Calcium-Bound S100P Protein Is a Promiscuous Binding Partner of the Four-Helical Cytokines

Abstract

S100 proteins are multifunctional calcium-binding proteins of vertebrates that act intracellularly, extracellularly, or both, and are engaged in the progression of many socially significant diseases. Their extracellular action is typically mediated by the recognition of specific receptor proteins. Recent studies indicate the ability of some S100 proteins to affect cytokine signaling through direct interaction with cytokines. S100P was shown to be the S100 protein most actively involved in interactions with some four-helical cytokines. To assess the selectivity of the S100P protein binding to four-helical cytokines, we have probed the interaction of Ca2+-bound recombinant human S100P with a panel of 32 four-helical human cytokines covering all structural families of this fold, using surface plasmon resonance spectroscopy. A total of 22 cytokines from all families of four-helical cytokines are S100P binders with the equilibrium dissociation constants, Kd, ranging from 1 nM to 3 µM (below the Kd value for the S100P complex with the V domain of its conventional receptor, receptor for advanced glycation end products, RAGE). Molecular docking and mutagenesis studies revealed the presence in the S100P molecule of a cytokine-binding site, which overlaps with the RAGE-binding site. Since S100 binding to four-helical cytokines inhibits their signaling in some cases, the revealed ability of the S100P protein to interact with ca. 71% of the four-helical cytokines indicates that S100P may serve as a poorly selective inhibitor of their action.

Interaction of S100A6 Protein with the Four-Helical Cytokines

“…An analysis of the free energy changes upon the S100A6-cytokine interactions (Figure 5), ∆G, shows that the average S100A6 affinities for the cytokines of different SCOP families in the fold decrease in the following order: short-chain cytokines > long-chain cytokines > interferons/IL-10 (∆G of −49.8 kJ/mol < −47.1 kJ/mol < −46.8 kJ/mol). The same tendency was observed previously for the S100P protein [47]. 2).…”

“…An analysis of the distribution of the predicted contact residues of the S100A6 dimer along its amino acid sequence within the models of its complexes with the four-helical cytokines (Figure 6B) shows that cytokines of the different structural families share the contact surfaces of S100A6, including Nand C-termini, helix α1, the 'hinge' region, and helices α3 and α4. These regions of the S100A6 dimer form a well-defined cytokine-binding site located between its subunits (Figure 6A), which is remarkably similar to that previously predicted for S100P interaction with the four-helical cytokines [47]. The qualitative difference between the predicted behavior of the S100A6 and S100P proteins is the more complete involvement in cytokine recognition of the helix α3 and the N-terminal half of helix α4 of S100A6.…”

“…The ClusPro docking server [59] was used to generate the models of the tertiary structures of the S100A6-cytokine complexes, according to [43,47]. The tertiary structure of the Ca 2+ -bound S100A6 dimer was taken from PDB [60] entry 1K9K (X-ray, chains A and B [61]), while the structures of the cytokines were either extracted from PDB, or predicted via AlphaFold2 (https://alphafold.ebi.ac.uk/; accessed on 1 June 2023 [62]) (Table S2).…”

“…We have studied the affinity of the Ca 2+ -loaded (1 mM CaCl 2 ) recombinant human S100A6 protein to the panel of 35 recombinant human four-helical cytokines (including the panel used in [47], extended with Flt3L, SCF, and IL-19), which covers all structural families in this fold (Table S1). The cytokines were immobilized on the surface of the SPR sensor chip via amine coupling, and 61 nM-8 µM solutions of S100A6 were passed over the surface at 25 • C. Whereas nine cytokines (Table S3) did not reveal noticeable effects in response to the S100A6, the SPR sensograms for 26 cytokines showed the S100A6 concentration-dependent effects, characteristic of association-dissociation processes (Figures 1-4).…”

“…The cytokines were immobilized on the surface of the SPR sensor chip via amine coupling, and 61 nM-8 µM solutions of S100A6 were passed over the surface at 25 • C. Whereas nine cytokines (Table S3) did not reveal noticeable effects in response to the S100A6, the SPR sensograms for 26 cytokines showed the S100A6 concentration-dependent effects, characteristic of association-dissociation processes (Figures 1-4). The kinetic SPR data were adequately described within the heterogeneous ligand model (1) (Figures 1-4, Table 2), which was earlier successfully used for the description of the S100-cytokine interactions [25,[43][44][45][46][47]63,64]. The data for IL-2 were described via the one-site binding scheme.…”