Noncanonical assembly, neddylation and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex

Daniel Horn-Ghetko; Linus V M Hopf; Ishita Tripathi-Giesgen; Jiale Du; Sebastian Kostrhon; D Tung Vu; Viola Beier; Barbara Steigenberger; J Rajan Prabu; Luca Stier; Elias M Bruss; Matthias Mann; Yue Xiong; Brenda A Schulman

doi:10.1038/s41594-024-01257-y

Noncanonical assembly, neddylation and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex

Nat Struct Mol Biol. 2024 Apr 11. doi: 10.1038/s41594-024-01257-y. Online ahead of print.

Authors

Daniel Horn-Ghetko^#¹, Linus V M Hopf^#^{1

2}, Ishita Tripathi-Giesgen^{1

2}, Jiale Du¹, Sebastian Kostrhon¹, D Tung Vu^{1

3}, Viola Beier¹, Barbara Steigenberger⁴, J Rajan Prabu¹, Luca Stier^{1

2}, Elias M Bruss^{1

2}, Matthias Mann³, Yue Xiong^{5

6}, Brenda A Schulman^{7

8}

Affiliations

¹ Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
² Department of Chemistry, TUM School of Natural Sciences, Garching, Germany.
³ Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
⁴ Mass Spectrometry Core Facility, Max Planck Institute of Biochemistry, Martinsried, Germany.
⁵ Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁶ Cullgen Inc., San Diego, CA, USA.
⁷ Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany. schulman@biochem.mpg.de.
⁸ Department of Chemistry, TUM School of Natural Sciences, Garching, Germany. schulman@biochem.mpg.de.

^# Contributed equally.

PMID: 38605244
DOI: 10.1038/s41594-024-01257-y

Abstract

Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, 'cullin-RING' and 'RBR', are individually found in several hundred human E3 ligases, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin-RING and RBR domains. Here, cryo-EM, biochemistry and cellular assays elucidate a 1.8-MDa hexameric human CUL9-RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9-RBX1 in trans. Our data show CUL9 as unique among RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrates are recruited to various upstream domains, while ubiquitylation relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.