpg incorporation (in cell translation assays) to compare the mechanisms by which Didemnin B and Ternatin-4 inhibit translation elongation. They found that, while binding to the same pocket of eEF1A and blocking accommodation after GTP hydrolysis, Didemnin B had an irreversible effect on protein synthesis, but Ternatin-4, while still a potent inhibitor, allowed more flexibility in complexes (increased disorder of regions in cryo-EM structures) that allowed increased sampling of on-pathway accommodated states (observed by smFRET), and reversibility of effects on protein synthesis in cultured cells (by Hpg incorporation). This is a straightforward study and the conclusions are well-supported by the data using appropriate techniques. The work will be of impact to the ribosome field, which may use these drugs in other mechanistic studies, and researchers wanting to employ the drugs to combat cancer and other diseases.

    We are thankful to Reviewer 3 for their assessment of this work as well-supported and impactful.

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  2. eLife Assessment:

    Juette and coworkers employed single-molecule fluorescence, cryogenic-electron microscopy structures, and in vivo measurements to investigate the mechanism whereby two natural products with potential as cancer therapeutics, didemnin B and ternatin-4, act. The compounds are shown to inhibit tRNA accommodation within the ribosomal A site during translation elongation by interfering with movement of eukaryotic elongation factor 1 alpha after its activation by the GTPase activation site of the ribosome, with the degree and nature of this restriction differing subtly between the two compounds, leading to more marked differences in their effects on global translation and cell growth. The compelling results of this interdisciplinary work solidify prior conclusions, particularly on didemnin B, and illuminate the similarities and differences on how these two drugs interfere with the normal functioning of the elongating ribosome in vitro and inhibit protein synthesis and cell growth in vivo. Some revisions of figures and text are required to clarify the results and the authors' interpretations.

    (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, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

    Was this evaluation helpful?
  3. Reviewer #1 (Public Review):

    The authors have employed a variety of techniques (single-molecule fluorescence kinetic and steady state measurements, cryo-EM structure determination, and in vivo measurements of protein synthesis and cell proliferation) to investigate the mechanism of action of two molecule products: Didemnin B and Ternatin-4. Both molecules have previously shown to target eEF1A and have potential as cancer therapeutics. In addition, the structure of Didemnin B, bound to eEIF1A and to an elongation complex, have previously been solved.

    The authors here show that both compounds disrupt the dynamic accommodation of tRNA driven by eEF1A and its activation by the GTPase activation center of the ribosomal large subunit, relying on previous assignment of the FRET intensities observed in pre-steady state single-molecule fluorescence experiments in which peptide-tRNA and incoming aminoacylated tRNA are labeled with donor and acceptor dyes, respectively. They further show that this inhibition is dose dependent for both compounds and sensitive to the A399V eEF1A mutant, which creates a steric clash with didemnin B in its usual binding site. Subsequent analysis of steady-state single-molecule FRET experiments shows that didemin B more strongly inhibits transitions between the intermediate (0.45) FRET state and the high (0.8) FRET states (though the authors choose to focus only on the effect of transitions from 0.45 to 0.8) previously assigned to the GTPase activated and fully accommodated conformations of the ternary complex, respectively. Further single-molecule experiments provide initial evidence that Didemnin B remains more stably bound to elongation complexes than does Ternatin-4.

    The authors then turn to cryo-EM structures of each compound bound to elongation complexes purified either from lysate or assembled from purified components. The structure of the Ternatin-4 complex shows additional density in the same binding cleft observed for Didemnin B in a prior structure reported elsewhere, with which the Didemnin B structures reported here also agree. This binding location provides structural evidence for both compounds effects on ternary complex dynamics, as well as their previously described effects on tRNA accommodation and elongation. Further comparison of the Didemnin B and Ternatin-4 structures reveals decreased electron density in the Ternatin-4 structure for elements of eEF1A (switch loops 1 and 2 and helix alpha2) , as compared to the Didemnin B structures. The authors interpret this as evidence for greater mobility of these elements, which might explain the more modest restriction of A-site tRNA dynamics they observe in the presence of Ternatin-4 (as opposed to Didemnin B). Certainly this decreased density (which might be more convincingly demonstrated using difference maps of the two structures) is consistent with that interpretation. That said, it is certainly not a smoking gun.

    Finally, the authors turn to in vivo measurements of protein synthesis and effects on cellular proliferation or survival in the presence of both compounds. Consistent with their single-molecule experiments, they observe more severe and durable inhibition of protein synthesis in the presence of Didemnin B, whereas Ternatin-4 exhibits more modest effects that are more rapidly restored upon removal of the drug in solution. Interestingly, Ternatin-4 appears to elicit similar, and perhaps more rapid, effects on cellular survival, increasing apoptosis more rapidly than Didemnin B, though these effects (like those on protein synthesis rates) are once again more sensitive to removal of the drug. The authors describe these results as evidence that Didemin-B "irreversibly inhibits" protein synthesis in cells. I find this assertion strange, given that the authors have previously measured a dissociation rate for this molecule from elongation complexes and they have not performed measurements to ensure that activity is not simply restored at timescales longer than their initial measurements. That said, I concede that this might be a semantic distinction if the vast majority of cells perish prior to dissociation of the drug. In either case, I would suggest the authors apply a somewhat more nuanced interpretation of these results lest they be misunderstood.

    Overall, this is a rigorous and well reasoned study that employs multiple complementary techniques to investigate the mechanism of action of compounds of potential therapeutic interest. In places, the higher order interpretation of the experimental data leaks into the results section (as opposed to being fully explored in the discussion) and is at times somewhat aggressive. Nonetheless, the results presented here illuminate important questions at the intersection of translational mechanism, cell proliferation, and cancer.

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

    The manuscript of Juette et al presents a combined structural and dynamic view of how a class of inhibitors (Didemins) block human ribosomal elongation. Prior work had shown that these cyclic peptide drugs bind to eEF1A in the ternary complex on the ribosome, between the Domains I and III of the factor, blocking the dissociation of the elongation factor from elongator tRNA and ribosome during decoding. Here the authors use beautiful single-molecule and structural approaches to probe the mechanisms of two related drugs-Didemnin B and Ternatin-4. Their results expand on prior observations of drug mechanism, and provide clarity for the similarities and differences on how the two drugs work both in vitro and in vivo. Using single-molecule tRNA-tRNA FRET, the authors show that the drugs (at saturating concentrations) block progression of the tRNA from a mid-FRET (GTPase activating) state to the fully accommodated (high FRET) state; they observe slightly more transitions to high FRET in the presence Ternatin-4 than Didemnin B (more below on this). These results are consistent with the idea that the drugs trap the ternary complex on the ribosome after GTP hydrolysis. Using the fraction of ribosomes that lead to accommodation, the authors performed a titration to determine the apparent Ki for the drugs (which were similar in the range of 5-10nM). They also performed clever washout experiments (always in presence of cycloheximide to block further conformational dynamics once a tRNA accommodates). These experiments probed the drug dissociation rate and showed marked differences between Didemnin-B (slow rate) and Ternatin-4 (faster rate). The authors then recapitulate the prior structural work (at lower resolution in RRL) using a reconstituted system. Their results show a similar structure as that solved previously, but with more disordered loops in the presence of ternatin-4, although the resolution here is moderate (3.2 and 3.8Å for the two drug complexes). Finally, the authors perform in vivo analyses of drug action on protein synthesis using clickable amino acid incorporation. They show that the two drugs block protein synthesis in a dose dependent manner, and that the effect of ternatin-4 can be reversed by washout of the drug, whereas that of didemnin-5 is poorly reversed, explaining differences in drug action despite the similar binding site.

    Overall, this is a rigorous and well performed study probing the mechanisms of drug action in human translation elongation. The combination of dynamics measurements and structure are particularly novel, and will complement ongoing investigations (and publications) by the Blanchard lab on human elongation in general.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    In this article, Juette et al employed single-molecule FRET, cryo-EM, and Hpg incorporation (in cell translation assays) to compare the mechanisms by which Didemnin B and Ternatin-4 inhibit translation elongation. They found that, while binding to the same pocket of eEF1A and blocking accommodation after GTP hydrolysis, Didemnin B had an irreversible effect on protein synthesis, but Ternatin-4, while still a potent inhibitor, allowed more flexibility in complexes (increased disorder of regions in cryo-EM structures) that allowed increased sampling of on-pathway accommodated states (observed by smFRET), and reversibility of effects on protein synthesis in cultured cells (by Hpg incorporation). This is a straightforward study and the conclusions are well-supported by the data using appropriate techniques. The work will be of impact to the ribosome field, which may use these drugs in other mechanistic studies, and researchers wanting to employ the drugs to combat cancer and other diseases.

    Was this evaluation helpful?