Poly(ADP-ribosyl)ation (PARylation) is a highly dynamic post-translational modification mediated by poly(ADP-ribose) polymerases (PARPs), influencing DNA damage response, transcription, and cell death. Previously, we showed that noncovalent interactions between PAR and the intrinsically disordered regions (IDPRs) of the p53 C-terminal domain (CTD) mediate the PARP1-dependent covalent modification of its target proteins. In this study, we test whether this mechanism also applies to noncovalent interactions involving the highly basic RGG IDPR of the FUS protein, as well as to domains lacking IDPRs, such as WWE, PBZ, and the macrodomain. We employed a chemical biology approach using a fluorescently labeled NAD+ analogue together with fusion constructs that contain defined ADP-ribose-binding domains and validated PARylation acceptors. We find that the p53-CTD, RGG, WWE, and PBZ domains support efficient covalent PARylation, whereas binding through the macrodomain protein Af1521 results in only very weak PARylation. These findings suggest that the mode of PAR binding influences how effectively proteins are directed toward covalent PARylation. This work broadens our molecular understanding of the interplay of noncovalent and covalent PARylation mechanisms and highlights the importance of distinct PAR-binding modes, which may inform future therapeutic strategies aimed at modulating PAR signaling in disease.
Fischbach et al. (Fri,) studied this question.