Fig 1.
Interactions of EGF with mS100A4 monitored using fluorescence spectroscopy.
(A) Fluorescence emission spectra of EGF illustrating the changes in the intrinsic tryptophan fluorescence that occur with increasing concentrations of mS100A4 in the micromolar range. (B) Changes in the fluorescence intensities measured at 345 nm as a function of the mS100A4 concentration were calculated according to Eq 1. The binding was fitted (solid red line) to a one-site binding model.
Fig 2.
Analysis of the 1H-15N HSQC Spectra of mS100A4 in Complex with EGF.
(A) Overlaid 1H-15N HSQC spectra of 0.5 mM 15N-labeled free mS100A4 (red) and mS100A4: EGF, molar ratio = 1:0.3 (green), 1:0.6 (blue), and 1:1 (white), with the intensity drop indicated by green boxes. (B) Bar graph representing the changes in the cross-peak intensities (I/I0) of free mS100A4 and mS100A4 in complex with EGF versus the mS100A4 residue number (1–101). In this plot, (I) represents the cross-peak intensity of mS100A4 in complex with EGF, and (I0) represents the initial intensity of free mS100A4. The black dashed line indicates the threshold of selected residues that exhibited a significant decrease in intensity.
Fig 3.
Analysis of the 1H-15N HSQC Spectra of EGF in Complex with mS100A4.
(A) Overlaid 1H-15N HSQC spectra of 0.5 mM 15N-labeled free EGF (red) and EGF: mS100A4, molar ratio = 1:0.3 (green), 1:0.6 (blue), and 1:1 (white), with cross-peak perturbation changes indicated by white boxes and the intensity drop indicated by blue boxes. (B) The weighted average of the chemical shift (15N and 1H) variations Δδ = [(δ1HN)2 + 0.2 (δ15N)2]1/2 of residues in EGF on complex formation with mS100A4. The horizontal line is an arbitrary line drawn to demarcate residues that exhibit significant chemical shift perturbations (>0.20 ppm).
Fig 4.
Model of the mS100A4-EGF Complex Determined using HADDOCK.
(A) The interface between mS100A4 and EGF. The hydrophobic residues of mS100A4 are yellow, and the hydrophobic residues of EGF are red. (B) Ribbon representation of the 20 best mS100A4-EGF complex overlap, with EGF as green. The mS100A4 homodimer is cyan.
Fig 5.
Analysis of the 1H-15N HSQC Spectra of mS100A4 in Complex with Amlexanox.
(A) Overlaid 1H-15N HSQC spectra of 0.5 mM 15N-labeled free mS100A4 (red) and mS100A4: EGF, molar ratio = 1:0.3 (green), 1:0.6 (blue), and 1:1 (white), with the intensity drop indicated by blue boxes. (B) The weighted average of the chemical shift (15N and 1H) variations Δδ = [(δ1HN)2 + 0.2 (δ15N)2]1/2 of residues in mS100A4 on complex formation with amlexanox. The horizontal dashed line is an arbitrary line drawn to demarcate residues that exhibit significant chemical shift perturbations. (C) Ribbon representation of mS100A4, with residues that exhibited a decrease in cross-peak intensity mapped (red) on the ribbon diagram.
Fig 6.
Model of the mS100A4-amlexanox Complex Determined using HADDOCK.
(A) The interface between mS100A4 and amlexanox. The hydrophobic residues of mS100A4 are colored red. (B) Ribbon representation of the mS100A4-EGF complex, with 10 overlaid amlexanox structures. The mS100A4 homodimer is blue.
Fig 7.
(A) A431 cells were treated with 10 nM EGF, 10 nM EGF + 5 μM mS100A4, 10 nM EGF + 25 μM mS100A4, 10 nM EGF + 50 μM mS100A4, or 10 nM EGF + 100 μM mS100A4, and cell proliferation was assessed using the WST-1 assay. The relative cell counts after treatment with mS100A4 are plotted as the fold induction, with serum-free medium and AG1478 as the controls (lanes 7–11). The data are expressed as the mean ± SD of 3 independent experiments. (B) Effects of amlexanox on mS100A4-mediated cell proliferation and EGFR signaling. A431 cells were treated with 10 nM EGF, 10 nM EGF + 100 μM mS100A4, 10 nM EGF + 100 μM mS100A4 + 5 μM amlexanox, 10 nM EGF + 100 μM mS100A4 + 10 μM amlexanox, or 10 nM EGF + 100 μM mS100A4 + 50 μM amlexanox. Cell proliferation was analyzed after 48 h. (C) Left panel: A431 cells were serum starved for 24 h and then incubated with or without 10 nM EGF in the presence or absence of 100 nM mS100A4 or 1 μM amlexanox for 1 h. The cell lysate was extracted from each treatment, and 1 mg of cell lysate was immunoprecipitated with an anti-EGFR antibody (Biovision). The amounts of immunoprecipitated EGF and EGFR were examined by Western blotting (upper plot). The amounts of phosphorylated and total EGFR in cell lysate were subsequently detected by Western blotting. Alpha-tubulin was used as an internal control (lower plot). Right panel: The quantitative result of EGFR-associated EGF was shown from two independent experiments.