Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules

  1. Yuzuru Itoh
  2. Vivek Singh
  3. Anas Khawaja
  4. Andreas Naschberger
  5. Minh Duc Nguyen
  6. Joanna Rorbach
  7. Alexey Amunts

  • Curated by eLife

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    Evaluation Summary:

    As a consequence of the bacterial origin of mitochondria, a range of medically relevant antimicrobials can affect not only bacteria but also human cells. For example, they may inhibit mitochondrial protein synthesis, giving rise to important side-effects during medical treatment, such as hearing loss or renal toxicity in patients treated with aminoglycosides. In this manuscript, the authors present the structure of the human mitochondrial small ribosomal subunit bound to one such antibiotics, streptomycin. This cryoEM-based structural analysis will be of interest to scientists in the infectious disease community as well as those interested in ribosome structural biology. It provides an important advance that could aid future medicinal chemistry efforts to improve the therapeutic potential of streptomycin derivatives.

    (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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Abstract

The mitoribosome regulates cellular energy production, and its dysfunction is associated with aging. Inhibition of the mitoribosome can be caused by off-target binding of antimicrobial drugs and was shown to be coupled with a bilateral decreased visual acuity. Previously, we reported mitochondria-specific protein aspects of the mitoribosome, and in this article we present a 2.4-Å resolution structure of the small subunit in a complex with the anti-tuberculosis drug streptomycin that reveals roles of non-protein components. We found iron–sulfur clusters that are coordinated by different mitoribosomal proteins, nicotinamide adenine dinucleotide (NAD) associated with rRNA insertion, and posttranslational modifications. This is the first evidence of inter-protein coordination of iron–sulfur, and the finding of iron–sulfur clusters and NAD as fundamental building blocks of the mitoribosome directly links to mitochondrial disease and aging. We also report details of streptomycin interactions, suggesting that the mitoribosome-bound streptomycin is likely to be in hydrated gem-diol form and can be subjected to other modifications by the cellular milieu. The presented approach of adding antibiotics to cultured cells can be used to define their native structures in a bound form under more physiological conditions, and since streptomycin is a widely used drug for treatment, the newly resolved features can serve as determinants for targeting.

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  1. Version published to 10.7554/elife.77460 on eLife
  2. eLife

    Evaluation Summary:

    As a consequence of the bacterial origin of mitochondria, a range of medically relevant antimicrobials can affect not only bacteria but also human cells. For example, they may inhibit mitochondrial protein synthesis, giving rise to important side-effects during medical treatment, such as hearing loss or renal toxicity in patients treated with aminoglycosides. In this manuscript, the authors present the structure of the human mitochondrial small ribosomal subunit bound to one such antibiotics, streptomycin. This cryoEM-based structural analysis will be of interest to scientists in the infectious disease community as well as those interested in ribosome structural biology. It provides an important advance that could aid future medicinal chemistry efforts to improve the therapeutic potential of streptomycin derivatives.

    (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.)

  3. eLife

    Reviewer #1 (Public Review):

    Antibiotics of different classes often have the unfortunate property of inhibiting mitochondrial protein synthesis in humans. To overcome these off-target activities, it would be helpful to have high-resolution views of how these antibiotics bind the mitochondrial ribosome to enable future medicinal chemistry efforts. However, to date, no such high-resolution views are available, even for the analogous bacterial system. In this manuscript, Itoh et al. present a cryo-EM reconstruction of the human mitochondrial small ribosomal subunit bound to streptomycin, at a local resolution of ~2.2 Å. With this resolution, the authors are able to define the binding interactions between the compound and the binding pocket in the ribosome. Notably, the antibiotic seems to adopt the gem-diol form in the streptose ring, and also to involve a nearby magnesium ion with one inner-sphere coordination to an rRNA phosphate group. This structure provides an important advance in our understanding of how streptomycin binds the mitochondrial ribosome that could aid future efforts to improve the therapeutic properties of streptomycin derivatives. There are a few issues the authors should address, however, as described below.

    First, the authors suggest that adding streptomycin to the human cells prior to ribosome preparation ensures the complex is more physiological. It's not clear that they would have observed a different result had they purified mitochondrial small subunits and subsequently added the antibiotic in vitro. Rather, the more important point the authors could make is that the off-rate of the antibiotic must be rather slow for it to remain bound through the purification steps. (It would help to know just how much washing was done and with what volumes and times.) Is there evidence for slow off-rates, for example from wash-out experiments?

    Second, the authors present an "unbound" structure in Figure 1c on the right. Where are the data for this structure? It also needs to be described and deposited.

    Third, it is quite puzzling how the central streptose ring could be so flexible, given that the first and third rings are so well constrained by the binding pocket. Could the authors tell us whether it is possible that there is a mixture of aldehyde and gem-diol? It would help to see the aldehyde form modeled and fit to the density. The authors should present correlations for density fit as well.

  4. eLife

    Reviewer #2 (Public Review):

    The overall aim of this work was to understand binding of the aminoglycoside antibiotic streptomycin to the human mitochondrial ribosome. This is an undesirable off-target effect that occurs presumably because the mitochondrial ribosome remains quite similar to the bacterial ribosome from which it evolved. Understanding this binding and comparing it to streptomycin-bound ribosomes from target bacteria may allow for the development of new antibiotic variants without this problematic off-target binding.

    To develop this understanding the authors chose to use cryogenic electron microscopy (cryo-EM), the now-standard method for determining the structures of large macromolecular complexes like this. Of particular note, rather than taking the more common route of isolating the ribosomes and then adding the ligand, they instead chose to treat actively-growing human cells with streptomycin prior to isolating the ribosomes. The fact that the streptomycin appears (exceedingly) clearly in the final map despite the fact that none was included in any of the purification steps is very strong evidence that this is a physiologically-relevant result. One obvious (and somewhat sobering) question this raises for me: what are the implications of this for the vast body of in vitro mammalian cell culture research carried out with a cocktail of penicillin and streptomycin added to the media for infection control?

    The resolution of the final map (2.23Å) represents a dramatic improvement over the previous-best 2.97Å map of the mitochondrial ribosome small subunit, and to the authors' credit, it is clear that a great deal of effort has gone into high-quality modelling throughout. I am very happy to see that since it remains sadly common for some authors to focus their efforts only on the region of their immediate interest. In this case, the focal site is the bound streptomycin - which is clear and unambiguous, and in my opinion fully supports the authors' conclusions regarding the details of binding, most notable of which is that the aldehyde group found on one of the streptomycin sugar residues becomes hydrated to the geminal diol form (two hydroxyl groups bound to the same carbon) prior to binding the ribosome.

  5. eLife

    Reviewer #3 (Public Review):

    In this manuscript, the authors set out to determine the structural basis of off-target binding to the human mitoribosome of the anti-tuberculosis antibiotic streptomycin using the latest advances in cryo-electron microscopy. The maps they have generated are of very high quality, enabling them to visualize streptomycin bound to the shoulder of the small subunit with the coordinated waters and magnesium molecules at an impressive overall nominal resolution of 2.3 A. The resolution of the maps represents a significant improvement compared to the previously published X-ray structures of streptomycin bound to the bacterial ribosome, allowing the authors to propose an improved model for the binding mode of streptomycin. In particular, the authors find that the density for a previously modeled aldehyde group in the bacterial ribosome-streptomycin complexes is poorly defined. In contrast to previous work, the authors interpret the density as consistent with a hydrated aldehyde, a model that is supported by previously published solution NMR spectroscopic analysis.

    As streptomycin is still widely used for anti-TB therapy, this structure and the methodological advances involved in achieving such high-quality maps will be of interest to investigators in the mitochondrial biology field and more widely within the community of researchers interested in structure-guided drug design applied to infectious disease to minimize treatment-related toxicities. The data clearly reveal how streptomycin binds to the mitochondrial small subunit but some aspects of the study require further clarification.

    1. The precise source of the streptomycin used in the study is not currently stated in the text. It is not clear if the compound was subjected to detailed mass spectrometry analysis to validate the composition assumed by the authors and to identify any potential chemical heterogeneity.

    2. Although the overall density strongly supports the presence of bound streptomycin, the map does not convincingly support the proposed hydrated aldehyde within the streptomycin streptose moiety. While the claim that the poor density may reflect increased mobility of the hydrated aldehyde as the authors suggest is completely reasonable (and is supported by previously published NMR data), this would need to be independently validated given the ambiguity in the map. A structure of streptomycin added in vitro to the mitochondrial small subunit would resolve whether the added molecule is indeed modified by the cellular milieu. While the authors suggest that adding the streptomycin to cultured cells rather than in vitro is an advantage of the work, the disadvantage is that it generates uncertainty that has not been resolved by the cryo-EM map alone about the chemical nature of the bound compound. The density might also be consistent with a potassium ion, for example.

    3. Given the high quality of the map, it is surprising that there is no discussion about the remainder of the mitochondrial small subunit, including ligands/spermine listed in Table 1. What is the rationale for GMPPNP and ATP, which are listed as ligands in Table 1?

  6. Version published to 10.1101/2022.02.02.478878v1 on bioRxiv