Reconstitution of the DTX3L-PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric...

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Ubiquitination and ADP-ribosylation are critical post-translational modifications involved in DNA damage response and viral infection.
• Enzymes catalyzing these modifications, like DTX3L-PARP9, are potential therapeutic targets.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying DTX3L-PARP9's enzymatic activities.
• To understand the structural basis for DTX3L-PARP9 complex formation and its interaction with ubiquitin.

Main Methods:

• Production of recombinant DTX3L and PARP9 proteins.
• Investigation of protein-protein interactions and stoichiometry using biophysical methods.
• Analysis of ADP-ribosylation activity and reversibility using in vitro assays.

Main Results:

• The DTX3L D3 domain (residues 230-510) mediates high-affinity binding to PARP9 (1:1 stoichiometry).
• DTX3L and PARP9 form higher-order oligomers through the DTX3L N-terminal region (residues 1-200).
• ADP-ribosylation of ubiquitin at Gly76 by DTX3L-PARP9 is reversible by Macrodomain hydrolases.

Conclusions:

• DTX3L-PARP9 utilizes intra- and inter-subunit interactions to regulate ubiquitination and ADP-ribosylation.
• The findings provide a structural framework for understanding DTX3L-PARP9 function in cellular signaling pathways.