Yashwanth Ashok, Carlos Vela-Rodríguez, Chunsong Yang, Heli I. Alanen, Fan Liu, Bryce M. Paschal, Lari Lehtiö; Reconstitution of the DTX3L–PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric assembly. Biochem J 11 February 2022; 479 (3): 289–304. doi: https://doi.org/10.1042/BCJ20210722
Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimerization and multimeric assembly
|Author:||Ashok, Yashwanth1; Vela-Rodríguez, Carlos1; Yang, Chunsong2;|
1Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland
2Department of Biochemistry and Molecular Genetics, University of Virginia, PO Box 800577, Charlottesville, VA 22908, U.S.A.
3Department of Structural Biology, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Deutschland
|Online Access:||PDF Full Text (PDF, 2.8 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe2022112867384
|Publish Date:|| 2022-11-28
Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L–PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD⁺-dependent ADP-ribosylation of ubiquitin by DTX3L–PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L–PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230–510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1–200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L–PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.
|Pages:||289 - 304|
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
The research was supported by the Academy of Finland grants 287063, 294085 and 319299 (to LL) and by an NIH grant No. R01CA214872 (to B.M.P.), and by Leibniz Association with the Leibniz-Wettbewerbs (to F.L.).
|Academy of Finland Grant Number:||
287063 (Academy of Finland Funding decision)
294085 (Academy of Finland Funding decision)
319299 (Academy of Finland Funding decision)
© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.