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Extended Data Fig. 6 | Mutagenesis analysis of bacterial STING cyclic
dinucleotide recognition and TIR activation. a, Table of bacterial STING
cyclic dinucleotide contacts tested with mutagenesis analysis. Residues were
selected according to contacts observed in the structure of the FsSTING–3′,3′-
cGAMP complex (FsSTING residues listed in parentheses) and tested in
SfSTING to allow analysis of the effect on both c-di-GMP binding and NADase
activity. b, Plate reader analysis of mutant SfSTING NAD+ cleavage activity in
the presence of 500 nM protein and increasing c-di-GMP concentration.
Mutant SfSTING variants with cyclic-dinucleotide-binding pocket mutations
require 10–1,000× greater c-di-GMP concentration for NADase activation. Data
are ± s.d. of n = 3 technical replicates and are representative of 3 independent
experiments. c–e, EMSA analysis demonstrating that SfSTING R234A and
D259A mutations reduce stable c-di-GMP complex formation compared
to SfSTING wild type (Fig. 2d). SfSTING(D259A) protein titration and
quantification confirms significantly reduced affinity for c-di-GMP. Data are
representative of three independent experiments. f, g, HPLC and plate reader
analysis of mutant SfSTING NAD+ cleavage activity in the presence of 500 nM
protein and 10 μM c-di-GMP (HPLC) or 500 nM protein and ± 20 μM c-di-GMP
(plate reader). Mutation of residues responsible for ligand recognition in the
cyclic-dinucleotide-binding domain (R234 and D259) and catalysis in the TIR
enzymatic domain (E84) disrupts SfSTING NADase activity and explains loss of
E. coli toxicity observed in Fig. 3d. One hundred μM of c-di-GMP was used for
SfSTING(D259A) plate reader NAD+ cleavage analysis to confirm complete loss
of c-di-GMP-induced activation. HPLC data are representative of three
independent experiments. Plate reader data are ± s.d. of n = 3 technical
replicates and are representative of 3 independent experiments. h, i, Analysis
of SfSTING toxicity in E. coli cells expressing normal c-di-GMP signalling
enzymes with and without nicotinamide (NAM) supplementation. NAM
supplementation is sufficient to partially alleviate SfSTING-wild-type-induced
NADase toxicity. Each line represents the average of two technical replicates
for each of four separately outgrown colonies. Data are representative of two
independent experiments.