Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function

Abstract

AbstractUbiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways like DNA damage response and infection, making them attractive targets for therapeutic intervention. DTX3L, an E3 ubiquitin ligase, forms a heterodimer with PARP9. The complex has ubiquitin ligase activity and also ADP-ribosylates the C-terminus of ubiquitin on Gly76. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. By using individually produced proteins, we have studied the interaction between DTX3L and PARP9. We identify that the D3 domain (230 – 510) of DTX3L mediates interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. Our results also suggest the formation of a higher molecular weight oligomer mediated by the N-terminus of DTX3L (1-200). Furthermore, we show that ADP-ribosylation of ubiquitin at Gly76 is a reversible modification that can be removed by several macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination in an inter-regulatory manner.