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

In a reaction that requires processing by E1 and E2 enzymes, the E3 ligase complex DTX3L-PARP9 ADP-ribosylates the carboxyl terminus of Ubiquitin and prevents its conjugation to substrate protein. This provides an NAD+-sensitive mechanism for regulating mono-Ub modification of certain substrates. DTX3L and PARP9 are coordinatedly expressed from a single, bidirectional promoter and can be isolated as a protein complex from multiple cell types. We used purified DTX3L and PARP9, prepared as individual proteins, to investigate how the complex assembles and how complex formation is related to E3 activity. We find that DTX3L binds PARP9 with nanomolar affinity and with an apparent stoichiometry of 1:1. Using a combination of deletion mutants and cross-linking mass spectrometry, we show the interaction involves the D3 domain of DTX3L (230 - 510) and a central region of PARP9 (491 - 630). We found evidence that the DTX3L-PARP9 heterodimer can assemble into a high molecular weight oligomer dependent on the D1 (1 - 100) and D2 (101 - 200) domains of DTX3L. We also show that ADP-ribosylation of Ubiquitin by DTX3L-PARP9 is reversible by several macrodomain-type hydrolases. Our data helps to provide a structural framework for understanding how ubiquitination and ADP-ribosylation are mediated by DTX3L-PARP9.