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PreprintCited by 11NF-κB Signaling PathwaysUbiquitin and proteasome pathwaysEnzyme Structure and Function

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Structural analysis shows that the BIR2 domain of E3 ligase XIAP binds across the RIP2 kinase dimer interface

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Lethier, Mathilde, Hons, Michael, Favier, Adrien, Brutscher, Bernhard, Erba, Elisabetta Boeri, Cusack, Stephen, Pellegrini, Erika·2022·Life Science Alliance
DOI 10.1101/2022.10.14.512215PMC PMC10485824

Preprint — published as 10.26508/lsa.202201784 · Paper page

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Abstract

NOD1 and NOD2 are innate immune system pattern recognition receptors that play a key role in the protection from bacterial infections and in the maintenance of gastro-intestinal homeostasis. Dysregulation of NOD signalling pathways promotes chronic inflammatory diseases such as inflammatory bowel disease. RIP2, which contains a kinase domain (RIP2K) and a CARD domain, is the immediate downstream signalling partner in the NOD pathway and inhibition of its kinase activity and ubiquitination are promising strategies to address these inflammatory diseases. However, recent work indicates that the phosphorylation activity of RIP2K is dispensable for signalling and that inhibitors of both RIP2K activity and RIP2 ubiquitination prevent the essential interaction between RIP2K and the BIR2 domain of XIAP, the key RIP2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined Native mass spectrometry, NMR, cryo-electron microscopy and Alphafold2 predictions to determine the structure of the RIP2K-XIAP BIR2 complex with 2:1 stoichiometry. The structure shows that complex formation requires that the kinase is in an active-like (i.e. αC-helix IN) and dimeric conformation and explains both inhibitory mechanisms. It also shows why phosphorylation of the kinase activation loop is dispensable for signalling and reveals the structural role of RIP2K-K209 residue in the RIP2K-XIAP BIR2 interaction. Importantly, our results clarify the features of the RIP2K conformation essential for its role as a scaffold protein for ubiquitination.

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Metadata

Authors
Lethier, M., Hons, M., Favier, A., Brutscher, B., Erba, E. B., Cusack, S., & Pellegrini, E.
Affiliations
  • European Molecular Biology Laboratory (DE)
  • Centre National de la Recherche Scientifique (FR)
Corresponding author
  • Cusack, Stephen
  • Pellegrini, Erika
ORCID
Venue
Life Science Alliance
Publisher
openRxiv
Published
2022-10-14
Volume
6
Issue
11
Pages
e202201784-e202201784
ISSN
2575-1077 (ISSN-L)
DOI
10.1101/2022.10.14.512215
PMC
PMC10485824
OpenAlex
W4386483311
Cited by
11
References
1
Citations by year
2026: 4 · 2025: 1 · 2024: 5 · 2023: 1
FWCI
1.39
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Life Science Alliance (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Francis Crick Institute
  • European Molecular Biology Laboratory (R01-GM129325, R35GM141603)
  • Agence Nationale de la Recherche (UAR 3518 CNRS-CEA-UGA-EMBL, ANR-17-EURE-0003, ANR-17-EURE-, ANR-17, ANR-10, ANR-10-INBS-0005)
  • European Synchrotron Radiation Facility
  • Centre National de la Recherche Scientifique (FR 2054, IR-RMN-THC Fr3050, ANR‐17‐EURE‐0003, UAR 3518 CNRS-CEA-UGA-EMBL)
  • French Infrastructure for Integrated Structural Biology (ANR-17-EURE-0003, ANR-10-INBS-0005, ANR-10-INBS-000502, ANR-10-INBS)
  • National Institutes of Health
  • University of California, San Francisco (ANR-10-INBS-0005-02, ANR-17-EURE-0003, R01-GM129325)
  • National Institute of General Medical Sciences (R35GM141603, R01GM129325)
  • National Institute of Allergy and Infectious Diseases
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
RIPK2, XIAP, E3 ubiquitin ligase, inflammatory bowel disease, scaffold proteins, protein-protein interaction, cryo-EM structure
MeSH terms
Biological Assay, Phosphorylation, Cryoelectron Microscopy, Ubiquitin-Protein Ligases, Ubiquitination

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