The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

  1. Alan Rodriguez Carvajal
  2. Carlos Gomez Diaz
  3. Antonia Vogel
  4. Adar Sonn-Segev
  5. Katrin Schodl
  6. Luiza Deszcz
  7. Zsuzsanna Orban-Nemeth
  8. Shinji Sakamoto
  9. Karl Mechtler
  10. Philipp Kukura
  11. Tim Clausen
  12. David Haselbach
  13. Fumiyo Ikeda

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Abstract

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.

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  1. eLife

    ###Reviewer #3:

    In this manuscript, Carvajal and coworkers prepared a recombinant LUBAC complex, composed of the full-length HOIP, HOIL-1L, and SHARPIN subunits, and analyzed its 3D structure by electron microscopy. This is the first report to show that the LUBAC complex has an elongated, asymmetric crescent-like structure, although it is low resolution. Moreover, the authors examined the intra- and inter-domain associations by cross-linking mass spectrometry, and investigated the oxyester-linked heterotypic branched ubiquitin chains produced through the E3 activity of HOIL-1L. These results are novel and intriguing; but unfortunately, this study has not provided detailed clarifications of the LUBAC structure and catalysis.

    Major comments:

    1. How about the EM structure from peaks I and III in Suppl. Fig. 1A? Peak I eluted in a higher molecular weight fraction than that of thyroglobulin (670 kDa). Is it possible to form a LUBAC complex consisting of trimers with 1:1:1 stoichiometry between the HOIP, HOIL-1L, and SHARPIN subunits? Peak III predominately includes HOIL-1L and SHARPIN, but lacks HOIP. Therefore, it seems possible to estimate the subunit organization in the 3D structure. Please clarify whether the 3D structure shown in Fig. 2B represents monomers or dimers with 1:1:1 stoichiometry between the HOIP, HOIL-1L, and SHARPIN subunits.

    2. On pages 7-8: The authors emphasize the interaction of the RBR domains of HOIP and HOIL-1L, based on their XL-MS analysis, and speculate that LUBAC may have a single catalytic center. However, since multiple interactions in-between LUBAC domains are detected (Figs. 3B-E), the authors need to explain why they focused on this particular interaction. It will be interesting to analyze the effect of E2 or E2~Ub.

    3. In Fig. 4B, why could the mixed LUBAC subunits generate a linear chain, but not an oxyester-linked branched Ub4? Does it form a high molecular weight complex in gel filtration? Please indicate the anti-ubiquitin blot in Figs. 4B and 4C to clarify the doublet migration in M1-Ub3.

    4. In Figs. 4E and 5A, it is interesting that Cezanne and vOTU could cleave ester-linked branched Ub4, although the molecular bases of these reactions were not revealed. Are the NH2OH-sensitive His-Ub3 and Ub2 generated by LUBAC, as shown in Fig. 5B, cleavable by Cezanne and vOTU? Please indicate that the Ub2 remaining after the OTULIN-treatment (Fig. 4E) is sensitive to NH2OH or not.

    5. Why did the NH2OH-treatment in Figs. 5F and 6C cause a drastic decrease in the linear ubiquitin level? The previous PNAS paper from Cohen's group showed a partial reduction in the molecular weight of the Ub chain bound to IRAK and Myd88 after NH2OH-treatment. In contrast, the current data seem to indicate that most of the LUBAC-generated ubiquitin chains were composed of an ester-linked Ub chain, but not a linear chain. Please indicate the lower molecular weight region of the immunoblot. It is surprising that GST-NEMO(250-412) almost non-specifically captured a variety of Ub chains. How about employing GST-NEMO-UBAN alone or M1-TUBE to specifically pull-down the linear polyubiquitin-containing chains?

    6. On page 11, 2nd paragraph, although the authors described that "the restriction analyses showed that the ubiquitin chains assembled by LUBAC contained non-linear di- and tri-ubiquitin chains", the di-ubiquitin is barely detectable in Fig. 6B.

    7. On the bottom of page12, the authors mentioned that "LUBAC with HOIL-1L T203A,R210A assembled ubiquitin chains more efficiently than WT-LUBAC, but less efficiently than HOIL-1L C460". However, in Fig. 6E, LUBAC with HOIL-1L T203A,R210A seems to have the most powerful E3 activity. Moreover, it is not clear if the partial impairment of branching activity is due to HOIL-1L T203A,R210A, since the upper band of Ub4 has a good signal. Therefore, the authors should reconsider the scheme shown in Fig. 7. The NH2OH-sensitive upper band of Ub3 did not react with an anti-linear ubiquitin antibody, in contrast to the pan-ubiquitin antibody. These results suggested that the upper band of Ub3 consists of two ester-linked branched ubiquitins on single ubiquitin. Does it bind HOIL-1L NZF? If not, then HOIL-1L NZF apparently does not contribute the ester-linked branched ubiquitination activity of LUBAC.

  2. eLife

    ###Reviewer #2:

    The manuscript by Carvajal et al. describes a study on the LUBAC complex. They build upon the striking and highly significant discovery that the HOIL-1 protein is an active ubiquitin E3 ligase with non-lysine esterification activity. This discovery was initially demonstrated by Kelsall et al. As the original findings by Kelsall et al. were quite unexpected, and in part contrary to a study from the Iwai lab, the findings presented here corroborating the former study are of great importance for the field.

    Testament to the challenges with structure determination of the LUBAC complex, little structural information is known, despite its discovery over 10 years ago, few structural insights have been obtained. Carvajal et al. report an insect-based expression and purification system for preparing recombinant LUBAC and present a low-resolution structure of the LUBAC complex consisting of sharpin, HOIL and HOIP at 1:1:1 stoichiometry. The structure is supported by mass photometry and most informatively, crosslinking mass spectrometry. However, the low resolution of the negative stain EM LUBAC structure does not allow placement of the individual subunits but does reveal an asymmetric elongated dumbbell shape. Complementary XL-MS data suggests the catalytic RBR modules from HOIP and HOIL-1 are in proximity. They build upon the work of Kelsall et al. by demonstrating that HOIL-1 retains its esterification activity when part of the LUBAC complex. This is notable as it allows prior LUBAC-associated function to be implicated with non-lysine ubiquitination. The manuscript implies that a major function of HOIL-1 esterification activity is to introduce ester branch points within linear Ub chains, and this is observed within cells after TNF stimulation. Intriguingly, at the end of the manuscript they propose that HOIP and HOIL-1 might undergo ubiquitin relay, reminiscent of that reported for MYCBP2 by the Virdee lab.

    Overall the manuscript is an important contribution. Some additional experiments should be carried out. Furthermore, the manuscript in its current form affords only a modest advance over the Kelsall et al. study. Additional experiments should also be carried out to address this as stated below.

    1. The grey unannotated regions (Figure 3) in sharpin, HOIL and even HOIP to a degree demonstrate anomalously promiscuous crosslinking. Could the authors comment and perhaps add some discussion to the paper? Does this suggest these unannotated regions are highly dynamic? Might this relate to the difficulty in solving higher resolution structures?

    2. Thr12 and Thr55 were identified as potential ester linkage sites within polyUb species. However, their mutation did not abolish formation of the hydroxylamine sensitive bands. The authors should state the observed ubiquitin sequence coverage in the MS experiment. Which regions were not covered?

    3. To confirm that the residual oligomeric Ub species after OTULIN treatment are exclusively ester-linked, a subsequent hydroxylamine treatment step should be performed.

    4. The authors hypothesise that a key function of the HOIL-1 esterification activity is to form heterotypic chains. Whilst this might be the case, the alternative hypothesis that HOIL-1 primes substrates via an ester linkage, which are then linearly extended by HOIP, is also equally valid. Particularly as multiple substrates have been reported to be modified with linear chains yet HOIP appears to be tailored to modify a Ub substrate exclusively. The authors should discuss this alternative hypothesis and also how and why both systems might be important.

    5. Perhaps in further support of substrates being the most abundant ester linked species, NEMO enriched linear chains from TNF treated cells show a much more pronounced collapse compared to the ester-linked Ub-Ub linkages produced in vitro in the absence of substrate. It would greatly strengthen the paper if they could add a recombinant substrate to the in vitro reaction (e.g. IRAK1/2 or MyD88). I am not sure about the feasibility of this.

    6. Finally, the suggestion that HOIP-HOIL Ub relay might be at play is exciting and implies that E3-mediated Ub relay might be a prevalent process. In principle it should be possible to test this by impairing E2 binding to the RING1 domain in HOIL in the LUBAC complex. A steric mutation (e.g. X to Arg) would be a more elegant approach than mutation of the zinc coordinating cysteine. If relay is at play then the LUBAC should still be able to form ester linkages.

  3. eLife

    ###Reviewer #1:

    Carvajal et al. provide a novel mechanistic insight to the function of HOIL-1L in the formation of heterotypic ubiquitin chains in the context of the full LUBAC complex. This expands on recent work suggesting HOIL-1L has the intrinsic ability to form oxyester-type linkages on its own, and nicely describes the phenomenon in the context of LUBAC both in vitro and in cells. Initial descriptions of the preparation of pure and stoichiometric LUBAC complex are clear and will be of utility to the field. The authors use negative stain EM to structurally characterize the complex, but conformational flexibility prevented the generation of a reliable 3D model for de novo model or docking of known components. The organization of the complex is also described by XL-MS, which enabled the authors to suggest positions the RBR domains of HOIP and HOIL-1L in proximity along with the NZF domain of HOIL-1L into a putative catalytic center. Visualization of a unique triUb or tetraUb conjugate is analyzed with gel-based assays to assess determinants associated with its formation or destruction. The unusual species are formed only in the presence of co-purified LUBAC containing catalytically active HOIL-1L, but without requirement for the previously suggested T12 acceptor residue within Ubiquitin. Further, the heterotypic chains are removed by treatment with hydroxylamine (a nucleophilic acceptor of oxyester-linked Ub) or treatment with Cezanne (a deubiquitinase with K11 linkage specificity) but not OTULIN, a deubiquitinase specific for Met1 linkages. The work is given cellular context by induction of LUBAC activity in response to TNF signaling in lysates of MEFs with wild-type or mutant HOIL-1L. Indeed, more hydroxylamine-sensitive Ubiquitin chains are formed (and immunoprecipitated by the Linear-chain binding NEMO construct) in the wild-type but not HOIL-1L catalytic mutant MEFs upon TNF stimulation.

    This clearly written and well-organized manuscript presents new insights into LUBAC assembly and its formation of heterotypic chains. While it is unfortunate that the seemingly well-behaved, monodisperse, stoichiometric complex could not be further structurally characterized, the biochemical characterization of heterotypic Ub formation is thorough and the study constitutes an impactful advance in our understanding of polyubiquitin formation, non-traditional chain linkages, and the LUBAC.

    My primary criticism is centered on the 3D structure presented - what does it really contribute to the study? The 2D analyses demonstrate the substantial flexibility of the complex, and projections generated from the 3D structure only marginally match the selected projections shown in Figure 2. If EM analyses are meant to support the biochemical reconstitution of the active LUBAC complex, then the 2D class averages are more than sufficient. Based on the 2D data, and the fact that there are many class averages that are not recapitulated by 2D projections (and vice versa) it is highly unlikely that the purified complex is consistent with a single 3D structure. If the authors were able to use negative stain of complexes, where individual subunits contained identifiable tags (e.g. GFP, MBP), to localize subunits and corroborate the XL-MS, perhaps a 3D model would be appropriate, but as it stands, I don't see the utility of the 3D density.

    One other issue has to do with the 2D XL-MS plots. I've always found these plots to be particularly uncompelling representations of 3D structures. In particular, circus plots such as Figure 3B are difficult to interpret. Is it possible to "weight" the quantity or confidence of observed crosslinks, such that the reader's attention would be drawn to the most important and obvious linkages? This could be accomplished by using different line widths, color shade, or the presentation of multiple plots at distinct cutoff values. Further, the pair-wise domain representation similarly gives the impression that a single domain (or even single residue) is caught crosslinking to almost every part of the opposing protein {a straight line in the plot which contains many dots) in several instances. This could similarly benefit from thresholding or a more cautious description. Can it truly be inferred that the red RBRs and green NZF of HOIP and HOIL-1L are forming a catalytic center, when grey linker-regions are over-represented in the plot? It may also be visually more appealing to make non-domain grey regions significantly smaller in thickness than known domains or even just a linking line, in all representations 3A-3E and 6D.

    I do not review anonymously, and I applaud the authors for publicly sharing their submitted manuscript on the bioRxiv preprint server. This practice enables others to benefit from findings presented in this research, as well as providing the authors with feedback from the community prior to completion of formal peer review. A postdoc in my lab, Randy Watson, helped me with this review.

    -Gabe Lander

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    This manuscript by Carvajal et al. provides novel insights into HOIL-1L's activity within the LUBAC complex in synthesizing heterotypic, branched ubiquitin chains through oxyester-bond formation. The authors successfully produced and isolated recombinant LUBAC, containing full length HOIL-1L, HOIP, and SHARPIN, and although the intrinsic flexibility prevented a higher-resolution 3D-structure determination, negative-stain EM combined with crosslinking mass spec revealed important new information about the architecture of this complex. Based on the observed spatial proximity of HOIL-1L's and HOIP's catalytic RBR domains, the authors propose an intriguing ubiquitin relay mechanism between these E3 ligases in LUBAC.

    The reviewers agreed that this work represents an important contribution to the field, as it corroborates and extends previous findings of HOIL-1L's non-lysine esterification activity. However, the advance and impact could be improved by some additional experiments to further strengthen the mechanistic conclusions.

  5. Version published to 10.1101/2020.05.27.117952 on bioRxiv