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

ABSTRACT: Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, "cullin-RING" and "RBR", are individually found in several hundred E3 ligases in humans, 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 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 amongst RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrate ubiquitylation relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Barbara Steigenberger

LAB HEAD: Brenda A. Schulman

PROVIDER: PXD047229 | Pride | 2024-04-09

REPOSITORIES: Pride

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			Action	DRS
	20211028_QEP1_coreQEP1_BaSt_LC12_16_SA_Orbi4030_Sample1.raw	Raw
	20211028_QEP1_coreQEP1_BaSt_LC12_16_SA_Orbi4030_Sample1_1_Crosslinks.txt	Txt
	20230202_EXPL6_AnWe_SA_LC12_18_Orbi4534_Sample01.raw	Raw
	20230202_EXPL6_AnWe_SA_LC12_18_Orbi4534_Sample01_Crosslinks.txt	Txt
	20230202_EXPL6_AnWe_SA_LC12_18_Orbi4534_Sample02.raw	Raw

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

Project description:Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, "cullin-RING" and "RBR", are individually found in several hundred E3 ligases in humans, 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 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 amongst RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects from deneddylation. Mono-ubiquitylation of TP53 relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.

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