Fig 2: MD simulation of the interactions of native S100A9 with OleA. (A) Binding of one OleA molecule per subunit of S100A9 dimer. S100A9 monomers are shown by the ribbon diagram in yellow and green colors, respectively. The amino acid side chains of S100A9, which form binding interactions with OleA, are shown by cyan sticks. OleA molecules are shown by magenta sticks. OleA forms hydrogen bonds with Glu 52 and Arg 85 and π–π stacking interactions with Trp 88. (B) B-factor presentation of the S100A9 dimer backbone shows that OleA (in black sticks) binds to the protein site with low mobility (shown in blue). The loops with high mobility are shown by thicker tubes with a color gradient from yellow to red, corresponding to increasing mobility. (C) S100A9 dimer surface presentation in space filling and colors based on the polarity of the amino acid residues: hydrophilic residues, blue; hydrophobic residues, brown; residues with intermediate properties, white. OleA (shown by sticks) binds to a shallow hydrophobic cavity on the S100A9 surface. (D) Detailed presentation of the OleA orientation and binding interactions (presented in yellow) highlighting: π–π stacking between Trp 88 and OleA, hydrogen bonding between Arg 85 and the carbonyl oxygen of OleA, and the proximity between Glu 52 and the ester group of OleA. (E) Number of hydrogen bonds between each OleA molecule and S100A9 dimer. OleA molecules dissociate when the number of hydrogen bonds falls to zero. Hydrogen bonds were defined as the interactions between the polar groups with an angle of less than 20° and less than 3.0 Å distance. (F) Root mean square deviation (RMSD) values demonstrating the mobility of each OleA molecule and S100A9 dimer. Ligand dissociation results in the rapid increase in the RMSD values.

Fig 3: Cytotoxic effect of the S100A9 amyloids in the SH-SY5Y cells is mitigated by OleA. (A) Viability of SH-SY5Y cells measured by the MTT assay and (B) ROS levels in the SH-SY5Y cells measured by DCFDA fluorescence. Cells were exposed to 20 μM S100A9 samples for 24 h. S100A9 was added in either native or amyloid forms, which were incubated for 24 and 48 h in PBS, pH 7.4, at 42 °C, respectively, prior the addition to the cells. S100A9 to OleA molar ratios were 1:0, 1:1, 1:2, 1:5, and 1:10 as indicated along x-axis. The control corresponds to the untreated cells and is shown by a single black bar, where the absence of S100A9 and OleA is indicated as (0:0). The experimental bars corresponding to the addition of native S100A9 are shown in black, the addition of 24 h aged S100A9 amyloids are shown in gray, and the 48 h aged amyloids are shown in white. Error bars indicate the standard error of the mean of the independent experiments carried out in triplicate. ***p < 0.001 and **p < 0.01 versus control (0:0). °p < 0.05 and °°p < 0.01 versus cell viability in the presence of native or the corresponding aggregated S100A9 without OleA, i.e., molar ratio (1:0). (C) Confocal microscopy imaging of the intracellular free Ca2+ levels in the SH-SY5Y cells exposed for 10, 30, and 60 min to 20 μM S100A9 amyloids incubated for 48 h in the absence or in the presence of OleA at the molar ratios of S100A9 to OleA indicated in the figure. Scale bars are 14 μm in all images.

Fig 4: Inhibition of S100A9 amyloid formation in the presence of increasing OleA concentrations observed by the ThT fluorescence assay and AFM. (A) S100A9 amyloid kinetics in the presence of increasing OleA concentrations monitored by the ThT assay. (B) Fitting of the initial parts of S100A9 amyloid kinetics with the nucleation-dependent polymerization model. (C) Ratios of the effective rate constants of S100A9 amyloid kinetics in the presence and absence of OleA (B0 is the effective rate constant in the absence of OleA, and B(c) is the rate constant at an OleA concentration of c). (D) Ratios of plateau intensities of S100A9 amyloid kinetics in the presence and absence of OleA (I0 is the plateau intensity in the absence of OleA, and I(c) is the plateau intensity at an OleA concentration of c). AFM images of S100A9 amyloid species without OleA and with increasing OleA concentrations after a 6 h (E–H) and 48 h (I–L) incubation, respectively. Amyloid fibril length distributions without OleA and with increasing OleA concentrations after a 6 h (M–P) and 48 h (Q–S) incubation, respectively, measured in AFM images. (T) Median length of S100A9 amyloid fibrils after a 6 h (black circles) and 48 h (red triangles) incubation, respectively, with increasing OleA concentrations. Molar ratios of S100A9 to OleA and the time of incubation are indicated in the figures. 100 µM S100A9, PBS, pH 7.4, and 42 °C. z-scale in the AFM images is indicated on the right with the bar color gradient from dark brown to light yellow. x,y-AFM scan sizes are 2 × 2 µm.

Fig 5: Far-UV CD spectra of native and amyloid S100A9 and the OleA effect on them. Spectra were recorded at 25 °C at the following conditions: 75 µM S100A9 in all samples and 1:2 S100A9 to OleA molar ratio, if OleA is added. PBS, pH 7.4. S100A9 fibrils were formed during the 48 h incubation at 42 °C. Labeling of the samples is shown in the legend.