Fig 1.
Integrative structural biology study of CagI.
A) Size exclusion chromatograms (A280) of CagI, CagIN and CagIC. MALS weight-averaged molar masses are indicated as dotted lines. B) Schematic representation of CagI predicted secondary structures (top) and cartoon representation of Alpha Fold (AF) model of the CagI monomer with helices coloured as in the schematic view. C) Comparison of CagI dimer theoretical SAXS curve with experimental curve. D) Cartoon depiction of the AF model of CagI dimer coloured as in A) fitted in the SAXS envelope obtained with DAMMIF. E) Comparison of CagIC and CagL (PDB ID: 4YVM) depicted as cartoon. CagI is coloured as in A). CagL secondary structure elements equivalent of those of CagI are coloured accordingly. Cysteine residues involved in disulfide bridges are coloured in dark blue and displayed as ball-and-stick.
Fig 2.
DARPin interactions with CagI.
A) Pull down assays of purified untagged CagI (top panels), CagIC (middle panels) or CagIN (bottom panels) with NTA bead-immobilized His8-tagged DARPins. “I” denotes input protein and “E” denotes elution. In control experiments proteins were mixed with the resin in the absence of DARPin and were not detected in the elution fraction. B) Representative SPR experiments using single-cycle injection mode on CM5 chips coated with CagI or with C) CagIC. DARPins K5 (green curves), K2 (orange curves) or K8 (red curves) were injected on the chips at increasing concentrations as follows. For full-length CagI experiments, concentrations were 0.5, 2.5, 12.5, 62.5 and 312.5 nM for K5 and K8. For K2, concentrations were 1, 3, 9, 27 and 81 nM. For CagIC experiments, K2 and K8 were injected at 0.05, 0.15, 0.45, 1.35 and 4 nM. For K5 concentrations used were 0.11, 0.33, 1, 3 and 9 nM. Fit curves obtained with binding model 1:1 are shown as dashed lines.
Table 1.
Dissociation constant (KD) expressed in nM obtained in Surface Plasmon Resonance experiments with immobilized CagI or CagIC and the DARPins as analytes.
Values were obtained using the 1:1 binding model (mean of two separate experiments except for values labeled with a *, for which a single multi-injection experiment was performed).
Fig 3.
Structures of CagI:DARPin complexes.
A) Overview of the structure of CagI:K2 and CagI:K5 complexes. The two structures of DARPin K2 (wheat) and K5 (slate) have been superimposed and are depicted as cartoons. The CagI molecule is displayed as cartoon and surface coloured according to secondary structure (α4 in cyan, α5 in orange and α6 in magenta). Side chains of cysteines 272 and 283 involved in disulfide bridges are shown as ball-and-stick with atoms coloured blue (carbons) and yellow (sulfur). For clarity, only the CagI molecule from the CagI:K2 complex is displayed. B) Two rotated views of the structural superimposition of the crystal structure of CagI204-307 from the CagI:K2 complex (green) and corresponding region of the AF model (grey) displayed as cartoon. The side chains of the cysteine residues 272 and 283 forming the disulfide bond are displayed as ball-and-stick with sulfur atoms coloured in yellow. C) Detailed view of the interface between DARPin K5 loops and CagI α5 with side chains involved in hydrogen bonds shown as ball-and-stick with atoms coloured as follows: nitrogen in blue, oxygen in red, carbon coloured as in A). Grey dashed lines indicate hydrogen bonds. D) Close-up view of the interface of CagI:K5 (left panel) and CagI:K2 (right panel) showing residues F125 in K2 and L125 in K5 binding to the CagI groove.
Table 2.
Data collection and refinement statistics.
Fig 4.
Structural basis for higher affinity of DARPin K2 on CagI.
A) Overview of the structures of the CagI:K2 and CagI:K5 complexes interfaces. The two DARPin structures K2 (wheat) and K5 (blue) have been superimposed and are depicted as cartoons. The CagI molecule and symmetry related CagI’ are displayed as cartoons and surfaces are coloured as in Fig 3. For clarity, only CagI molecules from the CagI:K2 complex are displayed. B) detailed view of region 2 interactions between DARPin K5 loop residues and CagI α4 and α6 with involved side chains displayed as ball and sticks with atoms coloured as follows: nitrogen in blue, oxygen in red, carbon as in A). Dashed lines indicate hydrogen bonds. C) SEC-MALS measurements of the CagI:K2 and CagI:K5 purified complexes.
Fig 5.
CagA translocation inhibition by DARPins.
A) H. pylori P12 [TEM-1-CagA] was co-incubated for 2.5 h with AGS cells in the absence or presence of the indicated DARPins at a concentration of 5 μM, and CagA translocation was determined by a TEM-1-CagA translocation assay. As controls, the secretion-deficient mutant P12ΔcagT [TEM-1-CagA] was used without pre-treatment, or P12 [TEM-1-CagA] was pre-incubated for 30 min with 100 μM cisplatin. Data are indicated in relation to untreated control, which was set to 100%, and they represent mean values and standard deviations of five independent experiments. (One-way ANOVA; Tukey post-hoc test; **, p<0.01; ***, p<0.001). B) H. pylori P12 [HiBiT-CagA] was either left untreated or pre-treated for 30 min with 5 μM of the indicated DARPins or 100 μM cisplatin in PBS/10% FCS at 37°C, 10% CO2, and the bacterial suspensions were used to infect AGS [LgBiT] cells for 2.5 h. Luminescence values were recorded and normalized to untreated control. Data are indicated as mean values with standard deviations resulting from four independent experiments. (One-way ANOVA; Tukey post-hoc test; *, p<0.05; ***, p<0.001). C) IL-8 levels were determined in AGS cell supernatants 4h after infection with P12 wild-type (wt), P12ΔcagT or P12 wt in the presence of 100 μM cisplatin, K5, K8 or K2 DARPins (5 μM). IL-8 concentrations were normalized to untreated P12 wt control, and are indicated as mean values and standard deviations of six independent experiments. (One-way ANOVA; Tukey post-hoc test; ***, p<0.001). D) Cell binding measurements by flow cytometry with H. pylori P12 [pHel12::gfp] pre-treated with DARPins at 5 μM, cisplatin at 100 μM, or left untreated. Data shown are mean values and standard deviations of median fluorescence intensities (MFI) normalized to untreated bacteria, resulting from three independent experiments.
Fig 6.
Cell binding and spreading to cagT4SS proteins and domains.
A) Dose-dependent AGS cell adhesion to CagL, CagI, CagIC and CagIN. Multiwell plates were coated with different amounts of the proteins as indicated on the Figure. Each assay point was derived from triplicate measurements. B) Representative images of adhered AGS cells on well-surfaces coated with 0.15 μg of indicated proteins. C) Effect of DARPin K2 or D) K11 on adhesion of AGS cells to CagL, CagI, CagIC. Multiwell plates were coated with 4 μg of each protein. After saturation with 1% BSA, the wells were incubated with 50 μL of the indicated concentration of K2 or K11 for 1 h at room temperature, and the cells were seeded in the presence of the inhibitor. The extent of adhesion was measured as previously described and expressed as percentage of adhesion to each protein in the absence of the inhibitor. Each assay point was derived from triplicate measurements.