S100B as an antagonist to block the interaction between S100A1 and the RAGE V domain

<div><p>Ca2⁺-binding human S100A1 protein is a type of S100 protein. S100A1 is a significant mediator during inflammation when Ca²⁺ binds to its EF-hand motifs. Receptors for advanced glycation end products (RAGE) correspond to 5 domains: the cytoplasmic, transmembrane, C2, C1, and V domains. The V domain of RAGE is one of the most important target proteins for S100A1. It binds to the hydrophobic surface and triggers signaling transduction cascades that induce cell growth, cell proliferation, and tumorigenesis. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction between S100A1 and the RAGE V domain. We found that S100B could interact with S100A1 via NMR ¹H-¹⁵N HSQC titrations. We used the HADDOCK program to generate the following two binary complexes based on the NMR titration results: S100A1-RAGE V domain and S100A1-S100B. After overlapping these two complex structures, we found that S100B plays a crucial role in blocking the interaction site between RAGE V domain and S100A1. A cell proliferation assay WST-1 also supported our results. This report could potentially be useful for new protein development for cancer treatment.</p></div

Analysis of the ¹H–¹⁵N HSQC spectra of S100B in complex with the unlabeled S100A1.

<p>(A) Overlapped NMR HSQC spectra of ¹⁵N-S100B in black and ¹⁵N labeled S100B complex with unlabeled S100A1 in a 1:1M ratio (red). Residues displaying significant intensity decreases and those with perturbed chemical shifts are framed by boxes (green and blue, respectively).</p> <p>(B) Bar plot showing the changes in the intensity ratio (I/I0) comparing the ¹⁵N S100B and the complex of ¹⁵N S100B with S100A1. The blue line shows the threshold of selected residues that display a notably reduced intensity.</p> <p>(C) Bar plot presenting the changes in chemical shift perturbation comparing free ¹⁵N S100B and the ¹⁵N S100B in complex with Ca²⁺-bound S100A1. The blue line shows the threshold of selected residues that display a notable perturbation.</p> <p>(D) Ribbon (stick) diagram of the S100B in red obtained using the PyMOL program. The residues that show chemical shift perturbations and intensity decreases are shown in blue.</p

Analysis of the ¹ H–¹⁵N HSQC spectrum of the RAGE V domain with unlabeled S100A1.

<p>(A) The superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N V domain in black and ¹⁵N V domain complex with S100A1 in red are in a molar ratio of 1:1. Cross-peaks displaying significantly decreased intensity are shown in green boxes.</p> <p>(B) Bar graph analysis representing the intensity change (I/I₀) of cross-peaks of ¹⁵N V domain to ¹⁵N V domain complex with S100A1 versus residue numbers of RAGE V domain (24–128). I₀ denotes the initial intensity of ¹⁵N V domain, and I denotes the intensity of ¹⁵N V domain complex with S100A1. The blue line represents the criterion of selected residues that exhibit significantly decreasing cross-peak signals, and the chosen residues are indicated by red bars.</p> <p>(C) Ribbon representing the structure of V domain and residues that exhibit significantly decreasing cross-peak signals are mapped on the structure in blue.</p

Analysis of the ¹H–¹⁵N HSQC spectra of S100A1 in complex with the unlabeled RAGE V domain.

<p>(A) Superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N S100A1 (in black) and ¹⁵N S100A1 in complex with unlabeled RAGE V domain (red). Cross-peaks display significantly decreased intensity, and those with perturbed chemical shifts are boxed in green and blue, respectively.</p> <p>(B) Bar plot showing the chemical shift variations in cross-peaks for S100A1 and the S100A1-V domain complex. The threshold (blue line) of the chosen residues (red) displays a notable perturbation.</p> <p>(C) Bar graph analysis representing the change in intensities (I/I₀) of cross-peaks of free ¹⁵N S100A1 to ¹⁵N S100A1 in complex with V domain against residue numbers of S100A1 (1–93). I₀ denotes the initial intensity of free ¹⁵N S100A1, and I denotes the intensity of ¹⁵N S100A1 in complex with V domain. The blue line represents the criterion of selected residues that exhibit a significantly decreasing cross-peak signal, and the chosen residues are displayed in red bars.</p> <p>(D) Ribbon representing the structure of homo-dimer. The monomers of S100A1 homodimer are colored in green and blue.</p> <p>(E) A monomer of S100A1 and residues that exhibit significantly decreasing cross-peak signals are mapped on the structure in magenta.</p

Analysis of WST-1 assays.

<p>A. SW-480 cells were treated with 10 nM, 50 nM, or 100 nM S100 A1 (blue); 100 nM S100 A1 + 100 nM S100B (green). Cell proliferation was analyzed using a WST-1 assay. The relative cell counts following treatment with S100A1 are plotted as the fold induction with serum-free media as the corresponding controls (red). The data are expressed as the mean ± SD from 4 independent experiments.</p

Curve with fluorescence intensity changes versus S100B concentrations obtained at a wavelength of 351 nm.

<p>K_d is calculated as 1.9 ±0.1 μM from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190545#pone.0190545.e005" target="_blank">Eq 4</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190545#pone.0190545.ref083" target="_blank">83</a>].</p

Hypothetical mechanism of the S100A1-RAGE signaling pathway.

<p>The extracellular V domain of the RAGE protein binds to the S100A1 dimer, facilitating intracellular dimerization of the cytoplasmic domain. This activates a signaling cascade (MAP kinase, NF-κB, and AP-1), leading to downstream cell proliferation or inflammation.</p

Superimposition of the complex between S100A1 (monomer in green) and the V domain (yellow), with the complex between S100A1 and S100B (red).

<p>It is demonstrated that S100B blocks the interaction between S100A1 and the V domain.</p

Analysis of the ¹H–¹⁵N HSQC spectra of S100A1 in complex with the unlabeled S100B.

<p>(A) Overlapped NMR HSQC spectra of ¹⁵N-S100A1 in black and ¹⁵N S100A1 complex with unlabeled S100B in a 1:1 ratio (red). Residues displaying significant intensity decreases and perturbed chemical shifts are shown by boxes (green and blue, respectively).</p> <p>(B) Bar plot presenting the changes in cross-peak chemical shift of free-form S100A1 and the S100A1-S100B complex. The blue line shows the threshold of selected residues (red) that display a notable perturbation.</p> <p>(C) Bar plot presenting the decrease in the cross-peak intensity ratio (I/I₀) of S100A1 and the S100A1-S100B complex. The threshold (blue line) of chosen residues indicates a notably reduced intensity. Selected residues are highlighted in red.</p> <p>(D) Ribbon (stick) diagram of S100A1 in green obtained using the PyMOL program. The residues that exhibit chemical shift perturbations and intensity decreases are shown in magenta.</p

Complex structure of S100A1 and RAGE V domain.

<p>(A) Ribbon diagram of the S100A1-V domain complex. Two monomers of S100A1 are shown in green and blue; the two RAGE V domains are shown in yellow. Interacting residues are shown in magenta and red.</p> <p>(B) Ribbon diagram of the S100A1-V domain complex showing with different angle.</p