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Cited by 270Ubiquitin and proteasome pathwaysNF-κB Signaling PathwaysEnzyme Structure and Function

NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly

Open access
Baek, Kheewoong, Krist, David T., Prabu, J. Rajan, Hill, Spencer, Klügel, Maren, Neumaier, Lisa-Marie, von Gronau, Susanne, Kleiger, Gary +1 more·2020·Nature
DOI 10.1038/s41586-020-2000-yPMC PMC7050210
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Abstract

Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation 1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8 2-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1 β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports a 3.7 Å cryo-EM structure of a chemically trapped neddylated CRL1β-TRCP–UBE2D~UB–IκBα substrate intermediate, showing NEDD8 as a nexus that organizes activation, catalytic, and substrate-scaffolding modules. NEDD8-dependent multivalent interactions and conformational changes explain nearly 2000-fold stimulation of substrate priming and why NEDD8, rather than ubiquitin, activates CRLs.

Key findings

  • NEDD8 massively stimulates CRL1β-TRCP/UBE2D-catalyzed ubiquitylation by nearly 2000-fold overall; for IκBα, K_m for UBE2D was 408 ± 57 nM with neddylated CRL1β-TRCP versus 4014 ± 956 nM unneddylated, and k_obs S0–S1 was 12.9 ± 1.7 s⁻¹ versus 0.05 ± 0.002 s⁻¹, a 2667-fold change (Extended Data Table 1). NEDD8 activates both substrate priming and chain elongation, but UB linkage to a substrate-linked UB is 10-fold slower, suggesting optimal catalysis of substrate priming.
  • The 3.7 Å structure of the trapped neddylated CRL1β-TRCP–UBE2D~UB–substrate intermediate shows NEDD8 nearly encircled by interactions, binding its linked CUL1 WHB domain in an “activation module” and positioning a “catalytic module” relative to the substrate-scaffolding module; RBX1 binds UBE2D~UB in the canonical RING-activated closed conformation, and the structurally observed distance between IκBα’s β-TRCP-bound phosphodegron and the UBE2D~UB active site is ≈22 Å.
  • NEDD8 must adopt the Loop-out conformation to both form the activation module and engage UBE2D in the catalytic module, acting as a coincidence detector; mutations disrupting activation-module, activation–catalytic, or activation–substrate-scaffolding interfaces impair substrate priming, and combining mutations is synergistic (e.g., NEDD8 I44A is ≈10-fold defective alone, but with UBE2D H32A is nearly 200-fold defective).
  • The same mutations impair UBE2D-mediated priming with neddylated CRL1FBW7 and neddylated CRL4CRBN/pomalidomide, and the structure rationalizes how NEDD8 Gln40 deamidation by bacterial effectors impairs CRL1-dependent ubiquitylation.

Methods

  • Cryo-EM: a chemically adjoined stable proxy for the UBE2D~UB–IκBα substrate intermediate was bound to a hyperactive neddylated CRL1β-TRCP; the final reconstruction was refined globally to 3.7 Å (focused refinement 3.46 Å), with model building/refinement using known PDB structures, RELION-3.0, COOT, and Phenix.refine.
  • Kinetics: rapid quench-flow pre-steady-state experiments and UBE2D titrations with radiolabeled peptide substrates (IκBα and β-catenin variants) estimated K_m and k_obs; pulse-chase fluorescent assays monitored substrate priming, and separate assays monitored intrinsic UBE2D~UB discharge to free lysine.
  • Samples: human recombinant proteins were expressed in insect cells or E. coli, including UBE2D2/3, RBX1, CUL1, CUL4, NEDD8, UB, SKP1, β-TRCP, CRBN, DDB1, and UBA1; peptides were >95% HPLC purity, and variants were generated by PCR, Quikchange, or synthesis.

Limitations

  • Stated: the authors cannot exclude that UB could be transferred from RING- and NEDD8-bound UBE2D in various orientations relative to the substrate-scaffolding module; different forms of ubiquitylation may involve alternative, presently elusive catalytic architectures; and the structural basis for ubiquitylation by other CRLs awaits future studies.
  • Evident: the structure relies on a chemically trapped proxy, a hyperactive RBX1 N98R mutant, monomeric β-TRCP1, SKP1∆∆, and engineered UBE2D variants, so it may not capture all native transient conformations or wild-type dynamics.
  • Evident: the biochemical assays are in vitro with model peptide substrates, some validation assays are qualitative, and several experiments report n = 2 technical replicates rather than independent biological replicates.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-020-2000-y

Cites

76 of 76 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 78 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    A well-resolved substrate scaffolding module in the cryo EM reconstructions resembles models based on crystal structures of substrate-bound SKP1-β-TRCP and the portion of an F-box-SKP1-CUL1-RBX1 complex including CUL1's N-terminal domain and the intermolecular C/R domain.

    “A well-resolved “substrate scaffolding module” resembles models based on crystal structures of substrate-bound SKP1-β-TRCP and the portion of an F-box-SKP1-CUL1-RBX1 complex including CUL1’s N-terminal domain and the intermolecular C/R domain7,20.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002). source

      Not checked in the free run.

    • cites [20] Wu, G. et al. Structure of a β-TrCP1–Skp1–β-catenin complex: destruction motif binding and lysine specificity of the SCFβ-TrCP1 ubiquitin ligase. Mol. Cell 11, 1445–1456 (2003). source

      Not checked in the free run.

  • Findingown data

    A cryo EM reconstruction at 3.7 Å resolution shows a proxy for a UBE2D~UB-IκBα substrate intermediate bound to a hyperactive version of neddylated CRL1β-TRCP.

    “we obtained a reconstruction at 3.7 Å resolution showing our proxy for a UBE2D~UB-IκBα substrate intermediate bound to a hyperactive version of neddylated CRL1β-TRCP”
    Quote in the paperSubstantial claim
  • Findingown data

    Concomitant local structural remodeling and large-scale CRL domain movements converge to juxtapose the substrate and ubiquitylation active site.

    “Concomitant local structural remodeling and large-scale CRL domain movements converge to juxtapose the substrate and ubiquitylation active site.”
    Quote in the paperSubstantial claim
  • Findingown data

    The chemically adjoined surrogates form a stable mimic of the transition state that avidly binds neddylated CRL1β-TRCP.

    “We chemically adjoined surrogates for these entities in a stable mimic of the transition state, which avidly binds neddylated CRL1β-TRCP”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The chemically adjoined surrogate complex is a stable mimic of the transition state and represents the ubiquitylation intermediate.
  • Findingcitation

    The structurally-observed approximately 22 angstroms between IκBα's β-TRCP-bound phosphodegron and the UBE2D~UB active site is compatible with spacing between this motif and potential acceptor lysines in many substrates.

    “The structurally-observed ≈22 angstroms between IκBα’s β-TRCP-bound phosphodegron and the UBE2D~UB active site is compatible with spacing between this motif and potential acceptor lysines in many substrates32”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] Low, T. Y. et al. A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7, rs8 (2014).

      Not checked in the free run.

  • Classified as probably not a substantial claim. 32
  • May combine two claims. 44
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    In the catalytic module, RBX1 binds UBE2D~UB in the canonical RING-activated closed conformation, where noncovalent UBE2D and UB interactions allosterically activate the thioester bond between them.

    “In the catalytic module, RBX1 binds UBE2D~UB in the canonical RING-activated “closed” conformation, where noncovalent UBE2D and UB interactions allosterically activate the thioester bond between them28–30.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [28] Dou, H., Buetow, L., Sibbet, G. J., Cameron, K. & Huang, D. T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat. Struct. Mol. Biol. 19, 876–883 (2012). source

      Not checked in the free run.

    • cites [29] Plechanovová, A., Jaffray, E. G., Tatham, M. H., Naismith, J. H. & Hay, R. T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012). source

      Not checked in the free run.

    • cites [30] Pruneda, J. N. et al. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Comparing kinetic parameters for peptide substrates with 13-, 9-, and 4-residue spacers confirms the three predictions, with the 4-residue peptide reducing the priming rate with neddylated CRL1β-TRCP to below quantifiable.

    “Comparing kinetic parameters for peptide substrates with 13-, 9-, and 4-residue spacers confirms these predictions, with the latter peptide reducing the priming rate with neddylated CRL1β-TRCP to below that we could quantify”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NEDD8 is nearly encircled by interactions, binding its linked CUL1 WHB domain in an activation module, and positioning a catalytic module relative to the substrate scaffolding module to juxtapose the active site and substrate.

    “NEDD8 is nearly encircled by interactions, binding its linked CUL1 WHB domain in an “activation module”, and positioning a “catalytic module” relative to the substrate scaffolding module to juxtapose the active site and substrate”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neddylated CRL1β-TRCP together with UBE2D optimally catalyzes substrate priming, as UB linkage to a substrate-linked UB is 10-fold slower.

    “However, 10-fold slower UB linkage to a substrate-linked UB suggests neddylated CRL1β-TRCP together with UBE2D optimally catalyzes substrate priming”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NEDD8 activates both substrate priming and chain elongation in CRL1β-TRCP/UBE2D-catalyzed ubiquitylation.

    “Quantifying individual rates for linkage of successive UBs during polyubiquitylation showed NEDD8 activates both reactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the cryo EM map of neddylated CRL1β-TRCP2 bound to a mimic of UBE2D2~UB-IκBα, NEDD8 is bound to the backside of UBE2D, but the donor UB was not visible.

    “its linked NEDD8 (encircled in yellow) is bound to the backside of UBE2D, but the donor UB (absent from region circled in orange) was not visible”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Contacts between NEDD8 and UBE2D backside resemble those described for free NEDD8 or UB binding to UBE2D's backside, and allosterically stimulate intrinsic reactivity of an isolated RING-UBE2D~UB subcomplex.

    “Contacts resemble those described for free NEDD8 or UB binding to UBE2D’s backside, and allosterically stimulating intrinsic reactivity of an isolated RING-UBE2D~UB subcomplex35–38.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [35] Ozkan, E., Yu, H. & Deisenhofer, J. Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases. Proc. Natl Acad. Sci. USA 102, 18890–18895 (2005). source

      Not checked in the free run.

    • cites [36] Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W. & Klevit, R. E. A. A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol. Cell 21, 873–880 (2006). source

      Not checked in the free run.

    • cites [37] Sakata, E. et al. Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity. Nat. Struct. Mol. Biol. 14, 167–168 (2007). source

      Not checked in the free run.

    • cites [38] Buetow, L. et al. Activation of a primed RING E3-E2–ubiquitin complex by non-covalent ubiquitin. Mol. Cell 58, 297–310 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    NEDD8 residues Leu2, Lys4, Glu14, Asp16, Arg25, Arg29, Glu32, Gly63 and Gly64 nestle in a concave CUL1 surface, vary in UB and account for nearly one-third of sequence differences between them.

    “NEDD8’s Leu2, Lys4, Glu14, Asp16, Arg25, Arg29, Glu32, Gly63 and Gly64, which nestle in a concave CUL1 surface, vary in UB and account for nearly one-third of sequence differences between them”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The mutations also impair UBE2D-mediated priming of a Cyclin E phosphopeptide substrate with neddylated CRL1FBW7, and of IKZF zinc finger 2 by neddylated CRL4CRBN/Pomalidomide.

    “the mutations also impair UBE2D-mediated priming of a Cyclin E phosphopeptide substrate with neddylated CRL1FBW7, and of IKZF zinc finger 2 by neddylated CRL4CRBN/Pomalidomide”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    No individual mutation is as detrimental as eliminating neddylation; mutation of each interface has a roughly 10-fold or greater effect on its own.

    “First, although no individual mutation is as detrimental as eliminating neddylation, mutation of each interface has a roughly 10-fold or greater effect on its own.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NEDD8 must adopt the Loop-out conformation to both form the activation module and to engage UBE2D in the catalytic module.

    “Our data show NEDD8 must adopt the Loop-out conformation to both form the activation module and to engage UBE2D in the catalytic module”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutations designed to destroy the activation module (NEDD8 Q40E) or hinder interactions between the activation and catalytic modules (NEDD8 I44A or UBE2D S22R) substantially impair substrate priming, as does swapping key NEDD8 residues at the interface with the substrate scaffolding module with those in UB.

    “Mutations designed to destroy the activation module (NEDD8 Q40E) or hinder interactions between the activation and catalytic modules (NEDD8 I44A or UBE2D S22R) substantially impair substrate priming, as does swapping key NEDD8 residues at the interface with the substrate scaffolding module with those in UB.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mutations designed to destroy the activation module or hinder interactions between the activation and catalytic modules actually disrupt those intended interfaces.

  • May combine two claims.

    Findingown data

    Substrate-independent UB transferase activity was impaired by a NEDD8 mutant disrupting the integrity of the activation module, and by UBE2D mutations hindering interactions with its covalently-linked UB, RBX1's RING domain or NEDD8.

    “Substrate-independent UB transferase activity was impaired by a NEDD8 mutant disrupting the integrity of the activation module, and by UBE2D mutations hindering interactions with its covalently-linked UB, RBX1’s RING domain or NEDD8”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the absence of other factors, neddylated CRL1β-TRCP's scaffolding module robustly bridges the substrate with the C/R domain, while NEDD8, its linked CUL1 WHB and RBX1's RING domain are relatively dynamic.

    “In the absence of other factors, neddylated CRL1β-TRCP’s scaffolding module robustly bridges the substrate with the C/R domain, while NEDD8, it’s linked CUL1 WHB and RBX1’s RING domain are relatively dynamic, apparently waving around”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For WT β-catenin with WT UBE2D, unneddylated CUL1-RBX1 has Km 4638±138 ×10^-9 M and kobs S0-S1 0.05 ± 0.005 sec^-1, giving a 2000-fold change.

    “| WT β-cat | WT UBE2D | CUL1–RBX1 | 4638±138 | 0.05 ± 0.005 | - | 1.1 x 104 | 2000 |”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    The dimer exacerbated structural heterogeneity, with minor differences presumably based on natural motions between the two protomers.

    “First, the dimer exacerbated structural heterogeneity, with minor differences presumably based on natural motions between the two protomers.”
    • Also stated in: § Extended Data · plain
      “Variations between the two protomers of the dimer also exacerbated sample heterogeneity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The final reconstruction of neddylated CRL1β-TRCP1∆D with RBX1 N98R bound to the proxies was refined to 3.7Å resolution.

    “final reconstruction refined to 3.7Å resolution shown on right”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For IκBα with WT UBE2D, neddylated CRL1β-TRCP has Km 408 ± 57 ×10^-9 M and kobs S0-S1 12.9 ± 1.7 sec^-1.

    “| IκBα | WT UBE2D | NEDD8-CUL1–RBX1 | 408 ± 57 | 12.9 ± 1.7 | 0.24 ± 0.04 | 3.2 x 107 | - |”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the cryo EM reconstruction of neddylated CRL1β-TRCP1∆D bound to the newly devised proxy, the phospho-IκBα peptide-substrate-bound β-TRCP-SKP1-CUL1-RBX1-NEDD8-UBE2D portion superimposes with the map for the dimeric complex, and the entire complex is visible including both NEDD8 and donor UB.

    “The phoshpo-IκBα peptide-substrate-bound β-TRCP-SKP1-CUL1-RBX1-NEDD8-UBE2D portion of this map superimposes with the map for the dimeric complex shown in e, but here the entire complex is visible, including both the NEDD8 (encircled in yellow) and donor UB (encircled in orange).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    NEDD8 is the entity displaying the highest local resolution in the map.

    “NEDD8, encircled in yellow, is the entity displaying the highest local resolution in the map.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    The UBE2D H32A mutation at the interface between the catalytic and substrate scaffolding modules affects substrate priming.

    “showing the effect of UBE2D H32A mutation at the interface between the catalytic and substrate scaffolding modules.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The UBE2D H32A mutation is at the interface between the catalytic and substrate scaffolding modules.

  • May combine two claims.

    Findingown data

    For WT β-catenin with WT UBE2D, neddylated CRL1β-TRCP has Km 214 ±30 ×10^-9 M and kobs S0-S1 4.8 ±0.46 sec^-1.

    “| WT β-cat | WT UBE2D | NEDD8-CUL1–RBX1 | 214 ±30 | 4.8 ±0.46 | 0.19 ±0.02 | 2.2 x 107 | - |”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    The dimerization domain agrees well with the prior crystal structure.

    “wherein the dimerization domain agrees well with the prior crystal structure27”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Tang, X. et al. Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination. Cell 129, 1165–1176 (2007). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    For medium β-catenin with WT UBE2D, neddylated CRL1β-TRCP has Km 372 ± 48 ×10^-9 M and kobs S0-S1 11 ±1.00 sec^-1.

    “| medium β-cat | WT UBE2D | NEDD8-CUL1–RBX1 | 372 ± 48 | 11 ±1.00 | 0.24 ± 0.04 | 3.0 x 107 | - |”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Reconstruction of the data yielded a focused refinement at 3.46 Å resolution and a global refinement at 3.7 Å resolution that superimposes well with lower resolution maps obtained during attempts to visualize substrate priming with neddylated wild-type dimeric CRL1β-TRCP.

    “Ultimately, reconstruction of the data yielded a focused refinement at 3.46 Å resolution and a global refinement at 3.7 Å resolution that superimposes well with lower resolution maps obtained during attempts to visualize substrate priming with neddylated wild-type dimeric CRL1β-TRCP.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The short β-catenin peptide has a lysine 5 residues upstream of the N-terminal phosphoSer in the degron.

    ““short” β-catenin peptide with a lysine 5 residues upstream of the N-terminal phosphoSer in the degron”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For medium β-catenin with S22R UBE2D, neddylated CRL1β-TRCP has Km 913±135 ×10^-9 M and kobs S0-S1 1.89 ±0.14 sec^-1, giving a 14.2-fold change.

    “| medium β-cat | S22R UBE2D | NEDD8-CUL1–RBX1 | 913±135 | 1.89 ±0.14 | 0.17 ±0.03 | 2.1 x 106 | 14.2 |”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For medium β-catenin with WT UBE2D, NEDD8 I44A-CUL1-RBX1 has Km 1087 ±69 ×10^-9 M and kobs S0-S1 3.60 ±0.27 sec^-1, giving a 9.1-fold change relative to neddylated WT.

    “| medium β-cat | WT UBE2D | NEDD8 I44A-CUL1–RBX1 | 1087 ±69 | 3.60 ±0.27 | 0.17 ±0.03 | 3.3 x 106 | 9.1 |”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    For medium β-catenin with H32A UBE2D, neddylated CRL1β-TRCP has Km 347 ± 53 ×10^-9 M and kobs S0-S1 8.15 ± 1.04 sec^-1, giving a 1.3-fold change.

    “| medium β-cat | H32A UBE2D | NEDD8-CUL1–RBX1 | 347 ± 53 | 8.15 ± 1.04 | 0.73 ± 0.09 | 2.3 x 107 | 1.3 |”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For medium β-catenin with WT UBE2D, UBylized NEDD8-CUL1-RBX1 has Km 1651±137 ×10^-9 M and kobs S0-S1 2.20 ±0.15 sec^-1, giving a 23.1-fold change.

    “| medium β-cat | WT UBE2D | UBylized NEDD8-CUL1–RBX1 | 1651±137 | 2.20 ±0.15 | 0.17 ±0.04 | 1.3 x 106 | 23.1 |”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For medium β-catenin with WT UBE2D, NEDD8 Q40E-CUL1-RBX1 has Km 1941 ±226 ×10^-9 M and kobs S0-S1 1.77 ± 0.13 sec^-1, giving a 33-fold change.

    “| medium β-cat | WT UBE2D | NEDD8 Q40E-CUL1–RBX1 | 1941 ±226 | 1.77 ± 0.13 | 0.07 ±0.01 | 9.1 x 105 | 33 |”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For medium β-catenin with WT UBE2D, UB-CUL1-RBX1 has Km 2056 ±170 ×10^-9 M and kobs S0-S1 1.0 ±0.03 sec^-1, giving a 61.2-fold change.

    “| medium β-cat | WT UBE2D | UB-CUL1–RBX1 | 2056 ±170 | 1.0 ±0.03 | 0.15 ±0.01 | 4.9 x 105 | 61.2 |”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust for rapid substrate ubiquitylation and fragile to enable ensuing cullin-RING functions.

    “show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust for rapid substrate ubiquitylation and fragile to enable ensuing cullin-RING functions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    NEDD8 Gln40 deamidation by an enteropathogenic and enterohemorrhagic Escherichia coli effector results in accumulation of the CRL1β-TRCP substrate IκBα as well as substrates of other CRLs.

    “NEDD8 Gln40 deamidation by an enteropathogenic and enterohemorrhagic Escherichia coli effector results in accumulation of the CRL1β-TRCP substrate IκBα as well as substrates of other CRLs24–26.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [24] Cui, J. et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010). source

      Not checked in the free run.

    • cites [25] Jubelin, G. et al. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways. PLoS Pathog. 6, e1001128 (2010). source

      Not checked in the free run.

    • cites [26] Morikawa, H. et al. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1. Biochem. Biophys. Res. Commun. 401, 268–274 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hijacking of CRL1β-TRCP enables HIV to evade host immunity.

    “hijacking of CRL1β-TRCP enables HIV to evade host immunity23”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Margottin, F. et al. A novel human WD protein, h-βTrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif. Mol. Cell 1, 565–574 (1998). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mutations impairing CRL1β-TRCP ubiquitylation of β-catenin promote tumorigenesis.

    “mutations impairing CRL1β-TRCP ubiquitylation of β-catenin promote tumorigenesis22.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Frescas, D. & Pagano, M. Deregulated proteolysis by the F-box proteins SKP2 and β-TrCP: tipping the scales of cancer. Nat. Rev. Cancer 8, 438–449 (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    Because ubiquitylation does occur with mutant substrates or enzymes at substantially lower rates, the authors cannot exclude that UB could be transferred from RING- and NEDD8-bound UBE2D in various orientations relative to the substrate-scaffolding module.

    “Because ubiquitylation does occur with mutant substrates or enzymes, albeit at substantially lower rates (Extended Data Table 1), we cannot exclude that UB could be transferred from RING- and NEDD8-bound UBE2D in various orientations relative to the substrate-scaffolding module.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    UBE2D3 contributes to stability of CRL4CRBN neomorphic substrates in cells.

    “Our assay for CRL4CRBN ubiquitylation of IKZF was established based on findings that ZF2 mediates tight immunomodulatory-drug dependent interaction sufficient to target degradation, UBE2D3 contributes to stability of CRL4CRBN neomorphic substrates in cells57,60,61.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [57] Koduri, V. et al. Peptidic degron for IMiD-induced degradation of heterologous proteins. Proc. Natl Acad. Sci. USA 116, 2539–2544 (2019). source

      Not checked in the free run.

    • cites [60] Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362 eaat0572 (2018). source

      Not checked in the free run.

    • cites [61] Lu, G. et al. UBE2G1 governs the destruction of cereblon neomorphic substrates. eLife 7, e40958 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Their UB counterparts would be predicted to repel CUL1, rationalizing a need for NEDD8 as a distinctive UB-like protein.

    “Their UB counterparts would be predicted to repel CUL1, rationalizing a need for NEDD8 as a distinctive UB-like protein.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The numerous requisite protein-protein interactions and conformational changes suggest there could be multiple routes to the catalytic architecture, where formation of interfaces successively narrows the range of options, akin to progression down a free energy funnel.

    “The numerous requisite protein-protein interactions and conformational changes suggest there could be multiple routes to the catalytic architecture (Figure 4b), where formation of interfaces successively narrows the range of options, akin to progression down a free energy funnel.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    ZF2 mediates tight immunomodulatory-drug dependent interaction sufficient to target degradation.

    “Our assay for CRL4CRBN ubiquitylation of IKZF was established based on findings that ZF2 mediates tight immunomodulatory-drug dependent interaction sufficient to target degradation, UBE2D3 contributes to stability of CRL4CRBN neomorphic substrates in cells57,60,61.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [57] Koduri, V. et al. Peptidic degron for IMiD-induced degradation of heterologous proteins. Proc. Natl Acad. Sci. USA 116, 2539–2544 (2019). source

      Not checked in the free run.

    • cites [60] Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362 eaat0572 (2018). source

      Not checked in the free run.

    • cites [61] Lu, G. et al. UBE2G1 governs the destruction of cereblon neomorphic substrates. eLife 7, e40958 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Perplexing neddylated CRL features incompatible with prior structures are now rationalized, including NEDD8-stimulated crosslinking between a CRL1β-TRCP-bound phosphopeptide and UBE2D, simultaneous NEDD8 linkage to a cullin and binding to the backside of RBX1-bound UBE2D, and catastrophic effects of bacterial effector-catalyzed NEDD8 Gln40 deamidation.

    “Second, perplexing neddylated CRL features incompatible with prior structures are now rationalized, including NEDD8-stimulated crosslinking between a CRL1β-TRCP-bound phosphopeptide and UBE2D4; simultaneous NEDD8 linkage to a cullin and binding to the backside of RBX1-bound UBE2D3,7,37; and catastrophic effects of bacterial effector-catalyzed NEDD8 Gln40 deamidation24–26”
    Quote in the paperSubstantial claim0 of 7 sources checked
    • cites [4] Saha, A. & Deshaies, R. J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008). source

      Not checked in the free run.

    • cites [3] Duda, D. M. et al. Structural insights into NEDD8 activation of cullin–RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008). source

      Not checked in the free run.

    • cites [7] Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002). source

      Not checked in the free run.

    • cites [37] Sakata, E. et al. Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity. Nat. Struct. Mol. Biol. 14, 167–168 (2007). source

      Not checked in the free run.

    • cites [24] Cui, J. et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010). source

      Not checked in the free run.

    • cites [25] Jubelin, G. et al. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways. PLoS Pathog. 6, e1001128 (2010). source

      Not checked in the free run.

    • cites [26] Morikawa, H. et al. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1. Biochem. Biophys. Res. Commun. 401, 268–274 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The architecture observed in the neddylated CRL1β-TRCP-UBE2D~UB-substrate complex structure may both stimulate intrinsic reactivity of the UBE2D~UB intermediate and place the catalytic center in proximity of the β-TRCP-bound substrate.

    “Thus, the architecture observed in the neddylated CRL1β-TRCP-UBE2D~UB-substrate complex structure may both stimulate intrinsic reactivity of the UBE2D~UB intermediate and place the catalytic center in proximity of the β-TRCP-bound substrate.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Ubiquitylation of IκBα by CRL1β-TRCP via UBE2D3 was monitored using a pulse-chase format that specifically detects CRLβ-TRCP-dependent UB modification from UBE2D to IκBα independently of effects on UBA1-dependent formation of the UBE23D~UB intermediate.

    “Ubiquitylation of IκBα by CRL1β-TRCP via UBE2D3 was monitored using a pulse-chase format that specifically detects CRLβ-TRCP-dependent UB modification from UBE2D to IκBα independently of effects on UBA1-dependent formation of the UBE23D~UB intermediate.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationown data

    While neddylated CRL1β-TRCP and UBE2D seem optimal for UB priming of peptide-like substrates, the limited effect of the mutations on linkage of subsequent UBs raises the possibility that different forms of ubiquitylation involve alternative, presently elusive, catalytic architectures.

    “While neddylated CRL1β-TRCP and UBE2D seem optimal for UB priming of peptide-like substrates, the limited effect of the mutations on linkage of subsequent UBs (Extended Data Table 1) raises the possibility that different forms of ubiquitylation involve alternative, presently elusive, catalytic architectures.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The RBX1 N98R variant is hyperactive toward UBE2D~UB.

    “used the RBX1 N98R variant that is hyperactive toward UBE2D~UB39.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Scott, D. C. et al. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157, 1671–1684 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The resultant negative charge from Gln40 deamidation would shatter the CUL1-NEDD8 interface, rationalizing how pathogenic bacterial effectors impair CRL1-dependent ubiquitylation.

    “This rationalizes how pathogenic bacterial effectors catalyzing Gln40 deamidation impair CRL1-dependent ubiquitylation12,24–26: the resultant negative charge would shatter the CUL1-NEDD8 interface.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [12] Yu, C. et al. Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8. Nat. Commun. 6, 10053 (2015).

      Not checked in the free run.

    • cites [24] Cui, J. et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010). source

      Not checked in the free run.

    • cites [25] Jubelin, G. et al. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways. PLoS Pathog. 6, e1001128 (2010). source

      Not checked in the free run.

    • cites [26] Morikawa, H. et al. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1. Biochem. Biophys. Res. Commun. 401, 268–274 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The activation module binds the catalytic module, solving the mystery of how neddylation helps CRL1β-TRCP recruit UBE2D in cells.

    “The activation module binds the catalytic module, solving the mystery of how neddylation helps CRL1β-TRCP recruit UBE2D in cells34.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Kawakami, T. et al. NEDD8 recruits E2-ubiquitin to SCF E3 ligase. EMBO J. 20, 4003–4012 (2001). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    In experiments comparing neddylated with unneddylated CRLs, the NEDD8 modification sites of CUL1 and CUL4 were mutated to Arg (CUL1 K720R and CUL4A K705R) to prevent low-level ubiquitylation of the NEDD8 consensus Lys during the ubiquitylation reactions.

    “in the unneddylated versions the NEDD8 modification sites of CUL1 and CUL4 were mutated to Arg (CUL1 K720R and CUL4A K705R) to prevent obscuring interpretation of the results by low-level ubiquitylation of the NEDD8 consensus Lys during the ubiquitylation reactions3.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Duda, D. M. et al. Structural insights into NEDD8 activation of cullin–RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

    Interpretationcitation

    The malleability of neddylated CRLs, coupled with numerous UB carrying enzyme partners, may underlie successful molecular glue or PROTAC-targeted protein degradation, while potential limits in their multivalent catalytic architectures may explain failures in such chemically-directed ubiquitylation of heterologous substrates.

    “The malleability of neddylated CRLs, coupled with numerous UB carrying enzyme partners, may underlie successful molecular glue or PROTAC-targeted protein degradation, while potential limits in their multivalent catalytic architectures may explain failures in such chemically-directed ubiquitylation of heterologous substrates45.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Schapira, M., Calabrese, M. F., Bullock, A. N. & Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discov. 18, 949–963 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The worst single mutation is NEDD8 substitution with UB (R72A), confirming the importance of NEDD8 and interactions between the activation and substrate scaffolding modules.

    “The worst “single” mutation is NEDD8 substitution with UB (R72A), confirming the importance of NEDD8 and interactions between the activation and substrate scaffolding modules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The mutations have comparatively little effect on chain extension, consistent with the neddylated CRL1β-TRCP-UBE2D~UB-substrate intermediate structure defining UB transfer from UBE2D directly to unmodified substrate.

    “Third, the mutations have comparatively little effect on chain extension, consistent with the neddylated CRL1β-TRCP-UBE2D~UB-substrate intermediate structure defining UB transfer from UBE2D directly to unmodified substrate.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The attenuated pulse-chase assay qualitatively, but exclusively, monitors NEDD8-activated substrate priming by CRL1β-TRCP and UBE2D.

    “in an attenuated pulse-chase assay format that qualitatively, but exclusively, monitors NEDD8-activated substrate priming by CRL1β-TRCP and UBE2D”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In sumoylation and neddylation intermediates, aromatic side-chains guide the lysine targets.

    “where aromatic side-chains guide the lysine targets (blue and green, respectively)39,73.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [39] Scott, D. C. et al. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157, 1671–1684 (2014). source

      Not checked in the free run.

    • cites [73] Yunus, A. A. & Lima, C. D. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nat. Struct. Mol. Biol. 13, 491–499 (2006). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The RBX1 RING sequence represents a compromise to meet requirements for its many different catalytic activities achieved with neddylation E2s, various UB carrying enzymes, and regulators including the inhibitor GLMN.

    “we considered that the RBX1 RING sequence represents a compromise to meet requirements for its many different catalytic activities achieved with neddylation E2s, various UB carrying enzymes, and regulators including the inhibitor GLMN72.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [72] Duda, D. M. et al. Structure of a glomulin–RBX1–CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface. Mol. Cell 47, 371–382 (2012). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Good geometry and map to model correlation was reached.

    “Further iterative manual model building and real space refinements were carried out until good geometry and map to model correlation was reached.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Only a "loop out" conformation is compatible for the CUL1-linked NEDD8 to bind the catalytic module, because "loop in" conformations would prevent noncovalent interactions with the UBE2D backside.

    “Only a “loop out” conformation is compatible for the CUL1-linked NEDD8 to bind the catalytic module, because “loop in” conformations in the structures shown in g would prevent noncovalent interactions with the UBE2D backside (cyan).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The CRL1β-TRCP RBX1 RING and CUL1 WHB domains, without or with covalently-linked NEDD8, are dynamic in the absence of other factors and are harnessed in catalytic architecture for substrate ubiquitylation with UBE2D.

    “CRL1β-TRCP RBX1 RING and CUL1 WHB domains, without or with a covalently-linked NEDD8, are dynamic in the absence of other factors and are harnessed in catalytic architecture for substrate ubiquitylation with UBE2D.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    The final reconstructions displayed clear main chain and side chain densities, enabling modeling and refinement of atomic coordinates.

    “The final reconstructions displayed clear main chain and side chain densities, that enabled us to model and refine the atomic coordinates.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In prior cullin crystals, the CUL1 region connecting the C/R and WHB domains forms a rod-like Helix-29 continuing into the WHB domain.

    “whereas in prior cullin crystals this forms the rod-like Helix-29 continuing into the WHB domain7.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    RBX1 N98R was previously shown to improve neddylated CRL and UBE2D-dependent substrate priming at the expense of other RBX1-dependent functions.

    “we mutationally introduced a second RBX1 linchpin residue (N98R) previously shown to improve neddylated CRL and UBE2D-dependent substrate priming at the expense of other RBX1-dependent functions (e.g. with UBE2M and UBE2R2)39.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Scott, D. C. et al. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157, 1671–1684 (2014). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    A point mutant version of RBX1 is hyperactive for substrate priming with UBE2D but defective for UB chain elongation with UBE2R-family E2s.

    “using a point mutant version of RBX1 that is hyperactive for substrate priming with UBE2D but defective for UB chain elongation with UBE2R-family E2s39.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Scott, D. C. et al. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157, 1671–1684 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Gel images are representative of independent technical replicates (n=2), and symbols from graphs show data from independent experiments (n=2).

    “Gel images are representative of independent technical replicates (n=2); The symbols from graphs show the data from independent experiments (n = 2)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Only a "loop out" conformation is compatible with the neddylated CRL activation module structure, because a "loop in" conformation would prevent noncovalent interactions with the CUL1 WHB domain.

    “Only a “loop out” conformation is compatible with the neddylated CRL activation module structure, because “loop in” conformation from the structures shown in g would prevent noncovalent interactions with CUL1 WHB domain (green).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationcitation

    Mutation of the noncatalytic cysteines hindered formation of the RING-activated, closed, active UBE2D~UB conformation.

    “Based on the structural locations of these cysteines, we presumed that their mutation hindered formation of the RING-activated, closed, active UBE2D~UB conformation28–30.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [28] Dou, H., Buetow, L., Sibbet, G. J., Cameron, K. & Huang, D. T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat. Struct. Mol. Biol. 19, 876–883 (2012). source

      Not checked in the free run.

    • cites [29] Plechanovová, A., Jaffray, E. G., Tatham, M. H., Naismith, J. H. & Hay, R. T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012). source

      Not checked in the free run.

    • cites [30] Pruneda, J. N. et al. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    RBX1's Trp35 serves as a multifunctional platform for either the RING domain in different orientations, or for the E2-linked NEDD8 during neddylation.

    “RBX1’s Trp35 is highlighted to show it serving as a multifunctional platform for either the RING domain in different orientations, or for the E2-linked NEDD8 during neddylation39.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Scott, D. C. et al. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157, 1671–1684 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The gold standard Fourier Shell Correlation curve shows overall resolution at 3.72Å at FSC=0.143.

    “Gold standard Fourier Shell Correlation curve showing overall resolution at 3.72Å at FSC=0.143.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Two loops in SKP1 are known to be flexible and not required for ubiquitylation activity, although they are required for CAND1-mediated substrate-receptor exchange.

    “removing two loops in SKP1 that are known to be flexible and not required for ubiquitylation activity (although they are required for CAND1-mediated substrate-receptor exchange)13,54”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Pierce, N. W. et al. Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153, 206–215 (2013). source

      Not checked in the free run.

    • cites [54] Schulman, B. A. et al. Insights into SCF ubiquitin ligases from the structure of the Skp1–Skp2 complex. Nature 408, 381–386 (2000). source

      Not checked in the free run.

68 of 68

  • The paper relies on this without support.

    Substrate priming involves fleeting simultaneous linkage of UBE2D’s active site, UB’s C-terminus, and the substrate.

    “Substrate priming involves fleeting simultaneous linkage of UBE2D’s active site, UB’s C-terminus, and the substrate”

  • The authors state this as an assumption.

    The chemically adjoined surrogate complex is a stable mimic of the transition state and represents the ubiquitylation intermediate.

    “We chemically adjoined surrogates for these entities in a stable mimic of the transition state, which avidly binds neddylated CRL1β-TRCP”

  • The paper relies on this without support.

    Mutations designed to destroy the activation module or hinder interactions between the activation and catalytic modules actually disrupt those intended interfaces.

    “Mutations designed to destroy the activation module (NEDD8 Q40E) or hinder interactions between the activation and catalytic modules (NEDD8 I44A or UBE2D S22R) substantially impair substrate priming”

  • The paper relies on this without support.

    UBE2D contains three additional cysteines (Cys21, Cys107, Cys111) besides the active-site cysteine.

    “However, UBE2D contains three additional cysteines (Cys21, Cys107, Cys111).”

  • The paper relies on this without support.

    UBE2D’s myriad substrates neither conform to a specific motif, nor do they or UBE2D display specific side-chains guiding lysine acceptors into the catalytic center.

    “However, UBE2D’s myriad substrates neither conform to a specific motif, nor do they or UBE2D display specific side-chains guiding lysine acceptors into the catalytic center.”

  • The paper relies on this without support.

    The NEDD8 Q40E mutation disrupts the activation module.

    “testing effect of NEDD8 Q40E, which would disrupt the activation module.”

  • The paper relies on this without support.

    The NEDD8 I44A mutation disrupts interactions between the activation and catalytic modules.

    “showing the effect on substrate priming of disrupting interactions between the activation and catalytic modules with NEDD8 I44A or UBE2D S22R mutation.”

  • The paper relies on this without support.

    The UBE2D S22R mutation disrupts interactions between the activation and catalytic modules.

    “showing the effect on substrate priming of disrupting interactions between the activation and catalytic modules with NEDD8 I44A or UBE2D S22R mutation.”

  • The paper relies on this without support.

    The “Ubiquitylized” NEDD8 mutant with six residues swapped for UB counterparts (L2Q K4F E14T D16E G63K G64E) disrupts interactions between the activation and substrate scaffolding modules.

    “showing the effect on substrate priming of disrupting interactions between the activation and substrate scaffolding modules, though CUL1 modification by a “Ubiquitylized” NEDD8 mutant with six residues swapped for UB counterparts (L2Q K4F E14T D16E G63K G64E).”

  • The paper relies on this without support.

    The UBE2D H32A mutation is at the interface between the catalytic and substrate scaffolding modules.

    “showing the effect of UBE2D H32A mutation at the interface between the catalytic and substrate scaffolding modules.”

  • The authors state this as an assumption.

    The covalently-linked chemical trap is a proxy for the IκBα substrate’s acceptor in the active site.

    “highlighting the covalently-linked proxy for the IκBα substrate’s acceptor in the active site”
11 of 11

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Ubiquitin E3 ligases serve as scaffolds between a target protein and an activated E2-Ub, binding both and catalysing transfer of ubiquitin from the E2 active site cysteine to a lysine on the target.

    “ubiquitin E3 ligases serve as scaffolds between a target protein and an activated ubiquitin E2 conjugating enzyme charged with a ubiquitin molecule (E2–Ub), by binding both molecules, then catalysing the transfer of ubiquitin from the active site cysteine of the E2 onto a lysine side chain of a target protein104–107.”

    From PROTAC targeted protein degraders: the past is prologue (2022) · cites this paper as [106]

  • The exact mechanism of ubiquitylation for MRLs is not as well understood as the mechanism for CRLs, for which substrate recruitment by multiple adaptors and the molecular details of Ub transfer from E2-Ub to the target have been extensively characterized at the atomic level of an entire CRL1 complex.

    “The exact mechanism of ubiquitylation for MRLs is not as well understood as the mechanism for CRLs, for which substrate recruitment by multiple adaptors and the molecular details of Ub transfer from E2–Ub to the target has been extensively characterized at the atomic level of an entire CRL1 complex106,117.”

    From PROTAC targeted protein degraders: the past is prologue (2022) · cites this paper as [106]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Arctica acquisition settings differ between Methods and Table 2

    Minor

    The Methods say the Arctica datasets were recorded at 73,000×, whereas Table 2 lists one Arctica dataset at 92,000×.

  • High-resolution Krios defocus range differs between Methods and Table 2

    Minor

    The high-resolution Krios dataset is described with a defocus range ending at −3.6 μm in the Methods, but its Table 2 column-the 130,000× dataset-ends at −3.3 μm.

2 of 2

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Authors
Baek, K., Krist, D. T., Prabu, J. R., Hill, S., Klügel, M., Neumaier, L.-M., von Gronau, S., Kleiger, G., & Schulman, B. A.
Affiliations
  • Max Planck Institute of Biochemistry (DE)
  • University of Illinois Urbana-Champaign (US)
  • University of Nevada, Las Vegas (US)
Corresponding author
Schulman, Brenda A.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2020-02-12
Volume
578
Issue
7795
Pages
461-466
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-020-2000-y
PMC
PMC7050210
OpenAlex
W3006502081
Cited by
270
References
77
Citations by year
2026: 33 · 2025: 33 · 2024: 49 · 2023: 42 · 2022: 39 · 2021: 46 · 2020: 27 · 2019: 1
FWCI
12.49
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion, other-oa)
Funders
National Institute of General Medical Sciences (R15GM117555)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
Keywords
NEDD8, Cullin-RING E3 ligases, ubiquitin transfer, ubiquitination, neddylation, protein ubiquitylation, IκBα, cryo-EM structure, protein-protein interaction, conformational change, E2 ubiquitin-conjugating enzyme
MeSH terms
NF-KappaB Inhibitor alpha, NEDD8 Protein, Humans, Models, Molecular, Phosphorylation, Protein Conformation, Substrate Specificity, Cryoelectron Microscopy, Ubiquitin, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination, Biocatalysis

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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