RT Journal Article
SR Electronic
T1 Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.06.14.448324
DO 10.1101/2021.06.14.448324
A1 Ashok, Yashwanth
A1 Vela-Rodriguez, Carlos
A1 Yang, Chunsong
A1 Alanen, Heli I.
A1 Liu, Fan
A1 Paschal, Bryce M.
A1 Lehtiö, Lari
YR 2021
UL http://biorxiv.org/content/early/2021/10/13/2021.06.14.448324.abstract
AB Ubiquitination 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.Competing Interest StatementThe authors have declared no competing interest.