Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in... Show moreAndrogen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly. Show less