Protein ADP-ribosylation is a covalent, reversible post-translational modification whereby ADP-ribose (ADPr) is transferred from nicotinamide adenine dinucleotide (NAD+) onto different amino acid residues of target proteins to regulate protein structure, stability, activity and molecular interactions. To further elucidate the cellular function of ADP-ribosylation, it is of importance to detect and decode the target protein landscape. This is however challenging due to the low abundance of ADP-ribosylation. In order to decipher the ADP-ribosylome including the ADPr acceptor sites, we recently co-developed a mass spectrometry-based approach that enriches ADP-ribosylated peptides with the help of the Af1521 macrodomain, which binds the terminal ADP-ribose units. To improve the enrichment and thus detection of ADP-ribosylated proteins in lysates, we aimed at improving the affinity of Af1521 to ADP-ribosylated peptides.
We thus evolved by random mutagenesis and in vitro selection an engineered Af1521 macrodomain with significantly increased affinity towards ADP-ribosylated peptides as verified by ELISA and Surface Plasmon Resonance (SPR). The crystal structure of the evolved macro domain revealed that two amino acid substitutions form an additional salt bridge forcing a rotation of the ribose and subsequently leading to less conformational flexibility and trapping of the ADP-ribose unit. The comparison of the conventional and the engineered Af1521 macro domain in the peptide enrichment of our proteomic ADP-ribosylome approach revealed that the identification rate of the ADP-ribosylated targets increased 2-fold with the mutated binder when assaying genotoxic stress condition that leads to mainly Serine-ADP-ribosylation. Further characterization of the evolved Af1521 domain as fusion protein with a Fc fragment confirmed the improved detection of cellular ADP-ribosylation by immuno blot and immunofluorescence, suggesting that our new engineered Af1521 macro domain can also serve as a valuable tool for the identification and detection of ADP-ribosylation.