A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1

  1. Joanna Koszela
  2. Anne Rintala-Dempsey
  3. Giulia Salzano
  4. Viveka Pimenta
  5. Outi Kamarainen
  6. Mads Gabrielsen
  7. Aasna L. Parui
  8. Gary S. Shaw
  9. Helen Walden

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Abstract

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin have been linked to early-onset Parkinson’s disease. Besides many other cellular roles, Parkin is involved in clearance of damaged mitochondria via mitophagy - a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognises its substrates for ubiquitination, and no substrate interaction site in Parkin has been reported. Here, we identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results explain the preferential targeting and ubiquitination of Miro1 by Parkin and provide a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation induced by mitochondrial damage. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson’s disease.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Parkin, a E3 ubiquitin ligase, is involved in the clearance of damaged mitochondrial via mitophagy. Upon mitochondrial damage, the activated Parkin ubiquitinates many mitochondrial substrates, leading to the recruitment of mitophagy effectors. However, the mechanism of substrate recognition by Parkin is still not known.

    In this manuscript, Koszela et al. utilized diverse biochemical assays and biophysical approaches, combined with AlphaFold prediction, to identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin that recognizes mitochondrial GTPase Miro1 via a stretch of hydrophobic residues and is critical for its ubiquitination activity on Miro1. This manuscript reveals the mechanisms by which Parkin recognizes and ubiquitinates substrate Miro1, providing a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation by mitochondrial damage. This study also provides insights into mitochondrial homeostasis and may facilitate new therapeutic approaches for Parkinson's disease.

    Major Comments:

    • The authors should expand the background introduction to include the biological function of Miro1, the domain architecture of Miro1 and more context of Miro1 K572 ubiquitination in mitophagy.
    • Figure 1B is confusing. Due to the presence of various bands, it is hard to assign specific bands in each lane. In addition, there are various unlabeled bands that makes things unclear. The authors should include loading controls to clearly discern pParkin, Ube1, Ube2L3, and all substrates.
    • In Figure 1B, it was not possible to identify the ubiquitination bands of E2 enzyme UBE2L3 and the E1 enzyme UBE1. Please indicate these bands on the gel.
    • Since ubiquitinated Miro1 and Mfn1 are similar in molecular weight (Fig. 1b), the authors should show a western blot against the Miro1 and Mfn1 tag as done in the supplementary information, At least for the competition assays involving both Miro1 and Mfn1.
    • The conclusion that Miro1 is pParkin's preferred substrate is not convincing. In the competition assay used to show substrate preference, Miro1 is at a five-fold higher concentration than the other substrates and 25-fold higher than FANCI/D2. This would ultimately drive pParkin's interaction with Miro1. This is further highlighted by the fact that it adding Mfn1 in excess has a similar effect. The competition assay should be done at equimolar concentrations of Miro1 and substrate. More convincing would be a competition assay where substrate ubiquitination is quantified at several different concentrations of Miro1.
    • In Figure 1F, it is unclear what is defined as "high" or "low" ubiquitination levels statistically. Some of the changes in ubiquitination levels are extremely subtle (ex. mitoNEET and FancI/D2 in the presence and absence of Miro1 and Mfn1). In some cases, I find it extremely difficult to tell if there is any change in the ubiquitination levels when comparing lanes containing excess of different substrates. I would like to see band quantifications of this experiment in triplicate to support the conclusions drawn from the competition assay.
    • The authors used both unmodified and phosphorylated Parkin for the crosslinking experiments and observe no difference in the intensity of the bands. However, this is not sufficient to draw any conclusion about the affinity between phosphorylated Parkin and Miro1 (which was done in lines 341-343). The authors should comment on why they did not test pParkin binding with Miro1, especially given the statement:

    "In our assays in the absence of pUb, pParkin must interact with its substrates without the action of pUb, likely through 158 transient, low affinity interactions"

    • The reference to Parkin115-124 as a "Substrate Targeting Region (STR)" is misleading. This would imply that this motif in Parkin is responsible for general substrate recognition when there is no direct evidence of this. In Figure 5F, the authors create a synthetic peptide based off the STR sequence. Although this sequence was effective in inhibiting the ubiquitination of Miro1, it was ineffective against Mfn1. This would indicate that Mfn1 relies on a completely different set of interactions for ubiquitination by Parkin. I suggest that the authors tone down the language in describing this region and rename this region (perhaps "Miro1 Targeting Region (MTR)"?).
    • The authors appear to confuse plDDT and PAE scores in Figure 5B. The PAE describes the expected positional error of each residue in the model. The plot should be colored in terms of Expected Position Error (Ångstrom), not plDDT scores.

    Minor Comments:

    • Figure 1A would benefit from a schematic showing the domain architecture. If the goal is to appreciate the length of the linker, then showing the actual amino acid length would be beneficial.
    • In Supplementary Figure 2D, the authors performed the MST experiment with His6-Smt3-tagged Parkin. The group had previously shown that the presence of the tag artificially interferes with autoubiquitination, potentially by forming intramolecular interactions. The SEC, Native Page, and ITC data of untagged Parkin with Miro1 provide sufficient evidence that the interaction between the two are weak. The authors should consider removing the MST data, since they are not congruent with the other experiments.
    • The ITC data in Supplementary Figure 2C look promising. It would be nice if the authors could try to quantify the Kd of their STR peptides to Miro1
    • Are STR peptides 1 and/or 2 unable to inhibit ubiquitination of other Parkin substrates besides Mfn1? Do these other substrates utilize the STR for recognition? AlphaFold modeling may provide some insight on Parkin recognition of other substrates.
    • The authors shold consider using AlphaFold3 to model the interaction of pParkin with Miro1 compares to unmodified Parkin.
    • Please label the protein names in Figure 4A for a better presentation.
    • Page 2, line 37. "...by a 65-residue flexible region (linker) to a unique to Parkin RING0 domain..." should be "...by a 65-residue flexible region (linker) to a unique Parkin RING0 domain...". The second "to" should be omitted.
    • Page 3, Line 48: "fulfill", not "fulfil"
    • Page 5, line 110. In sentence, "...phosphorylation at Ser65 of Parkin...", it is better to explicitly state that this phosphorylation happens on the Parkin Ubl domain.
    • Page 7, line151. Figure 1F should be Figure 1G.
    • Page 11, line 241. In sentence "...Miro1 residues R263, R265 and D228...", do the authors mean R261 and not R265?

    Significance

    Parkin is an E3 ubiquitin ligase that is activated to ubiquitinate diverse substrates on the mitochondrial membrane in response to mitochondrial damage, thereby recruiting mitophagy effectors. This study reveals the mechanisms by which Parkin recognizes and ubiquitinates Miro1, providing insights into mitochondrial homeostasis and facilitating new therapeutic approaches for Parkinson's disease.

    Readers with a background in protein ubiquitination and mitochondrial homeostasis might be interested in this study. My expertise includes protein ubiquitination and structural biology. However, I do not have sufficient expertise to evaluate the NMR experiments in this manuscript.

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    Referee #2

    Evidence, reproducibility and clarity

    Koszela et al. have submitted this manuscript demonstrating the molecular mechanism of interaction between Parkin and one of its known substrates, Miro1. While the interaction and ubiquitination of Miro1 by Parkin (and it's role in mitochondrial quality control) has been known since 2011, as demonstrated by the Schwarz group and others, the mechanism of action has remained unknown. The ability of Parkin to ubiquitinate multiple proteins upon mitochondrial damage has indeed led many groups to speculate that Parkin is a promiscuous E3 ligase upon activation; this manuscript tries to provide a rationale for the interaction with one of its known substrates through a combination of biochemical and biophysical studies.

    The authors demonstrate that Miro1 is efficiently ubiquitinated in in vitro biochemical assays in comparison to a few mitochondrial and non-mitochondrial proteins in an attempt to show that Miro1 is a preferred substrate for Parkin. Cross-linking coupled with mass spectrometry, SAXS and NMR experiments were used to provide compelling evidence for a direct and specific interaction between Parkin and Miro1. Molecular modelling using Colabfold and biochemical assays with mutants of the proposed interaction site were then used to provide further proof for the specificity of the interaction. This interaction is shown to occur between the conserved a.a.115-122 (referred to in this study as STR; located in the linker connecting the Ubl to RING0) and the EF domain of Miro1. Interestingly, the authors show that peptides corresponding to 115-122 competitively inhibit ubiquitination of Miro1 by Parkin. Overall, this article constitutes an important addition to our understanding of Parkin's mechanism of action. However, some of the key claims remain unsubstantiated, as described below.

    Major issues:

    1. In line 151 the authors claim, 'these data strongly support the hypothesis that Miro1 is the preferred substrate of pParkin...'. Arguably, the biggest issue with this study is the lack of substantial proof that Miro1 is the preferred parkin substrate in a cellular or physiological context. This claim cannot be made based on a biochemical assay with three other proteins. The Harper group has performed in-depth proteomics studies on the kinetics of Parkin-mediated ubiquitination and proposed that VDACs and Mfn2 (among a few others) are most efficiently ubiquitinated upon mitochondrial damage in induced neurons (Ordureau et al, 2018,2020). Interestingly, neither of these papers have been mentioned by the authors in this manuscript. The Trempe group has shown that Mfn2 is efficiently targeted by Parkin through mitochondrial reconstitution assays and proximity ligation assays (Vranas et al, 2022). The authors need to substantiate their claim through cellular or mitochondrial assays to prove that Miro1 is the preferred physiological substrate of Parkin. Cellular experiments also account for cellular abundance and proximity of Parkin to the substrate, which is not possible in biochemical assays of the kind presented here. In the absence of strong experimental proof for this claim, these claims should be tampered down to Miro1 being "the preferred substrate compared to the other proteins in this assay", and the manuscript should focus more on the molecular mechanism of interaction between Miro1 and Parkin.
    2. In addition to the point above, the authors do not describe the rationale for specifically choosing Mfn1 and MitoNEET for their comparison with Miro1 as substrates. Interestingly, Miro1, MitoNEET and Mfn1 are not among the most efficiently ubiquitinated substrates of Parkin (Ordureau et al, 2018). Additionally, the authors have used a construct of Mfn1 that lacks the full HR1 domain for their assays. Previously, it has been shown that the HR1 of mitofusins is targeted by Parkin (McLelland et al. 2018). Can the authors prove that their Mfn1 construct is as efficiently ubiquitinated as full-length Mfn1 by Parkin? If it is not possible to obtain soluble full-length Mfn1 or other membrane proteins for these assays, then I strongly recommend the authors should perform mitochondrial reconstitution assays as others have performed previously (Vranas et al, 2022) and use this opportunity to also report the ubiquitination kinetics of multiple mitochondrial substrates compared to Miro1 to make a more compelling case for substrate preference.
    3. The authors show that both pParkin-Miro1 and Parkin-Miro1 complexes can be captured by chemical cross-linking. It is well-established in the field that pUbl binds to RING0 (Gladkova et al, 2018) (Sauve et al, 2018) while non-phosphorylated Ubl binds RING1 (Trempe et al, 2013). The Komander group has also shown that the ACT (adjacent to the STR) element binds RING2 in the activated Parkin structure (Gladkova et al, 2018). This suggests that STR could occupy different positions in the Parkin and pParkin. The authors have only reported the cross-link/MS data and model of the Parkin-Miro1 complex. Arguably, the pParkin-Miro1 data is just as, if not more, relevant given that pParkin represents the activated form the ligase. The authors need to robustly establish that Miro1 binds to the STR element in both cases by demonstrating the following:

    A. Mass spectrometry data from cross-linked pParkin-Miro1 complex suggesting the same interaction site.

    B. Colabfold modelling with the pParkin structure to show that Miro1 would bind to the same element.

    1. Does Parkin only bind to Miro1, or can it bind to Miro2 as well? Are there differences between the binding site and Ub target sites between the two proteins? The author should also show experimentally if both proteins get ubiquitinated as efficiently by Parkin and if the STR element is involved in recognizing both proteins. Interestingly, the Harper group reports that Miro2 gets more efficiently ubiquitinated than Miro1 (Ordureau et al, 2018).
    2. In Figure 5D, the level of unmodified Miro1 seems to be similar in assays with WT or I122Y Parkin, though the former seems to form longer chains while the latter forms shorter chains. Is there an explanation for this? Perhaps, the authors need to perform this assay at shorter time points to show that there is more unmodified Miro1 remaining when treated with I122Y Parkin (and similarly for the L221R mutant of Miro1)? Also, why is the effect of Miro1 L221R and Parkin I122Y not additive?

    Minor comments:

    1. The authors should report the full cross-linking/MS data report from Merox including the full peptide table and decoy analysis report.
    2. The authors should report statistics for the fit of the Colabfold model to the experimental SAXS curve.
    3. Why is the Parkin-Miro1 interaction only captured by NMR and not by ITC? The authors should at least attempt to show the interaction of the STR peptide with Miro1 by an orthogonal technique like ITC.
    4. The authors should report the NMR line broadening data quantitatively i.e. reporting the reduction in signal intensity for the peaks upon peptide Miro1 binding to quantitatively demonstrate that the 115-122 peak intensity reduction is more significant than other regions.
    5. Figure 4 (structure figure) and B (PAE plot) should be annotated with the names of domains and elements in Parkin and Miro1 to make these figures clearer and more informative.

    Referees cross-commenting

    I am in agreement with reviewers 1 and 2. Both of them raise valid and interesting points in their reviews.

    Specifically, I would like to highlight the following:

    1. Reviewer 1 makes a very good point (5/6) highlighting that L119A does not impair Parkin recruitment in the previously reported study. I second this concern and believe that the authors need to re-frame their discussion and make it much more nuanced with regards to the role of Miro1-Parkin interaction in mitophagy (if any at all). Additionally, the authors should also note that previous studies in the field from the Youle group (Narendra et al, 2008) and multiple other groups have shown a complete absence of Parkin recruitment to healthy mitochondria. Parkin recruitment to healthy mitochondria hence remains a controversial idea at best, with no evidence for it outside of Parkin overexpression systems (Safiulina et al, 2018) which can also lead to artifacts. The discussion should take all major studies/observations into account to propose a more nuanced picture of the role of Parkin-Miro1 interaction. Perhaps, this interaction plays more of a role in mitochondrial quarantine (Wang et al. 2011) as suggested by the Schwarz group than in Parkin recruitment?
    2. Reviewer 3 raises a valid concern about the lack of quantification in ubiquitination assays and alludes to the difficulty in visualizing ubiquitination of multiple proteins. That was a concern I also had but did not include in my review. Perhaps, the authors should also show western blots for each of the protein (in a time course experiment) demonstrating the difference in ubiquitination kinetics of each of proteins instead of busy SDS-PAGE gels for the assay.

    Significance

    The key strength of this study is the strong biophysical evidence of a direct interaction between Parkin and Miro1 and the discovery of the Miro1 binding site on Parkin. The biophysical and biochemical experiments in this study have been well-designed and executed. The evidence for a specific interaction between Parkin and Miro1 has been provided through multiple approaches. The authors should be commended for this effort. The biggest limitation of this study is the lack of proof that Miro1 is the preferred Parkin substrate in a cellular/physiological context since in biochemical assays Parkin can ubiquitinate multiple proteins non-specifically. Substrate preference claims need to be established in more physiologically relevant experimental settings.

    Overall, the study represents a mechanistic advance in terms of our understanding of the interaction between Parkin and one of its substrates i.e. Miro1, showing that Parkin can indeed specifically bind its substrates before targeting them for ubiquitination. This might also inspire others to investigate the molecular mechanism of action of Parkin with other substrates. This paper would likely appeal specialized audiences i.e. biochemists and structural biologists studying Parkin in mitochondrial quality control.

    Reviewer expertise: Expert biochemist and biophysicist with a number highly cited works in the field of mitochondrial quality control and Parkin.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Koszela et al. explores the substrate preference of the Parkinson's disease associated ubiquitin E3-ligase, Parkin. They conclude that Miro1 is a preferred substrate of Parkin and go on to further characterize a binding site of Parkin to Miro1 using a range of biochemical approaches. This site is identical to previously reported (see point 2). The experimental work is strong with many high-quality assays supporting their ideas; however, there are several major points that should be considered:

    1. The majority (perhaps all) of their biochemical work on Miro1 uses a truncated form of Miro1 lacking the first GTPase domain. It isn't at all clear why this is the case, as no justification is given. Moreover, functional full-length Miro1 has been purified in several papers (e.g. PMID: 33132189). If ubiquitination kinetics are different between the full-length and truncated form of Miro1, this would call into question the significance of the findings in vivo, where the truncation does not exist.
    2. As the manuscript is currently written, there are areas which do not do justice to previous work. Firstly, the authors state throughout the manuscript that no previous work has identified a binding interface between Parkin and one of its substrates, e.g., in the abstract "no substrate interaction site in Parkin has been reported". This is not true as a recent paper already described the binding interface (DOI: 10.1038/s44318-024-00028-1). "we identify a conserved region in the flexible linker", again this interface is identical to that identified previously. Therefore, this study does not "identify" this interface. Given the timing, it is likely that this discovery has been "scooped" by the previous study, but since the present study goes much further in the biochemical characterization of the interface, it would not diminish the paper's importance to rewrite it, giving proper credit where due. Secondly, the authors spend a large part of their discussion speculating on the significance of non-activated Parkin being able to bind Miro e.g., "Importantly, our results suggest that Parkin can interact with Miro1 independently of its activation state, as Parkin phosphorylation does not detectably increase its interaction with Miro1...". Again, this was already known as Parkin has been shown to be recruited to mitochondria upon Miro1 overexpression in the absence of PINK1 (DOI: 10.15252/embj.201899384 and DOI: 10.15252 /embj. 2018100715). The further biochemical characterisation of the Parkin-Miro1 interaction is important and therefore, in both cases the work contained within the manuscript is still a significant contribution, which should, however, be properly discussed in the light of published work.
    3. The Miro L221R mutation is used to disrupt Miro-Parkin interaction. Yet, this non-conservative mutation in the midst of a folded domain might have other effects, like affecting calcium binding or preventing the folding of the domain. This is not tested. The complementary Parkin-I122Y used for the same purpose decreases but does not abolish Parkin-Miro1 binding. Parkin-L119A is proposed to abolish the Parkin-Miro1 interaction. The inclusion of this mutant might be important to fully ascertain the role of Parkin-Miro1 binding in Miro1 ubiquitination.
    4. The effect of Miro competition on other substrates' ubiquitylation is marginal and its reproducibility is questionable (whether mitoNEET ubiquitylation is affected at all in figure 1G is unclear. This blot is anyway over processed with an unnaturally uniform grey background). If the authors wish to make a point about it, these experiments should be repeated and quantified. Moreover, since the model is that the specific Miro-Parkin interaction is involved, the mutants above should be used in the same competition experiments and shown to be unable to compete.
    5. Related to the previous point, one important factor about the kinetics that the authors do not discuss is how any of it relates to mitophagy in vivo. There very well might be a slight intrinsic preference at a given concentration of substrate and Parkin; however, how this plays out in the cell is not clear, e.g., Miro1 may be many times more, or less, abundant than Mfn1, and so a preference might not have much of an effect. So ubiquitination kinetics would need to be considered in a broader cellular context.
    6. Related to the above point, the authors state "Parkin translocation was diminished upon L119A mutation, supporting the importance of the Parkin Miro1-interacting site in mitophagy.". However, the study (not cited but which this reviewer assumes to be DOI: 10.1038/s44318-024-00028-1 since the L119A mutation has only ever been used here) finds no change in Parkin recruitment upon damage. So, it cannot be used to support "the importance of the Parkin Miro1-interacting site in mitophagy".

    Referees cross-commenting

    The reviews align well together with many overlaping point and similar assessment of the significance. Reviewer 2 brings in interesting points pertaining to literature that we were not aware of, explaining why we didn't make these points.

    One comment on reviewer's 3 last major points

    It does not appear that there is a confusion between pIDDT and PAE scores. The plot is coloured according to PAE (which is a residue x residue 2D matrix, figure 4B), while the protein ribbon is coloured according to pIDDT, which is a 1D per-residue confidence score.

    Significance

    This study provides an in-depth in vitro assessment of a specific binding interface between the E3-ligase Parkin and one of its substrate Miro1. Although this interface has been recently described, this study goes well beyond previous knowledge by showing that the interface is important for complete Miro ubiquitylation by Parkin, therefore showing that interactions involving unstructured linkers participate in substrate recognition by the E3-ligase. The importance of this interaction remains to be assessed in vivo. This study is of interest to basic mitochondrial dynamics, quality control and mitophagy researcher as well as translational Parkinson's Disease researchers.

    The reviewer's expertise is in mitochondrial membrane dynamics.

  5. Version published to 10.1101/2024.06.03.597144v1 on bioRxiv