Cryo-EM structure of the chain-elongating E3 ligase UBR5

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   Dec 20, 2022

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   The authors do not wish to provide a response at this time.

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   Dec 20, 2022

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   Referee #3

   Evidence, reproducibility and clarity

   The manuscript presented the Cryo-EM structure of HECT E3 UBR5. Using Alphafold2 model of UBR5, the authors were able to dock and refine the structure model of full length UBR5. Interestingly, UBR5 exists as a homodimer and could potentially assemble into a larger oligomer based on SEC and Cryo-EM data. The antiparallel arrangement of the homodimer suggests that the C-terminal HECT domain could transfer ubiquitin in trans or in cis configuration. The tetrameric model reveals a ring-like structure with a large central cavity, presumably to accommodate large proteins/complexes. Using AKIRIN2 as a substrate, the authors demonstrated …

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   Referee #3

   Evidence, reproducibility and clarity

   The manuscript presented the Cryo-EM structure of HECT E3 UBR5. Using Alphafold2 model of UBR5, the authors were able to dock and refine the structure model of full length UBR5. Interestingly, UBR5 exists as a homodimer and could potentially assemble into a larger oligomer based on SEC and Cryo-EM data. The antiparallel arrangement of the homodimer suggests that the C-terminal HECT domain could transfer ubiquitin in trans or in cis configuration. The tetrameric model reveals a ring-like structure with a large central cavity, presumably to accommodate large proteins/complexes. Using AKIRIN2 as a substrate, the authors demonstrated that UBR5 did not ubiquitinate AKIRIN2, but prefers ubiquitin modified AKIRIN2 as the substrate for ubiquitin chain elongation. Indeed, they observed that UBR5 preferentially ubiquitinates pre-ubiquitinated non-cognate substrate and free ubiquitin hinting that UBR5 is a chain elongating E3. Lastly they showed that UBR5 HECT contains a plug-loop that blocks C-lobe rotation and suggested that conformational change is necessary for ubiquitin transfer.

   Comments:

   1. Homodimerization and oligomerization are the novel aspect of this study but the manuscript lacks validation of the structure. The authors should provide biochemical/mutagenesis analysis to support the dimerization interface observed in the structure. Also the model showed that SBB2 is involved in the tetramerization interface, could the authors verify this by designing a SBB2 deletion mutant?
   2. Would be useful to show the docking of Alphafold2 model onto the Cryo-EM map prior to further model building and refinement in the supplementary data.
   3. Please show the SDS-PAGE of purified UBR5 used for Cryo-EM study.
   4. Figure referencing is not in order. For example Figure 1J was described before Figure 1I. Figure 2A,B mentioned after Figure 2C. Also some Figures are not properly referenced in the main text, e.g. p10 when describing ubiquitin chain formation of UbAKIRIN2 and UbSecurin. Please check throughout the manuscript.
   5. Figure legends are missing for Figure 1H-1J
   6. It was stated in p10 that there was no binding between UBR5 and UbSecurin in Figure S3C, but Figure S3C showed faint FAM-UbSecurin across the fractions. It would be useful to repeat this with FAM-UbSecurin alone to ensure the faint bands are background signal.
   7. In p10, it was stated that UBR5 and UbAKIRIN2 interaction was enhanced in the presence of ubiquitin. How did the authors come to this observation? The sucrose gradients (Figure 3B) showed that UBR5 co-elutes with both AK2 and UbAK2. This reviewer is unclear whether the intensity of the bands can be used to evaluate the strength of the binding affinity. Was the experiment performed at the same protein component concentration/condition? The UBR5 appears to elute at different fractions from the two experiments.
   8. The authors suggested that the UBA domain might bind ubiquitin and promote ubiquitination of UbAKIRIN2. It is noteworthy that prior studies on several HECT E3s showed that HECT domain alone can catalyze free ubiquitin chain assembly. Could it be possible that the HECT domain of UBR5 alone could catalyze the extension of UbAKIRIN2, UbSecurin or UbdeltaGG?
   9. In Figures 3A and S3B, does the ubiquitin chain elongation occur only on the fused ubiquitin?
   10. Figure 4E mentioned in the main text but is missing.
   11. It is not clear from Figure 4 whether the plug-loop is blocking the rotation of C-lobe. The overlaid Figure 4 is quite busy, would be useful to show the UBR5 HECT domain alone with other HECT domain presented in the same orientation but in separate panels. With the plug-loop in the current configuration, does it block E2 binding and transthiolation reaction? Figure 5B seemingly suggested that plug-loop is blocking transthiolation but it is hard to visualize. The authors could enlarge Figure 5B and color the plug-loop differently.

   Significance

   The manuscript provides the structural insight into the organization of UBR5. While the Cryo-EM data largely agrees with the Alphafold2 UBR5 model, this should not take away the significant effort in obtaining the large UBR5 protein structure. The structure reveals an unexpected homodimerization of UBR5 and in the assembly of larger oligomer. The biochemical analyses suggest that UBR5 is proficient in ubiquitin chain elongation. Overall the study provides a structural framework for understanding how UBR5 could function as an ubiquitin ligase. The findings will be of interest to scientist in the ubiquitin field and in understanding UBR5 biology.

   Limitation: aside from the structure, the study lacks detailed mechanisms of how UBR5 catalyzes ubiquitin transfer. While few models for ubiquitin transfer were proposed, the study lacks suitable substrates to investigate the mechanism. The study showed that UBR5 can elongate ubiquitin chain, but it is not clear whether UBR5 could transfer ubiquitin to substrate.

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3. 

   Review Commons

   Dec 20, 2022

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   Referee #2

   Evidence, reproducibility and clarity

   This manuscript reported the CryoEM ring-like structure of the full-length human E3 assembly ligase UBR5, showing its assembly into a tetramer. The authors identified critical determinants for antiparallel homodimer and tetrameric assembly. They further described AKIRIN2 as UBR5 substrate and provided evidences of a preferential interaction and activity of UBR5 towards monoubiquitinated proteins. Based on these findings, they proposed UBR5 as chain-elongating E3 ligase.

   CryoEM data are solid, and the model interpretation of the tetrameric structure provides a precise description of the domain composition of the protein that well fit …

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   Referee #2

   Evidence, reproducibility and clarity

   This manuscript reported the CryoEM ring-like structure of the full-length human E3 assembly ligase UBR5, showing its assembly into a tetramer. The authors identified critical determinants for antiparallel homodimer and tetrameric assembly. They further described AKIRIN2 as UBR5 substrate and provided evidences of a preferential interaction and activity of UBR5 towards monoubiquitinated proteins. Based on these findings, they proposed UBR5 as chain-elongating E3 ligase.

   CryoEM data are solid, and the model interpretation of the tetrameric structure provides a precise description of the domain composition of the protein that well fit with biochemical data. Additional experiments are suggested to corroborate few statements of the authors.
   We believe they are realistic in terms of time and resource.

   1. Authors should address the importance of tetramerization by mutating SBB2 at the tetramerization interface and comparing the mutant with wild type in mass photometry and ubiquitination assays. In silico analysis of the interaction interfaces (e.g by using PISA software) could be useful to select amino acids to be mutated. The authors suggested a role for oligomerization in catalysis and mutants are needed in order to define the real "functional unit" of the enzyme.
   2. The authors used sucrose gradient sedimentation assay to prove UBR5 and substrate interaction (Fig. 3). Control experiment that showed UBR5 protein sedimentation in presence of GFP only is instead in Supplementary Fig. 3D. Unfortunately, in that panel the signal of UBR5 is not visible. Main figure should be revised showing proper controls of the experiment.
   3. The authors need to better clarify the features of the AKIRIN-UBR5 interaction. According to the data, the enzyme is equally active on both AKIRIN-Ub and Securin-Ub, suggesting a Ub-specific engagement. What would be a correct explanation of these results? Is the UBA domain directly involved in this process? Testing the activity of a UBA-impaired mutant should help to solve this issue.
   4. The authors identified a 25 aa sequence, called Plug loop, preceding the HECT domain. In the structure it is inserted between N and C-lobe subdomains of the HECT and appears to lock the enzyme in an open L-conformation. These structural findings are interesting, but no supported by experimental data. Which is the effect of the Plug loop deletion in a ubiquitination assay? Without further validation the last chapter of the results remains purely speculative and may better fit in the discussion.
   5. The datasets are clearly affected by preferential orientation as showed by the angular distribution and 2D classes (reason why the authors correctly performed data collection with tilt). A comment on this is required in the experimental section. In addition, it is not clear whether the presented maps (Fig 1 and 2) derive from merging of the two datasets or only the model has been built using the two different datasets.
   6. As a general comment, authors should enlarge panels in which structural details are described, highlighting the side chain residues involved in binding interfaces. Fig. 5 and Fig. 6 are particularly small and incomplete. Most of the structural figures miss key labels needed for a proper understanding. E.g. among the others, numbering of the helix composing the armadillo domain.

   • The overall organization of the figures is quite confusing. Pag. 7 Figure 2C should represent a "box stabilized by three zinc ions mediated by two histidine and seven cysteine residues" according to text citation, but none of these details is highlighted in the corresponding figure. The eye in Figure 1,2,4 does not mean much if a proper box is not linked to the actual site to be seen. In addition, arrows indicating the rotation axis is hard to interpret. Few panels miss the legend. Figure 1A and many other panels miss the reference in the text. More details below.

   Additional points:

   • Mass Photometry data need additional comments and labels. Please comment on the MP concentration used to analyze the samples. Being a dynamic system, you are probably seeing an equilibrium of species at 10 nM in MP. For better completeness of MP figures, labels that includes counts, % of species and sigma should be added to the nice representation of oligomers. Which condition/fraction represent the MP data showed in 1B?
   • If Alphafold models are mentioned and used for model building, it would be nice to provide at least a pLDDTscore and ptm score. Since some details of the AF model are described in the text, an additional superposition of the AF model with the final model derived by EM would be useful to the community.
   • A simple workflow describing the cryoEM data processing that includes how many particles have been used in each step is required, at least in the methods section. The authors need to show the cryoEM 2D classes of the dimer as well.
   • Please add the domain boundaries in Figure 1A and highlight the domains on the alignment included in Supplemental Table 1.
   • Pag. 8 please decide which abbreviation to use, either UBR or Ubr.
   • Page 8, line 192. I found annoying to find the same sentence used by competitors who posted a bioRxiv paper 3 days before the one we are reviewing (doi.org/10.1101/2022.10.31.514604 page 4, line 135).
   • In supp. 1C legend, "high concentration of NaCl" is a bit vague
   • Complementary to Supp Fig 2A, a zoom in of the density map with traced model would be beneficial to show the actual map quality obtained.
   • Pag. 6 lines 133-134, the helix residues involved in homodimerization are cited in the text, but not highlighted in the Figure 1.
   • Figure 1 legend, panels H-I-J description are missing.
   • Figure 3, panel B, meaning of the asterisk is not reported in the figure legend.
   • Figure 4, 5 panels from A to E are cited in the text while figure reported only 4.

   Referees cross-commenting

   I think all the reviews are fairly consistent and agree with the comments raised by my colleagues with the one exception of Point 3 of Reviewer 1. The issue is certainly important yet the experiment suggested is not clear. I personally have troubles designing an informative experimental set-up.

   Significance

   This paper presents the intriguing Cryo-EM structure of the full-length HECT E3 ligase UBR5. As it stands, this work provides evidence of the existence of a tetrameric RING-like conformation that could represent the functional unit of the catalysis. Very little validation of the features identified in the Cryo-EM structure is given, thus the paper remains quite descriptive, but in any case interesting and informative for the ubiquitin field.

   Considering that UBR5 is a quite competitive subject in these days (e.g. at least one additional Cryo-EM structure was posted in BioRxiv, doi.org/10.1101/2022.10.31.514604), I would positively consider this manuscript for publication if the authors reply in full to the issues raised.

   My field of expertise: Ubiquitin regulation and interactions, biochemistry, biophysics and Cryo-EM.

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4. 

   Review Commons

   Dec 20, 2022

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   Referee #1

   Evidence, reproducibility and clarity

   This manuscript describes cryo EM structural analyses of human E3 ligase Ubr5 in dimeric and tetrameric states. Ubr5 belongs to structurally poorly characterized family of Hect E3 ligases and has important biological functions, e.g. in targeting transcription factors for proteasomal degradation. The manuscript therefore addresses an important subject of basic science and biomedical interest. Using in vitro ubiquitylation assays the authors show that purified Ubr5 forms ubiquitin chains on a substrate (akirin2), but also on a non-substrate (securin), provided the proteins are covalently fused to ubiquitin. The authors interpret this …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   This manuscript describes cryo EM structural analyses of human E3 ligase Ubr5 in dimeric and tetrameric states. Ubr5 belongs to structurally poorly characterized family of Hect E3 ligases and has important biological functions, e.g. in targeting transcription factors for proteasomal degradation. The manuscript therefore addresses an important subject of basic science and biomedical interest. Using in vitro ubiquitylation assays the authors show that purified Ubr5 forms ubiquitin chains on a substrate (akirin2), but also on a non-substrate (securin), provided the proteins are covalently fused to ubiquitin. The authors interpret this as a preference of Ubr5 for ubiquitin chain elongation over initiation. Consistently they show that Ubr5 forms free ubiquitin chains linked by Lys48 in vitro. Whilst the structures of full-length Ubr5 are very interesting and important, this manuscript appears to be at a premature stage. The structural interpretation and models lack experimental validation and remain speculative. The presented activity assays are interesting but do not quite link up with the structural part which leaves the manuscript somewhat disconnected. In my view this manuscript requires considerably more work to correlate structure with function, as suggested below, but holds the potential of turning into a highly insightful story.

   Conceptual comments:

   1. Key observation is that a Ubr5 dimer assembles into higher-order oligomers. The authors speculate that this is functionally relevant, e.g. by the possibility of substrate ubiquitylation occurring within the central cavity of the ring shaped tetramer or ubiquitylation in cis and trans. However, neither significance of Ubr5 oligomerisation nor dynamics/determinants in solution is investigated.
      • In line 100, the authors state that individual oligomeric species could not be separated but do not show data. Why can the species not be separated? Do they exchange? Can exchange be controlled by ionic strength/pH/temperature...?
        The authors also suggest that the tetramer is transient (e.g. line 165). What is the evidence for this? Subunit exchange may be tested by mixing different species, e.g, containing labels/tags etc.
      • The authors should design structure-based mutations, particularly within the small, tetrameric interface, and measure oligomerisation state of the mutants to correlate their cryo EM analyses with oligomeric states observed in solution.
      • The authors should also subject individual oligomers (if required, by mutational stabilization of particular states) in activity assays to test their hypotheses.
   2. Lines 160-163: "We performed extended 3D classification of the tetramer, which allowed us to confidently dock two models of UBR5 dimers. This revealed that the tetrameric assembly of UBR5 is formed by SBB2 domains of two opposite dimers (Figure 1I)." The idea that the SBB2 domains make up tetrameric interface should be experimentally validated.
   3. Lines 311 onward: The authors speculate that oligomeric arrangements of Ubr5 allow for substrate modification in trans and cis, expanding the substrate repertoire. As part of results section, this should be experimentally addressed. Depending on the exchange behaviour of subunits within oligomers, it may be possible to use mixing experiments. Alternatively, they authors may consider comparing Ubr5 ubiquitylation efficiency towards substrates of different sizes, which may allow for better interpretation of the distance restraints they defined.
   4. Figure 1J suggests that MLLE domain was modelled, yet the authors note in the text (line 190) that this domain is disordered.
   5. The interpretation of the in vitro experiments comparing ubiquitylation of ubiquitin fused akirin2 (substrate) and ubiquitin fused securin (non-substrate) require a re-evaluation: The fact that even ubiquitin fused securin is efficiently ubiquitylated by Ubr5 in vitro shows that a fused ubiquitin molecule is sufficient to recruit a protein for modification by Ubr5 (likely via the UBA domain) in vitro. The specificity of Ubr5 for certain substrates in the cell must therefore follow different (unknown) mechanisms. It is possible that observed, additional affinity between Ubr5 and akirin2 (which is independent of ubiquitin) contributes to this. The available data, however, are insufficient to suggest a hierarchy of interactions (suggested in line 235-237). The interpretation should also be adapted in Figure 6 in which an order of binding events is postulated.
   6. Fluorescently labelled deltaGG-ubiquitin is used to monitor free chain formation by Ubr5. Why is this setup used rather than a simple assay with full-length ubiquitin? The rationale/benefit should be clarified.
   7. Conformation/functional significance of the plug loop should be validated by mutagenesis.
   8. The mass spec data should be presented in a compact supplementary figure or table, in addition to comprehensive data table in Suppl. Table 2.
   9. Statements without data backup should be phrased as hypotheses or experimentally validated, e.g.,
      • line 27:"Using cryo-EM processing tools, we observe the dynamic nature of the domain movements of UBR5, which allows the catalytic HECT domain to reach engaged substrates."
      • line 31:"This preference for ubiquitinated substrates permits UBR5 to function in several different signalling pathways and cancers".
      • line 78: "This striking feature allows the positioning of substrate binding sites in close proximity to the catalytic HECT domain in cis or trans, expanding its substrate-recruiting capacities."

   Methods section:

   • The authors should provide information on how Ubr5 sequence was optimized (line 477).
   • The authors should explain why different versions of Ubr5 were used for cryo-EM and activity assays.
   • Given the importance of oligomerisation, the authors should explain which fraction of the gel filtration was used for activity assays. Depending on the reversibility of oligomerisation and if oligomerisation impacts activity, the authors should also specify what concentrations these fractions had and/or which concentration they were concentrated to.
   • The authors should specify how and at which position cysteine was introduced for labelling of the deltaGG-ubiquitin version.
   • The authors should specify which concentrations mass photometry measurements were performed at.
   • A description of mass spectrometry measurements is missing.

   Results/figures etc:

   • Lines 88-91 require more precision: "...highly conserved" - compared to what? Some quantification would help here. "...the length is an average across all species". What is meant by "all species" (all species shown, all metazoans?)?
   • Figure 1B shows appears to also show a monomer peak. The authors should label it and comment on it. Can the authors comment on the right shoulder of tetramer peak which was not fitted?
   • Figure Legends 1H, 1I, and 1J are missing
   • In Figures 1G, 2A and 2B, colour labelling positive/negative is swapped.
   • Line 215: "... have observed the formation of free chains". Here, a figure/figure reference is needed.
   • Figure 5 C, D, Figure 6: The way models are drawn is very difficult to understand. Maybe the authors could find clearer way to illustrate their hypotheses?

   Referees cross-commenting

   Reviewer 2 noticed that the manuscript contains a sentence that, in paraphrased form, may have been adopted from a competing manuscript published on Biorxiv some days earlier. According to the conventions of good scientific practice, the competing manuscript should be cited here.

   Significance

   see above

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5. 

   Version published to 10.1101/2022.11.03.515015v1 on bioRxiv

   Nov 4, 2022