Extended Data Fig. 6: Purification of the cADPR isomer from ThsB′ TIR domain and comparison to 1′′–2′ gcADPR.
From: Viruses inhibit TIR gcADPR signalling to overcome bacterial defence
a, Purified ThsB′ (methods) was incubated with NAD+ and the reaction products were filtered and analyzed by HPLC. b, Y-axis zoom of (a). The predicted ThsA-activating ThsB′ reaction product (ThsB′ cADPR isomer) is indicated with an arrow. c, HPLC analysis of the ThsB′ cADPR isomer reaction after addition of cmTad1 followed by concentration and heat denaturation demonstrates that cmTad1 is able to purify and enrich the predicted ThsB′ cADPR isomer peak. This peak was further isolated by HPLC fractionation. d, HPLC analysis shows that the cmTad1-purified ThsB′ cADPR isomer migrates as a unique peak with a small amount of residual NAD+. ThsB′ cADPR isomer is distinct from 1′′–2′ gcADPR and separates from relevant molecule standards at pH = 6.8. (e-f) ThsA NADase activation curves of 1′′–2′ gcADPR (e) and 1′′–3′ gcADPR (f). ThsA activation is ~50–100× more sensitive to 1′′–3′ gcADPR than 1′′–2′ gcADPR.
