9IPU

cryo-EM structure of the RNF168(1-193)/UbcH5c-Ub ubiquitylation module bound to H1.0-K63-Ub3 modified chromatosome

Summary for 9IPU

• Entry DOI: 10.2210/pdb9ipu/pdb
• EMDB information: 60781
• Descriptor: Histone H1.0, DNA (171-MER), ZINC ION, ... (11 entities in total)
• Functional Keywords: rnf168, h1.0, ubiquitylation, nucleosome, chromatosome, nuclear protein
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 14
• Total formula weight: 282994.27

Authors

Ai, H.S.,Deng, Z.H.,Liu, L. (deposition date: 2024-07-11, release date: 2024-10-02, Last modification date: 2025-04-16)

Primary citation

Shi, Q.,Deng, Z.,Zhang, L.,Tong, Z.,Li, J.B.,Chu, G.C.,Ai, H.,Liu, L.
Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally Defined K63-Polyubiquitylated Linker Histone H1.
Angew.Chem.Int.Ed.Engl., 64:e202413651-e202413651, 2025

PubMed Abstract: The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

PubMed: 39363740
DOI: 10.1002/anie.202413651

Experimental method

ELECTRON MICROSCOPY (4.3 Å)