Fig. 5: The ART-domain has a distorted catalytic centre and has no ADP-ribosyltransferase activity on cell lysates.

a The ART-domain of conformation I (residues 3057–3223) is shown in cartoon representation (the ART-domains of all three conformations together are shown in Supplementary Fig. 14a) The residues are coloured in rainbow from the N-terminus (blue) to the C-terminus (red). The C-terminus occupies the gap between the ß-sheets and is hydrogen bonded (dotted line) with T3132. This gap is the NAD⁺ binding site in active ADP-ribosyltransferases of the R-S/T-E type. The residues R-T-E of the ART-domain are shown in magenta. These residues did not form an intact catalytic centre with R3107 pointing away from the centre. b Western blot analysis of ADP-ribosylation with anti-pan-ADP-ribose binding reagent (refer to Supplementary Fig. 14 for the Western blot together with the Ponceaus S loading control). HEK293T cell lysates (lanes 1–7 and 9) were incubated with NAD⁺ (lanes 2–7 and 9) in the presence of LifA (lanes 3 and 6), the ART-domain (lanes 4 and 7) or Iota Ia (lane 9). Lane 8 shows the ART-domain alone and M is the molecular weight standard. The molecular weights are indicated on the left. Samples were taken after 1 h (lanes 2–4 and 9) and after 20 h (lanes 5–7) at 30 °C. HEK-cell lysates showed NAD⁺ dependent ADP-ribosylation probably due to cellular ADP-ribosyltransferases. Addition of LifA (lanes 3 and 6) or the ART-domain (lanes 4 and 8) did not affect the ADP-ribosylation pattern. In contrast, addition of the Iota Ia, a known ADP-ribosyltransferase that acts on actin showed increased ADP-ribosylation after 1 h at 30 °C with a prominent band (black arrowhead) at the expected molecular weight of actin. The ART-assay was repeated three times independently (n = 3). Source data are provided as a Source Data file.