Extended Data Fig. 7: Activity and oligomerisation of TIR mutants.
From: Cyclic nucleotide-induced helical structure activates a TIR immune effector
a, Dynamic Light Scattering analysis confirms the oligomerisation of Y115A and D45AL46A in presence of 1:1.5 protein:cA3 molar ratio. Experiment in technical triplicates for each condition. b, NADase activity comparison of the Y115A and D45AL46A mutants with the WT for a 3-fold serial dilution in protein concentrations (0.16, 0.5, 1.5, 4.5, 13.5 µM). 27 µM cA3 was used to activate TIR-SAVED incubated with 500 µM ɛNAD+ substrate. c, Enzymatic properties of Y115A mutant. Based on a NAD range concentration experiment, the initial rate of fluorescent ADP ribose production was calculated and fitted following a Michaelis-Menten model. In these experiments, as used previously for the WT, 0.5 µM Y115A was mixed in presence of 1 µM cA3 to hydrolyse 25, 75, 225, 500, 1000, 1500, 2000 and 3000 µM ɛNAD+. The right panel compares the final Michaelis-Menten parameters of Y115A with the WT protein. Data are the means of three experiments with Standard deviation indicated.
