Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans

  1. Dipti Lenka
  2. Shakti Dahe
  3. Odetta Antico
  4. Pritiranjan Sahoo
  5. Alan R. Prescott
  6. Miratul M. K. Muqit
  7. Atul Kumar

  • Curated by eLife

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    eLife assessment

    This is a valuable manuscript describing the competitive binding between the RING2 and phosphorylated Ubl domains within Parkin involved in the regulation of Parkin activity. The evidence supporting this conclusion is incomplete, as it primarily relies on a single biochemical assay and does not utilize more stringent, quantitative biophysical approaches to probe this competitive binding. This work will be of interest to the research communities focused on the molecular basis of ubiquitin ligase regulation, PINK-PARKIN-regulated mitophagy, and mitochondrial quality control.

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Abstract

Loss of function Parkin mutations lead to early-onset of Parkinson’s disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by PINK1. Herein, we show a competitive binding mode of the phospho-Ubl and RING2 domains on the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can directly complex with unmodified Parkin, leading to the activation of autoinhibited Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme’s kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, crystal structure reveals a new donor ubiquitin binding pocket that plays a crucial role in function. This study provides new molecular insights into Parkin activation, which may aid the development of Parkin activators as a therapeutic strategy against Parkinson’s.

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  1. eLife

    eLife assessment

    This is a valuable manuscript describing the competitive binding between the RING2 and phosphorylated Ubl domains within Parkin involved in the regulation of Parkin activity. The evidence supporting this conclusion is incomplete, as it primarily relies on a single biochemical assay and does not utilize more stringent, quantitative biophysical approaches to probe this competitive binding. This work will be of interest to the research communities focused on the molecular basis of ubiquitin ligase regulation, PINK-PARKIN-regulated mitophagy, and mitochondrial quality control.

  2. eLife

    Reviewer #1 (Public Review):

    Summary:

    The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated. Still, the impact of their results builds on recent structural studies and the stated impact is based on these prior works.

    Strengths:

    (1) After reading through the paper, the major findings are:
    - RING2 and pUbl compete for binding to RING0.
    - Parkin can dimerize.
    - ACT plays an important role in enzyme kinetics.

    (2) The use of molecular scissors in their construct represents a creative approach to examining inter-domain interactions.

    (3) From my assessment, the experiments are well-conceived and executed.

    Weaknesses:

    (1) The manuscript, as written, is NOT for a general audience. Admittedly, I am not an expert on Parkin structure and function, but I had to do a lot of homework to try to understand the underlying rationale and impact. This reflects, I think, that the work generally represents an incremental advance on recent structural findings.

    (2) To this point, it is hard to understand the impact of this work without more information highlighting the novelty. There are several structures of Parkin in various auto-inhibited states, and it was hard to delineate how this is different.

    (3) As noted, I appreciated the use of protease sites in the fusion protein construct. It is unclear how the loop region might affect the protein structure and function. The authors worked to demonstrate that this did not introduce artifacts, but the biological context is missing.

    (4) While it is likely that the binding is competitive between the Ubl and RING2 domains, the data is not quantitative. Is it known whether the folding of the distinct domains is independent? Or are there interactions that alter folding? It seems plausible that conformational rearrangements may invoke an orientation of domains that would be incompatible. The biological context for the importance of this interaction was not clear to me.

    (5) What is the rationale for mutating Lys211 to Asn? Were other mutations tried? Glu? Ala? Just missing the rationale. I think this may have been identified previously in the field, but not clear what this mutation represents biologically.

    (6) I was confused about how the phospho-proteins were generated. After looking through the methods, there appear to be phosphorylation experiments, but it is unclear what the efficiency was for each protein (i.e. what % gets modified). In the text, the authors refer to phospho-Parkin (T270R, C431A), but not clear how these mutations might influence this process. I gather that these are catalytically inactive, but it is unclear to me how this is catalyzing the ubiquitination in the assay.

    (7) The authors note that "ACT can be complemented in trans; however, it is more efficient in cis", but it is unclear whether both would be important or if the favored interaction is dominant in a biological context.

    (8) The authors repeatedly note that this study could aid in the development of small-molecule regulators against Parkin to treat PD, but this is a long way off. And it is not clear from their manuscript how this would be achieved. As stated, this is conjecture.

  3. eLife

    Reviewer #2 (Public Review):

    This manuscript uses biochemistry and X-ray crystallography to further probe the molecular mechanism of Parkin regulation and activation. Using a construct that incorporates cleavage sites between different Parkin domains to increase the local concentration of specific domains (i.e., molecular scissors), the authors suggest that competitive binding between the p-Ubl and RING2 domains for the RING0 domain regulates Parkin activity. Further, they demonstrate that this competition can occur in trans, with a p-Ubl domain of one Parkin molecule binding the RING0 domain of a second monomer, thus activating the catalytic RING1 domain. In addition, they suggest that the ACT domain can similarly bind and activate Parkin in trans, albeit at a lower efficiency than that observed for p-Ubl. The authors also suggest from crystal structure analysis and some biochemical experiments that the linker region between RING2 and repressor elements interacts with the donor ubiquitin to enhance Parkin activity.

    Ultimately this manuscript challenges previous work suggesting that the p-Ubl domain does not bind to the Parkin core in the mechanism of Parkin activation. The use of the 'molecular scissors' approach to probe these effects is an interesting approach to probe this type of competitive binding. However, there are issues with the experimental approach manuscript that detract from the overall quality and potential impact of the work.

    The competitive binding between p-Ubl and RING2 domains for the Parkin core could have been better defined using biophysical and biochemical approaches that explicitly define the relative affinities that dictate these interactions. A better understanding of these affinities could provide more insight into the relative bindings of these domains, especially as it relates to the in trans interactions.

    I also have concerns about the results of using molecular scissors to 'increase local concentrations' and allow for binding to be observed. These experiments are done primarily using proteolytic cleavage of different domains followed by size exclusion chromatography. ITC experiments suggest that the binding constants for these interactions are in the µM range, although these experiments are problematic as the authors indicate in the text that protein precipitation was observed during these experiments. This type of binding could easily be measured in other assays. My issue relates to the ability of a protein complex (comprising the core and cleaved domains) with a Kd of 1 µM to be maintained in an SEC experiment. The off-rates for these complexes must be exceeding slow, which doesn't really correspond to the low µM binding constants discussed in the text. How do the authors explain this? What is driving the Koff to levels sufficiently slow to prevent dissociation by SEC? Considering that the authors are challenging previous work describing the lack of binding between the p-Ubl domain and the core, these issues should be better resolved in this current manuscript. Further, it's important to have a more detailed understanding of relative affinities when considering the functional implications of this competition in the context of full-length Parkin. Similar comments could be made about the ACT experiments described in the text.

    Ultimately, this work does suggest additional insights into the mechanism of Parkin activation that could contribute to the field. There is a lot of information included in this manuscript, giving it breadth, albeit at the cost of depth for the study of specific interactions. Further, I felt that the authors oversold some of their data in the text, and I'd recommend being a bit more careful when claiming an experiment 'confirms' a specific model. In many cases, there are other models that could explain similar results. For example, in Figure 1C, the authors state that their crystal structure 'confirms' that "RING2 is transiently displaced from the RING0 domain and returns to its original position after washing off the p-Ubl linker". However, it isn't clear to me that RING2 ever dissociated when prepared this way. While there are issues with the work that I feel should be further addressed with additional experiments, there are interesting mechanistic details suggested by this work that could improve our understanding of Parkin activation. However, the full impact of this work won't be fully appreciated until there is a more thorough understanding of the regulation and competitive binding between p-Ubl and RIGN2 to RORB both in cis and in trans.

  4. eLife

    Reviewer #3 (Public Review):

    Summary:

    In their manuscript "Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans", Lenka et al present data that could suggest an "in trans" model of Parkin ubiquitination activity. Parkin is an intensely studied E3 ligase implicated in mitophagy, whereby missense mutations to the PARK2 gene are known to cause autosomal recessive juvenile parkinsonism. From a mechanistic point of view, Parkin is extremely complex. Its activity is tightly controlled by several modes of auto-inhibition that must be released by queues of mitochondrial damage. While the general overview of Parkin activation has been mapped out in recent years, several details have remained murky. In particular, whether Parkin dimerizes as part of its feed-forward signaling mechanism, and whether said dimerization can facilitate ligase activation, has remained unclear. Here, Lenka et al. use various truncation mutants of Parkin in an attempt to understand the likelihood of dimerization (in support of an "in trans" model for catalysis).

    Strengths:

    The results are bolstered by several distinct approaches including analytical SEC with cleavable Parkin constructs, ITC interaction studies, ubiquitination assays, protein crystallography, and cellular localization studies.

    Weaknesses:

    As presented, however, the storyline is very confusing to follow and several lines of experimentation felt like distractions from the primary message. Furthermore, many experiments could only indirectly support the author's conclusions, and therefore the final picture of what new features can be firmly added to the model of Parkin activation and function is unclear.

    Major concerns:

    (1) This manuscript solves numerous crystal structures of various Parkin components to help support their idea of in trans transfer. The way these structures are presented more resemble models and it is unclear from the figures that these are new complexes solved in this work, and what new insights can be gleaned from them.

    (2) There are no experiments that definitively show the in trans activation of Parkin. The binding experiments and size exclusion chromatography are a good start, but the way these experiments are performed, they'd be better suited as support for a stronger experiment showing Parkin dimerization. In addition, the rationale for an in trans activation model is not convincingly explained until the concept of Parkin isoforms is introduced in the Discussion. The authors should consider expanding this concept into other parts of the manuscript.

    2a. For the in trans activation experiment using wt Parkin and pParkin (T270R/C431A) (Figure 3D), there needs to be a large excess of pParkin to stimulate the catalytic activity of wt Parkin. This experiment has low cellular relevance as these point mutations are unlikely to occur together to create this nonfunctional pParkin protein. In the case of pParkin activating wt Parkin (regardless of artificial point mutations inserted to study specifically the in trans activation), if there needs to be much more pParkin around to fully activate wt Parkin, isn't it just more likely that the pParkin would activate in cis?

    2ai. Another underlying issue with this experiment is that the authors do not consider the possibility that the increased activity observed is a result of increased "substrate" for auto-ubiquitination, as opposed to any role in catalytic activation. Have the authors considered looking at Miro as a substrate in order to control for this?

    2b. The authors mention a "higher net concentration" of the "fused domains" with RING0, and use this to justify artificially cleaving the Ubl or RING2 domains from the Parkin core. This fact should be moot. In cells, it is expected there will only be a 1:1 ratio of the Parkin core with the Ubl or RING2 domains. To date, there is no evidence suggesting multiple pUbls or multiple RING2s can bind the RING0 binding site. In fact, the authors here even show that either the RING2 or pUbl needs to be displaced to permit the binding of the other domain. That being said, there would be no "higher net concentration" because there would always be the same molar equivalents of Ubl, RING2, and the Parkin core.

    2c. A larger issue remaining in terms of Parkin activation is the lack of clarity surrounding the role of the linker (77-140); particularly whether its primary role is to tether the Ubl to the cis Parkin molecule versus a role in permitting distal interactions to a trans molecule. The way the authors have conducted the experiments presented in Figure 2 limits the possible interactions that the activated pUbl could have by (a) ablating the binding site in the cis molecule with the K211N mutation; (b) further blocking the binding site in the cis molecule by keeping the RING2 domain intact. These restrictions to the cis parkin molecule effectively force the pUbl to bind in trans. A competition experiment to demonstrate the likelihood of cis or trans activation in direct comparison with each other would provide stronger evidence for trans activation.

    (3) A major limitation of this study is that the authors interpret structural flexibility from experiments that do not report directly on flexibility. The analytical SEC experiments report on binding affinity and more specifically off-rates. By removing the interdomain linkages, the accompanying on-rate would be drastically impacted, and thus the observations are disconnected from a native scenario. Likewise, observations from protein crystallography can be consistent with flexibility, but certainly should not be directly interpreted in this manner. Rigorous determination of linker and/or domain flexibility would require alternative methods that measure this directly.

    (4) The analysis of the ACT element comes across as incomplete. The authors make a point of a competing interaction with Lys48 of the Ubl domain, but the significance of this is unclear. It is possible that this observation could be an overinterpretation of the crystal structures. Additionally, the rationale for why the ACT element should or shouldn't contribute to in trans activation of different Parkin constructs is not clear. Lastly, the conclusion that this work explains the evolutionary nature of this element in chordates is highly overstated.

    (5) The analysis of the REP linker element also seems incomplete. The authors identify contacts to a neighboring pUb molecule in their crystal structure, but the connection between this interface (which could be a crystallization artifact) and their biochemical activity data is not straightforward. The analysis of flexibility within this region using crystallographic and AlphaFold modeling observations is very indirect. The authors also draw parallels with linker regions in other RBR ligases that are involved in recognizing the E2-loaded Ub. Firstly, it is not clear from the text or figures whether the "conserved" hydrophobic within the linker region is involved in these alternative Ub interfaces. And secondly, the authors appear to jump to the conclusion that the Parkin linker region also binds an E2-loaded Ub, even though their original observation from the crystal structure seems inconsistent with this. The entire analysis feels very preliminary and also comes across as tangential to the primary storyline of in trans Parkin activation.

  5. Version published to 10.21203/rs.3.rs-2859540/v4 on Research Square
  6. Version published to 10.21203/rs.3.rs-2859540/v3 on Research Square
  7. Version published to 10.21203/rs.3.rs-2859540/v2 on Research Square
  8. Version published to 10.21203/rs.3.rs-2859540/v1 on Research Square