Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

  1. Shachin Patra
  2. Cheng-Wei Lin
  3. Manas K. Ghosh
  4. Steven M. Havens
  5. Seth A. Cory
  6. David H. Russell
  7. David P. Barondeau

  • Curated by eLife

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    Summary: This study provides support for a proposed allosteric regulatory mechanism in a human iron-sulfur cluster biosynthesis protein that is linked to the human genetic disorder, Friedrich's Ataxia. In an approach guided by inspection of a structure of the human enzyme, the authors successfully converted a bacterial homolog lacking allosteric regulation into a system that behaves similarly to the human one. The work provides validation of the roles of accessory proteins in activating iron-sulfur cluster biosynthesis machinery. It also could open novel routes for therapeutic intervention in genetic disorders of this process in humans.

    The major concerns about the study center on the significance of the form of the human enzyme structure used as the basis for designing the mutagenesis/activity experiments in the bacterial enzyme. To bolster the underlying framework for the experiment design, the description of the existing human enzyme structures and how exactly they were used to select sites for mutagenesis in the bacterial counterparts should be improved to include more detail and balanced perspective. Experiments are suggested to show that activity enhancement upon addition of accessory proteins is specific to those factors, along with a more comprehensive discussion of the errors and reproducibility in activity measurements. Finally, the significance of the work would be elevated if the authors could use a similar approach to install activating mutations in the human enzyme - particularly if these could overcome the requirement for frataxin.

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Abstract

Iron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share ∼60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreich’s ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5’-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.

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  1. eLife

    Reviewer #3:

    From the technical perspective this manuscript provides clear results that are consistent with, but do not prove, what this reviewer believes is the main objective of the work; to establish the relevance of the open structure of the eukaryotic cysteine desulfurase complex. This reviewer has no good basis to either accept or reject the open structure as having physiological relevance. This could well be the case but it is not clear from my (limited) knowledge of the published literature that the relevance of the open structure is generally accepted. From this perspective I believe the manuscript is sound from the technical approach and experimental implementation but suffers from a lack of clarity about the case for and against the relevance of the open structure. If this is a point of controversy in the field the topic should be discussed in depth and the position of the authors more clearly articulated.

  2. eLife

    Reviewer #2:

    In this manuscript, Barondeau and co-workers test a hypothesis for the role of the protein frataxin in iron-sulfur cluster assembly, seeking, inter alia, to explain the observation that mutations in the gene encoding this protein are associated with the incurable neurodegenerative disease, Friederich's ataxia. Their notion is that, whereas the bacterial versions of the sulfur-providing cysteine desulfurase are stable homodimers - in which the interactions between the monomers help to organize the mobile loop harboring the key cysteine residue that serves as general acid and nucleophile in the C-S-cleavage reaction that mobilizes the sulfur for incorporation into the cluster - the human enzyme (i) has a dimer interface that has been weakened through evolution, (ii) can be monomeric or form non-optimal dimeric forms, and (iii) can be driven to adopt the optimally active dimer form by intervention of accessory proteins (e.g., frataxin). Their approach was to perturb a bacterial (E. coli) cysteine desulfurase (IscS) by structure-guided mutagenesis in an attempt to introduce into it the behavior of the human enzyme, specifically its activation by accessory proteins (here CyaA and FXN). The experiments were successful in this goal. I like this paper and believe that it is interesting and important. I would point out two aspects that perhaps leave room for improvement.

    1. In principle, it would have been a more powerful test of their hypothesis had they been able to perturb the human enzyme to get a constitutively active form, no longer dependent on the binding of the accessory proteins, either instead of, or in addition to, the converse perturbation of the bacterial system. Perhaps this approach was precluded by difficulties associated with the human enzyme?

    2. The second criticism is that the effects on quinonoid form decay and activity are rather modest. However, I believe that important biological effects can arise from even such modest regulation of enzyme activity levels.

  3. eLife

    Reviewer #1:

    This study presents a detailed and focused study of the structural basis for a regulation strategy used by a human iron-sulfur cluster biosynthesis system, elucidated by artificial installation of new amino acids into a bacterial system that lacks the allosteric elements of the human enzyme. The work includes quaternary structure analysis and activity assays of variant bacterial proteins. It is performed competently and supports the conclusions. But the focus may be too narrow for a general audience. To bring the work over the bar, the authors could test whether installing the bacterial residues into human NFS1 restores activity without frataxin (inactivated in the human genetic disorder Friedrich's Ataxia). Furthermore, some elements of the study could be presented more clearly/rigorously to communicate the significance of the work to a general audience. These suggestions are listed below.

    1. It would be useful for an unfamiliar reader to include a diagram of the bacterial and human iron-sulfur cluster biogenesis pathway. It would also be helpful to depict the mechanism of the IscS/NFS1 cysteine desulfurase reaction - essentially a picture to go along with the description of the PLP-dependent transformations described in paragraph 2.

    2. In the first paragraph of the results section - I would be interested to see more details about the selection of the three residues targeted for mutagenesis. For example, did the authors inspect the interfaces of existing crystal structures of these complexes? Did they create sequence alignments for multiple eukaryotic/prokaryotic cysteine desulfurases and select sites conserved in bacterial proteins but not eukaryotic ones? More description of the experimental or bioinformatics basis for selecting these three sites would be important for convincing the reader that the basis for this work is sound.

    3. The structural basis for the dimer interaction and the enhanced activity isn't completely clear - how do the changed interactions enhance the enzyme activity? A good description of the different quaternary forms and why they are more/less active is given on page 4-5 - but perhaps another link could be made between the exact residues targeted for substitution and the features of the system important for catalysis.

    4. On page 10, the authors describe changes in IscS quaternary structure as a function of concentration. What is the estimated copy number or concentration inside the cell? Which concentration ranges would be most physiologically relevant?

    5. Addition of any helper protein appears to increase the proportion of variant IscS dimer and activity. Is there any reason to believe that this phenomenon is simply a crowding effect? If the same amount of an unrelated protein is added - does the activity/dimer fraction change compared to variant IscS alone?

    6. I found the color scheme in Figure 1 hard to follow - could the authors keep the subunit colors consistent and use text labels directly on the figure panels for the subunits and forms (open, ready, etc). I also don't think the "Clash!!" labels are necessary. A more effective approach might be to use zoomed-in insets for each clash.

    7. In Figures 4-6 - could the authors include a more complete description of the error bars? What kind of error is shown? Are the replicates different experiments done on different days? These presentations might also benefit from showing the actual data points on top of the bars/error bars.

  4. eLife

    Summary: This study provides support for a proposed allosteric regulatory mechanism in a human iron-sulfur cluster biosynthesis protein that is linked to the human genetic disorder, Friedrich's Ataxia. In an approach guided by inspection of a structure of the human enzyme, the authors successfully converted a bacterial homolog lacking allosteric regulation into a system that behaves similarly to the human one. The work provides validation of the roles of accessory proteins in activating iron-sulfur cluster biosynthesis machinery. It also could open novel routes for therapeutic intervention in genetic disorders of this process in humans.

    The major concerns about the study center on the significance of the form of the human enzyme structure used as the basis for designing the mutagenesis/activity experiments in the bacterial enzyme. To bolster the underlying framework for the experiment design, the description of the existing human enzyme structures and how exactly they were used to select sites for mutagenesis in the bacterial counterparts should be improved to include more detail and balanced perspective. Experiments are suggested to show that activity enhancement upon addition of accessory proteins is specific to those factors, along with a more comprehensive discussion of the errors and reproducibility in activity measurements. Finally, the significance of the work would be elevated if the authors could use a similar approach to install activating mutations in the human enzyme - particularly if these could overcome the requirement for frataxin.

  5. Version published to 10.1101/2020.10.06.326603 on bioRxiv