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A eukaryotic-like ubiquitination system in bacterial antiviral defence - Nature

  • ️Corbett, Kevin D.
  • ️Wed Jul 17 2024

Data availability

Final coordinates and structure factors for all structures have been deposited at the Research Collaboratory for Structural Bioinformatics PDB (https://www.rcsb.org) under the accession codes 8TYX (DUBBilC(E33A)–UblBilA form 1), 8TYY (DUBBilC(E33A)–UblBilA form 2), 8TZ0 (E1BilD–E2BilB–UblBilA form 1) and 8TYZ (E1BilD–E2BilB–UblBilA form 2). Raw diffraction images have been deposited at the SBGrid Data Bank (https://data.sbgrid.org) under the accession codes 1039 (DUBBilC(E33A)–UblBilA form 1), 1040 (DUBBilC(E33A)–UblBilA form 2), 1041 (E1BilD–E2BilB–UblBilA form 1) and 1042 (E1BilD–E2BilB–UblBilA form 2). Sequence data were downloaded from the IMG database of bacterial genomes (https://img.jgi.doe.gov/). Source data are provided with this paper.

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Acknowledgements

We thank the staff at The Protein Facility of the Iowa State University Office of Biotechnology for assistance with Edman degradation, R. Sorek for sharing information before publication, and members of the laboratory of K.D.C. for helpful discussions. This work was financed by the National Institutes of Health (NIH) grant R35 GM144121 (to K.D.C.), the Howard Hughes Medical Institute Emerging Pathogens Initiative (to K.D.C.), the NIH Director’s New Innovator Award DP2 AT012346 (to A.T.W.), a Mallinckrodt Foundation Grant (to A.T.W.), the Boettcher Foundation’s Webb-Waring Biomedical Research Program (to A.T.W.), the Pew Biomedical Scholars Program (to A.T.W.) and NIH grants R01 GM116897 (to H.Z. and R.T.S.), R01 GM151191 (to H.Z. and R.T.S.) and S10 OD023498 (to H.Z.). L.R.C. is supported by the University of California San Diego Molecular Biophysics Training Grant (T32 GM139795); J.C. is supported by a Pfizer-Cell Signaling San Diego graduate fellowship; and H.E.L. is supported as a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are financed by the National Institute of General Medical Sciences from the NIH (P30 GM124165). The Eiger 16M detector on beamline 24-ID-E is financed by an NIH Office of Research Infrastructure Programs High-End Instrumentation grant (S10 OD021527). This research used resources of the Advanced Photon Source, a US Department of Energy Office of Science User Facility operated for the US Department of Energy Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357.

Author information

Author notes

  1. These authors contributed equally: Lydia R. Chambers, Qiaozhen Ye

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

    Lydia R. Chambers

  2. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

    Qiaozhen Ye, Minheng Gong, Huilin Zhou & Kevin D. Corbett

  3. Department of Bioengineering, University of California San Diego, La Jolla, CA, USA

    Jiaxi Cai

  4. Department of Biochemistry, University of Colorado Boulder, Boulder, CO, USA

    Hannah E. Ledvina & Aaron T. Whiteley

  5. School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA

    Raymond T. Suhandynata

  6. Department of Molecular Biology, University of California San Diego, La Jolla, CA, USA

    Kevin D. Corbett

Authors

  1. Lydia R. Chambers

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  2. Qiaozhen Ye

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  3. Jiaxi Cai

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  4. Minheng Gong

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  5. Hannah E. Ledvina

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  6. Huilin Zhou

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  7. Aaron T. Whiteley

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  8. Raymond T. Suhandynata

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  9. Kevin D. Corbett

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Contributions

L.R.C. designed experiments, cloned expression vectors, purified proteins, determined crystal structures of E1BilD–E2BilB–UblBilA, carried out E1BilD–E2BilB–UblBilA coexpression and activity assays, and wrote the paper with K.D.C. Q.Y. designed experiments, cloned expression vectors, purified proteins, determined crystal structures of DUBBilC–UblBilA, carried out DUBBilC activity assays, and wrote the paper with K.D.C. J.C. designed experiments and carried out MS analysis of UblBilA-associated proteins. M.G. cloned expression vectors, purified proteins and assisted with protein crystallization. H.E.L. carried out bioinformatics analysis. H.Z. oversaw MS analysis. A.T.W. designed experiments and oversaw bioinformatics analysis. R.T.S. designed experiments and oversaw MS analysis. K.D.C. designed experiments, oversaw structural and biochemical assays, and wrote the paper with L.R.C. and Q.Y.

Corresponding author

Correspondence to Kevin D. Corbett.

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The authors declare no competing interests.

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Nature thanks Ivan Dikic, Ryan Jackson and Malcolm White for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Comparison of Type I and Type II BilABCD operons.

(a) Schematic of a typical ubiquitination pathway, with ubiquitin-like protein (Ubl) in orange, E1 in yellow, E2 in blue, optional E3 in light gray, and target in gray. An E1 protein mediates adenylation of the Ubl C-terminus followed by generation of an E1~Ubl thioester linkage. This thioester linkage is transferred to E2, then to a lysine residue on a target (optionally with the help of an E3). (b) Schematics of CEHH/6C operons in defense islands identified by the PADLOC server31. See Supplementary Table 1 for additional information. (c) Operon schematics of a Type I BilABCD operon from Collimonas sp. OK41224, and a Type II BilABCD (CEHH) operon from E. aridi TW10 (Supplementary Table 1). Noted under each gene are the conserved PFAM domain annotations for that gene.

Extended Data Fig. 2 Sequence analysis of BilABCD proteins.

(a) Unrooted average distance tree assembled from E1BilD proteins from Type I BilABCD operons24 and Type II BilABCD operons (Supplementary Table 1). Branches representing proteins from Type I and Type II operons are marked. Specific E1BilD proteins from Collimonas sp. OK412 (Type I) and E. aridi TW10 (Type II) are indicated with red stars and labeled. Scale bar: 1 substitution per site. (b) Unrooted average distance tree assembled from E2BilB proteins from Type I BilABCD operons24 and Type II BilABCD systems (Supplementary Table 1). Branches representing proteins from Type I and Type II operons are marked. Specific E2BilB proteins from Collimonas sp. OK412 (Type I) and E. aridi TW10 (Type II) are indicated with red stars and labeled. Scale bar: 1 substitution per site. (c) Unrooted average distance tree assembled from DUBBilC proteins from Type I BilABCD operons24 and Type II BilABCD operons (Supplementary Table 1). Scale bar: 1 substitution per site. Branches representing proteins from Type I and Type II operons are marked. Specific DUBBilC proteins from Collimonas sp. OK412 (Type I) and E. aridi TW10 (Type II) are indicated with red stars and labeled. Scale bar: 1 substitution per site. (d) Unrooted evolutionary tree of all UblBilA proteins in Type II BilABCD operons (Supplementary Table 1). Specific examples are labeled and their domain architectures (inferred from sequence analysis) noted. LLPS NTD: N-terminal domain predicted to undergo liquid-liquid phase separation; CC NTD: N-terminal domain predicted to form a coiled-coil. Inset: Sequence logo from bacterial Type II UblBilA proteins (Supplementary Table 1). Type II UblBilA homologs possess up to nine residues C-terminal to the highly conserved glycine (G97 in E. aridi BilA).

Extended Data Fig. 3 Structural parallels between bacterial and eukaryotic ubiquitination machinery.

(a) Domain architecture of E. aridi E1BilD, E2BilB, and UblBilA. IAD: inactive adenylation domain; AAD: active adenylation domain; CYS: E1 catalytic cysteine-containing domain; Ubl: Ubiquitin-like domain. (b) Crystal structure (Form 2) of the E. aridi E1BilD–E2BilB–UblBilA complex, with domains colored as in (a). (c) Closeup view of the E1BilD adenylation active site with bound UblBilA C-terminus. Conserved active site residues are shown as sticks and labeled. (d) View equivalent to (c) showing 2Fo-Fc composite-omit electron density at 1.5σ. (e) Closeup view of the E1BilD-E2BilB binding interface. The zinc ion (gray sphere) is coordinated by E1BilD residues C340, C343, C491, and C493. (f) Closeup view of the catalytic cysteine residues of E1BilD (C417; brown) and E2BilB (C138; blue) with 2Fo-Fc composite-omit electron density at 2.0σ. (g) Domain architecture of H. sapiens E1NAE1-UBA3, E2UBE2M, and UblNEDD8 (PDB ID 2NVU)37. CC: coiled-coil domain; UFD: ubiquitin-fold domain. (h) Crystal structure of the H. sapiens E1NAE1-UBA3–E2UBE2M–UblNEDD8 complex (PDB ID 2NVU)37, with domains colored as in panel (e). (i) Closeup view of the E1UBA3 adenylation active site with bound UblNEDD8 C-terminus. Conserved active site residues are shown as sticks and labeled, and bound ATP is shown as sticks. (j) Closeup view of the E1BilD-E2BilB binding interface. The UBA3 UFD is shown in gray. The zinc ion (gray sphere) is coordinated by UBA3 residues C199, C202, C343, and C346.

Extended Data Fig. 4 Type II Bil E2BilB protein structure and homodimer formation.

(a) Top: Sequence alignment of E. aridi E2BilB (residues 133–158) and Collimonas sp. OK412 E2BilB (residues 108–135), with the four residues of the originally identified CEHH motif in 6C/CEHH operon proteins shown in blue highlights. Of the four residues, only C138 and H151 are highly conserved across both Type I and Type II E2BilB proteins. Bottom: Structure of E. aridi E2BilB, with the four residues of the CEHH motif shown as sticks and labeled. (b) Crystallographic dimer of E2BilB in the Form 2 structure. (c) Closeup of the non-crystallographic E2BilB dimer in the Form 1 structure, oriented equivalently to panel (b). (d) Closeup view of the E2BilB dimer interface (rotated 90° from panel (b)). Residues involved in the interface from one protomer are colored blue, shown in sticks, and labeled. The same residues from the dimer-related protomer are shown as sticks and colored gray. Mutant 1 and Mutant 2 are two multi-site mutants of E2BilB used in panels (e)-(f). (e) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of purified E. aridi E1BilD (1–507, C417A)–E2BilB (1–181, wild type or mutant)–UblBilA (17–97) complexes. Thin lines indicate protein concentration as measured by differential refractive index (dRI; left axis), and thick lines indicate molecular weight in kDa (right axis). Dotted horizontal lines indicate the expected molecular weight of a 1:1:1 complex (85.35 kDa) and a 2:2:2 complex (170.7 kDa). Data for three constructs is shown: wild-type E2 (black/gray), mutant 1 (F36R/I48A/I59K/F83E; light blue/dark blue), and mutant 2 (F36R/I38A/F46K/I48A/I59K/F83E; yellow/brown). (f) SDS-PAGE analysis of purified complexes analyzed by SEC-MALS. For gel source data, see Supplementary Fig. 1. This experiment was independently performed three times, with consistent results.

Source Data

Extended Data Fig. 5 Biochemical and structural analysis of E. aridi DUBBilC.

(a) N-terminal sequencing (Edman degradation) of DUBBilC-cleaved UblBilA-GFP fusion (C-terminal fragment), showing the evaluated value from each of five cycles of degradation. The inferred N-terminal sequence of the fragment is AGIGS. This analysis was performed once. (b) Closeup view of the DUBBilC(E33A)–UblBilA (Form 1) active site, with proteins colored as in Fig. 3d. Active site residues of DUBBilC and glycine 97 of UblBilA are labeled. (c) View equivalent to panel (b), showing 2Fo-Fc composite omit map density at 1.5 σ. (d) Comparison of E. aridi DUBBilC–UblBilA (left) to two similar structures, Caldiarchaeum subterraneum Rpn11-homolog bound to ubiquitin-homolog (center)48 and Schizosaccharomyces pombe Sst2 bound to a ubiquitin K63-linked ubiquitin (right)49. Overall Cα r.m.s.d. values for DUBBilC versus its homolog are in Table 1.

Source Data

Extended Data Fig. 6 Identification of UblBilA targets.

(a) SDS-PAGE analysis of Ni2+ affinity-purified His6-UblBilA Δ97 and associated proteins in native conditions, after coexpression with E1BilD and E2BilB. Bands representing His6-UblBilA, E1BilD, and E2BilB are marked. E2BilB WT: wild type; Mutant 1: F36R/I48A/I59K/F83E; Mutant 2: F36R/I38A/F46K/I48A/I59K/F83E. Red asterisk indicates a likely UblBilA-E2BilB conjugate that is more abundant when E2BilB is mutated. For gel source data for panels (a)-(d), see Supplementary Fig. 1. (b) SDS-PAGE analysis of Ni2+ affinity-purified His6-UblBilA (full-length (FL) or Δ97) and associated proteins in native conditions, after coexpression with E1BilD (wild type or C417A mutant; indicated as “C-A”), E2BilB (wild type or C138A mutant; indicated as “C-A”), and DUBBilC (wild type or E33A mutant). Bands representing His6-UblBilA, E1BilD, E2BilB, and DUBBilC are marked. (c) Anti-His6 western blot analysis of the experiment shown in panel (b), showing His6-UblBilA-target conjugates. (d) SDS-PAGE gel (visualized by Coomassie Blue staining) of His6-tagged UblBilA Δ97 (wild type or V95K mutant) coexpressed with E1BilD and E2BilB, then purified in denaturing conditions. (e) Extracted Ion Chromatograms (EICs) of transition ions of the properly cleaved, unmodified UblBilA(Δ97, V95K) residues 76–92, using a 10 ppm m/z tolerance. RT: retention time.

Extended Data Table 1 Crystallographic data collection and structure determination

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Supplementary information

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Chambers, L.R., Ye, Q., Cai, J. et al. A eukaryotic-like ubiquitination system in bacterial antiviral defence. Nature 631, 843–849 (2024). https://doi.org/10.1038/s41586-024-07730-4

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  • Received: 24 September 2023

  • Accepted: 18 June 2024

  • Published: 17 July 2024

  • Issue Date: 25 July 2024

  • DOI: https://doi.org/10.1038/s41586-024-07730-4