DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on protein substrates

Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3 ' hydroxyl of the adenosine diphosphate (ADP)-ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3 similar to ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2 similar to ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad -like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.