PT  - JOURNAL ARTICLE
AU  - Wang, Xiangyi S
AU  - Jiou, Jenny
AU  - Cerra, Anthony
AU  - Cobbold, Simon A
AU  - Jochem, Marco
AU  - Mak, Ka Hin Toby
AU  - Corcilius, Leo
AU  - Silke, John
AU  - Payne, Richard J
AU  - Goddard-Borger, Ethan D
AU  - Komander, David
AU  - Lechtenberg, Bernhard C
TI  - The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-protein substrates in vitro
AID  - 10.26508/lsa.202503243
DP  - 2025 Jun 01
TA  - Life Science Alliance
PG  - e202503243
VI  - 8
IP  - 6
4099  - http://www.life-science-alliance.org/content/8/6/e202503243.short
4100  - http://www.life-science-alliance.org/content/8/6/e202503243.full
SO  - Life Sci. Alliance2025 Jun 01; 8
AB  - HOIL-1 is a RING-between-RING-family E3 ubiquitin ligase and a component of the linear ubiquitin chain assembly complex. Although most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has also been reported to ubiquitinate hydroxyl groups in protein serine and threonine sidechains and glucosaccharides, such as glycogen and its building block maltose, in vitro. However, HOIL-1 substrate specificity is currently poorly defined. Here, we show that HOIL-1 is unable to ubiquitinate lysine but can efficiently ubiquitinate serine and a variety of model and physiologically relevant di- and monosaccharides in vitro. We identify a critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 that enables this O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains. We use HOIL-1’s in vitro non-proteinaceous ubiquitination activity to produce preparative amounts of different ubiquitinated saccharides that can be used as tool compounds and standards in the rapidly emerging field of non-proteinaceous ubiquitination. Finally, we report an engineered, constitutively active HOIL-1 variant that simplifies in vitro generation of ubiquitinated saccharides.Atomic structures and diffraction data have been deposited in the PDB with accession codes 9EGV (HOIL-1 RING2/Ub) and 9EGW (HOIL-1 RING2/Ub-maltose). All other data supporting the conclusions are available in the article, supplementary material, and source data.