Fig. 1: Characterization of engineered Af1521 macro domain.

a Pull-down experiment using unmodified and ADP-ribosylated H2B peptides (biotinylated and bound to streptavidin Sepharose beads) of WT Af1521 and eAf1521 under conditions with increasing salt concentrations. Input lanes are the same for all measured conditions. The proteins were detected with SDS–PAGE followed by Coomassie staining. The experiment was repeated independently (n = 3) with similar results. b Sequence alignment of WT Af1521 and eAf1521. Point mutations in eAf1521 are highlighted. The binding region is underlined. c Structural comparison of the eAf1521 (mustard) and WT Af1521 (turquoise; PDB ID: 2BFQ). d Upper panel: detailed representation of the proximal ribose of the ADPr with the point mutations Y145R and K35E in its vicinity. The C-1′-linked oxygen is shown in purple. Lower panel left: surface rendering of the WT Af1521 ADPr-binding site. The I144 side chain is highlighted. Lower panel right: corresponding view for the eAf1521 macro domain, with the I144 side chain rotated to entrap the diphosphate moiety. e Pull-down experiment using unmodified and ADP-ribosylated H2B peptides (biotinylated and bound to streptavidin Sepharose beads) of WT Af1521-K35E-Y145R, eAf1521-E35K-R145Y, and eAf1521-I144G under increasing salt concentration. Input lanes are the same for all measured conditions. The proteins were analyzed by SDS–PAGE followed by Coomassie staining. The experiment was repeated independently (n = 2) with similar results. Source data are provided as a Source data file.