RT Journal Article
SR Electronic
T1 The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-protein substrates in vitro
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202503243
DO 10.26508/lsa.202503243
VO 8
IS 6
A1 Wang, Xiangyi S
A1 Jiou, Jenny
A1 Cerra, Anthony
A1 Cobbold, Simon A
A1 Jochem, Marco
A1 Mak, Ka Hin Toby
A1 Corcilius, Leo
A1 Silke, John
A1 Payne, Richard J
A1 Goddard-Borger, Ethan D
A1 Komander, David
A1 Lechtenberg, Bernhard C
YR 2025
UL http://www.life-science-alliance.org/content/8/6/e202503243.abstract
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.