9O4M

Crystal structure of Ubiquitin Carboxy Terminal Hydrolase L1 Q209C mutant covalently crosslinked to ubiquitin genetically encoded with N6-(6-bromohexanoyl)-L-lysine

Summary for 9O4M

• Entry DOI: 10.2210/pdb9o4m/pdb
• Descriptor: Polyubiquitin-C, Ubiquitin carboxyl-terminal hydrolase isozyme L1, HEXANOIC ACID, ... (4 entities in total)
• Functional Keywords: uchl1, genetic code expansion, covalent trapping, unnatural amino acid, ubiquitin, hydrolase
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 4
• Total formula weight: 67102.84

Authors

Pannala, N.,Patel, R.,Das, C. (deposition date: 2025-04-08, release date: 2025-07-30)

Primary citation

Das, C.,Pannala, N.M.,Patel, R.S.,Anit, A.S.,Bhattacharya, D.,Teron, K.N.,Drown, B.,Fasan, R.
Internal Ubiquitin Electrophiles for Covalent Trapping and Inhibition of Deubiquitinases.
Chembiochem, :e202500318-e202500318, 2025

PubMed Abstract: The ubiquitin (Ub) system governs vital cellular processes in eukaryotic biology through an intricate network of Ub-protein interactions. While semisynthetic C-terminal Ub electrophiles (UbEs) are widely used to study Ub transfer and deubiquitinase (Dub) activity, they are limited to probing the active site while leaving other functionally important sites unexplored. Building on previously identified multivalent interaction interfaces and potential allosteric sites which are key to understanding their dynamic nature, here we report the development of genetically encoded Ub-based probes to covalently tether Ub-protein interactions in a proximity driven manner at distal locations away from the active site. This study demonstrates that UbEs with internal electrophiles maintain conformational changes observed with their C-terminal counterparts while circumventing their limitations in capturing distal binding-site complexes, an emerging feature in Ub-mediated regulation. Genetically encoding these electrophiles further demonstrate rational variation as activity-based probes (ABP), leading to a Met1-diUb ABP showing preference for OTULIN over other Met1 cleaving Dubs. Taken together, our study introduces genetically encoded Ub-based probes to explore the structural and biochemical significance of Ub-Dub interactions beyond the canonical S1 site, overcoming some limitations of traditional Ub C-terminal electrophiles.

PubMed: 40626928
DOI: 10.1002/cbic.202500318

Experimental method

X-RAY DIFFRACTION (2 Å)