The intrinsically disordered protein TgIST from Toxoplasma gondii inhibits STAT1 signaling by blocking cofactor recruitment

Signal transducer and activator of transcription (STAT) proteins communicate from cell-surface receptors to drive transcription of immune response genes. The parasite Toxoplasma gondii blocks STAT1-mediated gene expression by secreting the intrinsically disordered protein TgIST that traffics to the host nucleus, binds phosphorylated STAT1 dimers, and occupies nascent transcription sites that unexpectedly remain silenced. Here we define a core region within internal repeats of TgIST that is necessary and sufficient to block STAT1-mediated gene expression. Cellular, biochemical, mutational, and structural data demonstrate that the repeat region of TgIST adopts a helical conformation upon binding to STAT1 dimers. The binding interface is defined by a groove formed from two loops in the STAT1 SH2 domains that reorient during dimerization. TgIST binding to this newly exposed site at the STAT1 dimer interface alters its conformation and prevents the recruitment of co-transcriptional activators, thus defining the mechanism of blocked transcription.


Supplementary Figure 1
The interaction between the secreted TgIST and STAT1 is IFN-g dependent. Western blot analysis of host proteins following immunoprecipitation (IP) of TgIST-Ty from U3A (STAT1-null) or U3A-STAT1 (STAT1 complemented) cells that were infected with Toxoplasma (vs. mock) for 17 hr, all without IFN-g treatment.
Two core components of the Mi-2/NuRD complex, metastasis-associated protein (MTA1) and histone deacetylase 1 (HDAC1), were co-precipitated with TgIST-Ty. TATA-binding protein (TBP) was used as a negative control. Representative blots of two independent experiments with similar results were shown here. for 24 hr were activated with IFN-g for 6 hr followed by staining for GFP (Alexa Fluor 488, green), STAT1 (Alexa Fluor 568, red) and DAPI (blue). Scale bar = 10 μm. Representative micrographs of two independent experiments with similar results were shown here. (b) Quantification of STAT1 in TgIST-expressing (green, GFP + ) cells. The intensity of STAT1 were separately measured for GFP + and GFPcells, then normalized to GFP-cells. One-way ANOVA by Dunnett's test for multiple comparisons was used to compare expression divergency between samples. Data presented as mean + s.d. from two independent experiments. The top of the bar represents the mean, and the error bars represent the standard deviation. (c) Western blot analysis of cells lysates from transfected HeLa cells in (a). Equal amounts of total protein were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with corresponding primary antibodies as indicated. Blots for a-Tublin and TATA-binding protein (TBP) were used as controls for the cytoplasmic and nuclear fractions, respectively. Representative blots of two independent experiments with similar results were shown here.

Supplementary Figure 4
Identification of the core STAT1 binding sequence in TgIST by limited trypsinization and mass spectrometery. (a) Purified TgIST-T2 complexed with STAT1cc was diluted to 10 µg in a 50 uL reaction volume. Dilutions of trypsin (1 mg/ml) from 1:20 to 1:1,280 (vol/vol %) were added to the TgIST-T2-STAT1cc complex and incubated for 5 min (5'), 10 min (10') or 15 min (15') as indicated. Reactions were stopped by addition of SDS sample buffer, followed by separation of samples by SDS-PAGE using 12% (the left and center gels) or 15% (the right gel) acrylamide gels. Resistant bands (numbered S1 -S6) from the samples treated with a 1:160 dilution of trypsin were cut from the gel and subjected to MS/MS analysis.
Representative gel image of two independent experiments with similar results were shown here. (b) Limited proteolysis and mass spectrometry (MS) analysis identified core regions in the repeats of TgIST that were protected by interaction with STAT1. Purified TgIST-T2 complexed with STAT1cc was treated with trypsin and resistant bands were isolated from SDS-PAGE gels for MS analysis. Regions identified from MS are shown as rectangles below the amino acids sequence of each repeat. S1 through S6 refer to partial degradation patterns detected by SDS-PAGE.

Repression of IRF1 by TgIST is independent of Mi-2/NuRD interaction. Cells infected with type I (a) and
type II (b) strains of T. gondii. HFF cells were plated on cover slips, infected for 6 hr with TgIST disruptant Type I (RH) or Type II (Pru) parasites, or strain complemented with wildtype TgIST (RH or PruDTgist/TgIST) or TgIST-T1 (RH or PruDTgist/TgIST-T1, lacks Mi-2/NuRD binding domain in its C-terminus). Cells were stimulated with IFN-g (100 U/ml) for the last 18 hr, followed by staninning with a-IRF1. Scale bar = 10 µm.
Representative micrographs of two independent experiments with similar results were shown here. (a) Electron density map of TgIST-R2 bound to phosphorylated STAT1 dimer (pSTAT1d). The two proteins were purified separately and crystallized together using a molar ratio of 1:2.1 (pSTAT1d: TgIST-R2). An additional density is seen located at the top of the pSTAT1d interface formed by two flexible loops (loop 1 and loop 2). Orange mesh represents the 2Fo-Fc map contoured at 1 σ, Green mesh represents the Fo-Fc map contoured at 3 σ. Black dots represents the putative TgIST-R2 binding path between loop1 and loop 2. TgIST-R2 and mutants were tested by copurification with STATcc. Mutants in TgIST are denoted above the gel. (b) His-tagged TgIST-R2 was tested by copurification with STATcc and its mutants. Mutants of STAT1cc are denoted above the gel. Eluted fractions were separated by SDS-PAGE gel and stained with Coomassie blue. One set of representative gels are shown here. Representative gel images of three independent experiments with similar results were shown here.

Supplementary Figure 10
The SH2 domain is conserved among STAT1 from different species and western blot analysis of proteins following immunoprecipitation of TgIST-Ty from HEK293T cells. Multiple sequence alignment of the STAT1 proteins from different species (a) and the superposition of their corresponding homology models (b). (a) STAT1 protein sequences were retrieved and imported into Jalview software (https://www.jalview.org/) and the multiple sequence alignment was performed by Muscle ( https://www.ebi.ac.uk/Tools/msa/muscle/) using default settings. The sequence accession numbers of STAT1 in Uniprot database (https://www.uniprot.org/ ) after the species name to the left of each sequence in the alignment. Color schemes are as follows, blue: hydrophobic residues; red: positively charged residues; magenta: negatively charged residues; green: polar residues; pink: cysteines; orange: glycines; yellow: prolines; and cyan: aromatic residues. (b) Homology models of the STAT1 sequences described above were built using the Swiss-model server (https://swissmodel.expasy.org/ ) based on the STAT1 dimer structure (PDB: 1BF5) as the template. Models were aligned and visualized by Pymol (https://pymol.org/2/ ). (c) HEK293T cells were transfected with TgIST constructs expressing the mature form of TgIST (M2), a truncated form containing both repeat TgIST-T2 (T2); a mutant where the core 7 amino acids in both repeats have been replaced with alanine TgIST-T2-M2 (T2-M2); and a truncated version lack both repeats TgIST-T3, also see schematic in Fig. 1d and 3b). Cells were infected for 23 hr, then treated ± IFN-g (100 U/mL) for additional 60 min prior to whole cell extract preparation. Membranes were incubated with corresponding primary antibodies as indicated and then IR dye-conjugated secondary antibodies. Visualization was performed using an Odyssey infrared imager. Representative blots of two independent experiments with similar results were shown here. (d) Label-free quantification by mass spectrometry of STAT1 immunoprecipitation (IP) from TgIST transfected HEK293T cells, corresponding to Fig. 7e. The peptides were quantified using the precursor abundance based on intensity. Then proteins were scaled using total peptide amount. (e) Relative fold change of CBP/p300 calculated from (d). Relative fold change was defined by using scaled abundance of CBP+p300 to divide the abundance of STAT1 in each sample. a Statistics for the highest-resolution shell are shown in parentheses. b Rmerge = Σhkl Σi |Ihkl,i -<Ihkl>|/ Σhkl Σi Ihkl,i and Rpim = Σhkl (1/(n-1)) 1/2 Σi | Ihkl,i -<Ihkl> |/ Σhkl Σi Ihkl,i , where Ihkl,i is the scaled intensity of the i th measurement of reflection h, k, l, <Ihkl> is the average intensity for that reflection, and n is the redundancy.