Structure of a RING E3 Trapped in Action Reveals Ligation Mechanism for the Ubiquitin-like Protein NEDD8
Open accessAbstract
Most E3 ligases use a RING domain to activate a thioester-linked E2∼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2∼UBL-target intermediate representing RBX1-UBC12∼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2∼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2∼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.
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The conformational dynamics of unneddylated CRLs would enable transitioning between the different conformations coordinating cycles of neddylation/deneddylation with CAND1-driven substrate-receptor exchange.
“it seems likely that the conformational dynamics of unneddylated CRLs (Extended Data Figure 2) would enable transitioning between the different conformations coordinating cycles of neddylation/deneddylation with CAND1-driven substrate-receptor exchange39,46–49.”
From NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly (2020) · cites this paper as [39]
RBX1's RING stacks on its C/R domain Trp35 side-chain, consistent with previously reported effects for RBX1 Trp35Ala mutation.
“On one side, RBX1’s RING stacks on its C/R domain Trp35 side-chain, consistent with previously reported effects for RBX1 Trp35Ala mutation39.”
From NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly (2020) · cites this paper as [39]
Substrate-independent activation of UBE2D~UB can be readily visualized using CUL1 complexed with a hyperactive RBX1 N98R mutant and high enzyme and lysine concentrations.
“Nonetheless, substrate-independent activation of UBE2D~UB can be readily visualized using CUL1 complexed with a previously-described hyperactive RBX1 N98R mutant39, and high enzyme and lysine concentrations.”
From NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly (2020) · cites this paper as [39]
RBX1 RING-dependent activity is limited in the discharge assay due to sequence constraints imposed by the requirements for binding to partners other than UBE2D.
“RBX1 RING-dependent activity is limited in this assay due to sequence constraints imposed by the requirements for binding to partners other than UBE2D39.”
From NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly (2020) · cites this paper as [39]
The chemical trap in the neddylated CRL1β-TRCP-UBE2D~UB-IκBα intermediate superimposes with consensus acceptors visualized in active sites of sumoylation and neddylation intermediates.
“The chemical trap superimposes with consensus acceptors visualized in active sites of sumoylation63 and neddylation39 intermediates”
From NEDD8 nucleates a multivalent cullin–RING–UBE2D ubiquitin ligation assembly (2020) · cites this paper as [39]
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References 79 · Cited by 201
- Authors
- Scott, D. C., Sviderskiy, V. O., Monda, J. K., Lydeard, J. R., Cho, S. E., Harper, J. W., & Schulman, B. A.
- Affiliations
- St. Jude Children's Research Hospital (US)
- Harvard University (US)
- Corresponding author
- Schulman, Brenda A.
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2014-06
- Volume
- 157
- Issue
- 7
- Pages
- 1671-1684
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC4247792
- OpenAlex
- W1964909962
- Cited by
- 201
- References
- 79
- Citations by year
- 2026: 15 · 2025: 10 · 2024: 19 · 2023: 12 · 2022: 10 · 2021: 15 · 2020: 25 · 2019: 20 · 2018: 18 · 2017: 13 · 2016: 20 · 2015: 18 · 2014: 6
- FWCI
- 7.79
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute on Aging (R01AG011085)
- National Cancer Institute (P30CA021765)
- National Institute of General Medical Sciences (R01GM069530)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Keywords
- RING E3 ligases, RBX1, Ubc12, NEDD8, cullin neddylation, CUL1, ubiquitin-like proteins, ubiquitination, protein structure
- MeSH terms
- NEDD8 Protein, Amino Acid Sequence, Carrier Proteins, Humans, Models, Molecular, Molecular Sequence Data, Ubiquitins, Crystallography, X-Ray, Cell Cycle Proteins, Catalytic Domain, Ubiquitin-Conjugating Enzymes, Cullin Proteins
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