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Interaction of S100A6 Protein with the Four-Helical Cytokines

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27 July 2023

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28 July 2023

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Abstract

S100 is a family of over 20 structurally homologous, but functionally diverse regulatory (calcium/zinc)-binding proteins of vertebrates. The involvement of S100 proteins in numerous vital (patho)physiological processes is mediated by their interaction with various (intra/extra)cellular protein partners, including cell surface receptors. Furthermore, recent studies revealed the ability of specific S100 proteins to affect cell signaling via direct interaction with cytokines. Previously, we have revealed binding of ca. 71% of the four-helical cytokines by S100P protein due to the presence in its molecule of a cytokine-binding site, which overlaps with the binding site for S100P receptor. Here we show that another S100 protein, S100A6 (pairwise sequence identity with S100P of 35%), specifically binds numerous four-helical cytokines. We have studied affinity of recombinant forms of 35 human four-helical cytokines covering all structural families of this fold to Ca2+-loaded recombinant human S100A6, using surface plasmon resonance spectroscopy. S100A6 recognizes 26 of the cytokines from all families of this fold with the equilibrium dissociation constants ranging from 0.3 nM to 12 µM. Overall, S100A6 interacts with ca. 73% of the four-helical cytokines studied to date with selectivity equivalent to that for S100P protein, with the differences limited to binding of Interleukin-2 and Oncostatin-M. The molecular docking study evidences presence in S100A6 molecule of a cytokine-binding site, analogous to that found in S100P. The findings argue the presence in some of the promiscuous members of S100 family of a site specific to a wide range of the four-helical cytokines. This unique feature of the S100 proteins potentially allows them to serve as universal inhibitors of signaling of the four-helical cytokines, which could be of value for reduction of severity of the disorders accompanied by excessive release of the cytokines.

Keywords:

Subject: Biology and Life Sciences - Biochemistry and Molecular Biology

1. Introduction

S100 is an evolutionary young family of structurally similar, but functionally diversified regulatory Ca²⁺-binding proteins of the EF-hand superfamily (for reviews, see [1,2,3,4]). The canonical Ca²⁺-binding motif of ‘EF-hand’ type (PROSITE [5] entry PDOC00018) is composed of a 12-residue Ca²⁺-coordinating loop flanked by two α-helices [6,7]. S100 proteins consist of a low-affinity non-classical N-terminal EF-hand motif and a high-affinity classical C-terminal EF-hand, which are connected by a flexible ‘hinge’ region [7,8]. Certain S100 proteins possess distinct Zn²⁺/Cu²⁺/Mn²⁺-binding sites [8,9]. S100 proteins are generally considered to be (homo/hetero)dimeric (except for monomeric S100G), whereas some of them tend to form higher order oligomers [2,10]. With the exception of the S100 fused-type proteins, human S100 family includes 21 members (78-113 residues; pairwise sequence identity calculated using Clustal Omega 2.1 [11] ranges from 16% to 61%), each of which has a unique set of functional activities, overall covering nearly all vital processes [1,2,3]. Some S100 proteins are associated with progression of oncological, inflammatory, autoimmune, neurodegenerative, cardiovascular, and pulmonary diseases, are used for diagnostic and prognostic purposes, and are considered as promising therapeutic targets [12,13,14,15,16,17,18]. The dysregulated expression of most S100 proteins in cancer is related to the fact that they are mainly encoded by the epidermal differentiation complex located on human chromosome 1 (locus 1q21), which is frequently rearranged in cancer [19,20]. The S100 genes located on chromosome 1 and their protein products are designated by Arabic numbers placed behind the ‘S100A’ (e.g., S100A1), while S100 genes originating from other chromosomes carry the stem symbols ‘S100’ followed by a single letter (e.g., S100P) [20,21]. The multifunctionality of S100 proteins is due to their tissue/cell-specific expression, ability to localize in the cytosol/nucleus/extracellular space, metal-binding properties, post-translational modifications, and propensity to recognize a wide range of targets, including enzymes, transcription factors, receptor/membrane proteins, lipids, and nucleic acids [1,2,3,22].

Despite the lack of a leader sequence, some of S100 proteins are secreted in a cell-dependent manner via the classical endoplasmic reticulum (ER)/Golgi pathway or several unconventional passive and active mechanisms [2,23,24]. When released into the extracellular space, certain S100 proteins exert a cytokine-like action due to interaction with specific cell surface receptors, such as RAGE, TLR4, ErbB1, ErbB3, ErbB4, IL-10R, integrin β1, neuroplastin-β, 5-HT_1B, 4-HT₄, SIRL-1, ALCAM, EMMPRIN, CD33, CD36, CD68, CD69, CD146 [2,22,25,26,27,28,29,30,31,32]. Moreover, some of the released S100 proteins interact with cytokines. For instance, S100A4 binds ErbB1/ErbB4 ligands [31,33], S100A13 interacts with IL1α/FGF1 [34,35], S100B binds FGF2 [36], S100A11/A12/A13 interact with soluble form of TNF [37]. Several S100 proteins recognize four-helical cytokines: S100A2/A6/P bind EPO [38], S100A1/A4/A6/B/P interact with IFN-β [22,39,40], distinct subsets of S100A1/A6/B/P bind to specific IL-6 family cytokines (IL-11, OSM, CNTF, CT-1, and CLCF1 [41]), whereas S100P binds 29 four-helical cytokines [42]. The S100 binding has been shown to affect the cytokine signaling in some cases: S100A4 enhanced the amphiregulin-mediated proliferation of embryonic fibroblasts [33], S100B inhibited FGF2-induced increase in proliferation of MCF-7 and MDA-MB468 cells [36], S100A12/A13 rescued Huh-7 cells from the cytotoxic effect of soluble TNF [37], and S100A1/A4/B/P proteins suppressed the IFN-β-induced inhibition of viability of MCF-7 cells [22,39,40]. Besides, S100-cytokine interactions can promote non-classical secretion of the cytokines as exemplified by S100A13 - IL1α/FGF1 interactions [34,35].

The vast majority of the established S100-cytokine interactions correspond to cytokines belonging to the superfamily of four-helical cytokines (SCOP [43] ID: 3001717), which is subdivided into three families: ‘Short-chain cytokines’ (SCOP ID 4000852), ‘Long-chain cytokines’ (SCOP ID 4000851) and ‘Interferons/interleukin-10 (IL-10)’ (SCOP ID 4000854). We have previously shown that S100P oncoprotein is poorly selective towards the four-helical cytokines of all structural families, interacting with 71% of them (29 out of the 41 cytokines studied) with the equilibrium dissociation constants, K_d, in the range from 1 nM to 3 µM (below the K_d value for S100P complex with V domain of RAGE) [42]. Using mutagenesis, we confirmed the presence of a cytokine-binding site in S100P molecule, overlapping with its RAGE-specific site. The ability of S100P to recognize multiple cytokines reflects its promiscuous nature, well established for certain members of S100 family, which readily share their binding partners [44,45]. Therefore, the specificity to many four-helical cytokines is also expected for other promiscuous representatives of S100 family. To explore this possibility, in this work we test the affinity of another promiscuous S100 oncoprotein [44,45], S100A6 (also known as calcyclin; pairwise sequence identity to S100P of 35%), specific to 6 four-helical cytokines shown in Table 1, to a panel of 35 four-helical cytokines from all their structural families (Table 2). Our findings greatly expand the long list of known extracellular target proteins of S100A6, which may be of value for deciphering its role in progression of many cancers (positive correlation between S100A6 expression and the disease stage, tumor size and/or metastasis is reported), myocardial infraction, acute coronary syndrome, chronic renal disease, primary biliary cholangitis, pulmonary fibrosis, systemic sclerosis of the lung, endometriosis, various eye pathologies, and other disorders (reviewed in [46]).

2. Materials and methods

2.1. Materials

Human S100A6 protein was prepared in E. coli as described in [39]. The samples of human cytokines are presented in Table 2. Protein concentrations were measured spectrophotometrically according to [47].

Sodium acetate and ethanolamine were from Bio-Rad Laboratories, Inc. HEPES and sodium chloride were purchased from PanReac AppliChem. CaCl₂, EDTA and TWEEN 20 were from Sigma Aldrich Co.

2.2. Surface plasmon resonance studies

SPR studies of S100A6 binding to the cytokines immobilized on the sensor chip surface (S100A6 was used as an analyte and the cytokines served as ligands) were performed at 25°C mainly as described in [42]. Cytokine (0.03-0.05 mg/ml) in 10 mM sodium acetate pH 4.5 buffer was immobilized on ProteOn™ GLH sensor chip surface of ProteOn™ XPR36 instrument (Bio-Rad Laboratories, Inc.) by amine coupling (up to 10 000 - 17 000 RUs). The remaining activated amine groups on the chip surface were blocked with 1 M ethanolamine solution. S100A6 (63 nM - 8 µM) solution in the running buffer (10 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 0.05% TWEEN 20, pH 7.4) was passed over the chip surface at a rate of 30 μl/min for 300 s, followed by passage of the running buffer for 1 200 – 2 400 s. The double-referenced SPR sensograms were described by either a single site binding model (S100A6 - IL-2 interaction) or a heterogeneous ligand model (1). The latter suggests existence of two populations of the ligand (L₁ and L₂) that bind a single analyte molecule (A):

(1)

Here, k_a and k_d are kinetic association and dissociation constants, respectively; K_d are equilibrium dissociation constants (k_d/k_a). The K_d and k_d values were evaluated for each S100A6 concentration using ProteOn Manager™ v.3.1 software (Bio-Rad Laboratories, Inc.). The resulting values are average of 3-5 estimates (the standard deviations are indicated). The ligand was regenerated by 20 mM EDTA solution pH 8.0 for 100 s. The free energy changes, accompanying the reaction were calculated as follows: ΔG_i = - RT ln(55.3/K_di), i=1,2.

2.3. Structural classification of cytokines

The cytokines investigated in this study were classified according to the SCOP 2 database (https://scop2.mrc-lmb.cam.ac.uk [43], build 1.0.6, updated on 06-29-2022) as belonging to the ‘All alpha proteins’ structural class, ‘4-helical cytokines’ fold (SCOP ID 2001054), ‘4-helical cytokines’ superfamily (SCOP ID 3001717). This superfamily comprises three families, namely ‘Short-chain cytokines’ (SCOP ID 4000852), ‘Long-chain cytokines’ (SCOP ID 4000851) and ‘Interferons/interleukin-10 (IL-10)’ (SCOP ID 4000854). CLCF1 and CT-1 were classified as the long-chain cytokines according to [48].

2.4. Structural modeling of the S100A6-cytokine complexes

The ClusPro docking server [49] was used to generate the models of tertiary structures of the S100A6-cytokine complexes, as previously described [38,42]. The structure of the Ca²⁺-loaded human S100A6 dimer was taken from PDB [50] entry 1K9K (X-ray, chains A and B [51]), while the tertiary structures of the four-helical cytokines were either extracted from PDB (including the complexes with their binding partners) or predicted using AlphaFold2 (https://alphafold.ebi.ac.uk/ [52]) (Table S1). Ten docking models were taken into account for each S100A6-cytokine pair. The contact residues included into five or more docking models were considered as the most probable residues of the binding site. Distributions of the predicted contact residues of S100A6 over its amino acid sequence within the models of S100A6 complexes with specific cytokines were calculated as described in [38]. The tertiary structures were visualized using the molecular graphics system PyMOL v.2.5.0 (https://pymol.org/2/).

3. Results and discussion

3.1. Selectivity of S100A6 binding to the four-helical cytokines

We have studied affinity of Ca²⁺-loaded (1 mM CaCl₂) recombinant human S100A6 protein to the panel of 35 recombinant human four-helical cytokines (includes the panel used in [42], extended by Flt3L, SCF and IL-19), which covers all structural families of this fold (Table 2). The cytokines were immobilized on the surface of SPR sensor chip by amine coupling, and 61 nM - 8 µM solutions of S100A6 were passed over the surface at 25ºC. Whereas 9 cytokines (Table S2) did not reveal noticeable effects in response to the S100A6 (data not shown), the SPR sensograms for 26 cytokines showed the S100A6 concentration-dependent effects, characteristic of the association-dissociation processes (Figure 1, Figure 2, Figure 3 and Figure 4). The kinetic SPR data were adequately described within the heterogeneous ligand model (1) (Figure 1, Figure 2, Figure 3 and Figure 4, Table 3), which was earlier successfully used for description of the S100-cytokine interactions [22,38,39,40,41,42,53,54]. The data for IL-2 were described by the one-site binding scheme. The respective equilibrium dissociation constants, K_d, range from 0.3 nM (in the case of THPO, similarly to S100P [42]) to 12 µM (for IL-2) (Table 3). For comparison, the reported SPR estimates of K_d values for the complexes of Ca2+-bound S100A6 with various extracellular fragments of its receptor, RAGE, are within the range from 28 nM to 13 µM [55,56], whereas the K_d value for V domain of RAGE measured by isothermal calorimetry is of 3 µM [57]. An analysis of the free energy changes upon the S100A6-cytokine interactions (Figure 5), ΔG, shows that the average S100A6 affinities for the cytokines of different SCOP families of the fold decrease in the following order: short-chain cytokines > long-chain cytokines > interferons/IL-10 (ΔG of -49.8 kJ/mol < -47.1 kJ/mol < -46.8 kJ/mol). The same tendency was observed previously for S100P protein [42].

The S100A6-cytokine complexes easily dissociated upon removal of Ca²⁺ by passage over the SPR chip of 20 mM EDTA solution, pH 8.0 (data no shown), which evidences an importance of the Ca²⁺-induced structural rearrangement within S100A6 molecule for the efficient interaction with the cytokines. Since Ca²⁺ binding induces solvent exposure of S100A6 residues of helices α2 and α3 and the ‘hinge’ between them [51], this region is likely involved in the cytokine recognition.

Since equilibrium homodimer dissociation constant for the Ca²⁺-loaded S100A6 does not exceed 0.5 µM [39], our estimates of its affinities to the cytokines (Table 3) mostly correspond to the dimeric state of S100A6. Meanwhile, serum S100A6 concentrations may be as low as 0.2 pM [58] (Table S3), which points out that in serum S100A6 may exist in monomeric form.

Since affinities of S100P monomer to the four-helical cytokines IL-11 and IFN-β exceed those for S100P dimer by 1.4-2.2 orders of magnitude [22,54,59], S100A6 monomerization is likely to improve its affinity to the four-helical cytokines. In this case, the K_d values for some of the S100A6-cytokine interactions (Flt3L, IL-3, IL-5, IL-9, IL-13, IL-15, IL-21, SCF, THPO; G-CSF, GH, GH-V, IL-31, PRL; IL-19, IL-20, IL-26) may reach nanomolar level or less (Figure 5), which is enough for the efficient cytokine binding to S100A6 at its serum concentrations reaching nanomolar level [58,60,61,62] (Table S3). Moreover, the local levels of extracellular S100A6 in damaged tissues expressing S100A6 should be even higher, further facilitating the S100A6-cytokine interactions.

Overall, S100A6 interacts with ca 73% of the four-helical cytokines studied to date (32 of the 44 cytokines, see Table 1, Table 3 and Table S2). The selectivity of S100A6 binding to the cytokines is equivalent to that of S100P [42], except for the interaction with IL-2 and OSM, which are specific only to S100A6 and S100P, respectively. Importantly, the revealed S100A6-cytokine interactions (Table 3) are non-redundant, since the S100A6-specific cytokines are mostly evolutionary distant from each other: the pairwise sequence identities within each of their SCOP families, calculated using Clustal Omega 2.1 (implemented in EMBL-EBI service [11]), ranges from 3% to 44% (the exception is GH – GH-V pair with the pairwise sequence identity of 93%). According to the IntAct [63] and BioGRID [64] databases, so far only S100P protein has been described as a soluble non-receptor extracellular target protein for the following cytokines specific to S100A6 (Table 3): IL-3, IL-5, IL-9, IL-13, IL-21, THPO, IL-22. Except for IL-22, they are short-chain four-helical cytokines with the highest average affinity to S100A6 (Figure 5).

3.2. Structural modeling of the S100A6-cytokine complexes

The docking models of the complexes between Ca²⁺-loaded S100A6 dimer and the S100A6-specific four-helical cytokines shown in Table 1 and Table 3 (excluding the heterodimeric IL-27/IL-35) were generated using ClusPro docking server [49] and analyzed as previously described [38,42]. The residues predicted to be involved in the interaction for 5 or more of the 10 docking models are listed in Table S4.

The S100A6 residues predicted to be key for recognition of the four-helical cytokines (for at least half of the cytokines) are I44 (16 cytokines out of 30) of the ‘hinge’ loop region between helices α2 and α3, R55 and D59 of helix α3 (17 cytokines), and I83 (21 cytokines) and Y84 (18 cytokines) of helix α4 (Figure 6A). An analysis of the distribution of the predicted contact residues of 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 N- and C-termini, helix α1, ‘hinge’ region, and helices α3 and α4. These regions of 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 [42]. The qualitative difference between the predicted behavior of S100A6 and S100P proteins is the more complete involvement into the cytokine recognition of the helix α3 and N-terminal half of the helix α4 of S100A6. In this sense, the predicted pattern of the contact residues of S100A6 is more reminiscent of that described for S100 proteins, with frequent involvement in target recognition of helix α1, ‘hinge’, and helices α3 and α4 [65]. Importantly, only 4 of the 11 residues of S100A6 previously shown to interact with V domain of RAGE using NMR [57] are predicted to interact with some of the cytokines (4 out of 30): R62 (GH-V, IL-11, IFN-β, IL-20), N63 (IL-11, IFN-β), K64 (IFN-β), N69 (IL-11) (Table S4). The RAGE-binding site of S100A6 reported in [66] is even more distinct from the predicted cytokine-specific site. Thus, the latter is notably different from the RAGE-binding site.

The molecular docking analysis indicates that the long-chain cytokines preferentially bind Ca²⁺-bound S100A6 dimer by the residues of N-terminus and helices α1 and α3 (Figure 7B). The cytokines of interferons/IL-10 family are predicted to bind S100A6 dimer via the residues of C-terminus and helices α2 and α3 (Figure 7C). Meanwhile, the predicted locations of the contact residues for the short-chain cytokines do not reveal clear regularities (Figure 7A).

Furthermore, unlike other families of the cytokines, this family tends to interact with only one chain of S100A6 dimer. Therefore, similarly to S100P binding to the four-helical cytokines [42], the putative location of the S100A6-binding site in the cytokines varies depending on structural family of the cytokine and the particular cytokine.

The cytokine residues predicted to interact with S100A6 for some of the short-chain cytokines (GM-CSF, IL-3, IL-13, IL-21, THPO), long-chain cytokines (GH, GH-V, IL-31, PRL) and cytokines of interferons/IL-10 family (IFN-ω1, IL-10, IL-20, IL-22, IL-24) overlap by more than 50% with the residues similarly predicted for interaction with S100P protein [42]. On the contrary, the putative S100A6-binding sites of specific short-chain cytokines (EPO, IL-5, IL-15), long-chain cytokines (LEP) and cytokines of interferons/IL-10 family (IFN-β, IL-26) do not intersect with the sites predicted for S100P binding [38,42]. Thus, despite the nearly identical selectivity of S100A6 and S100P proteins with regard to binding of the four-helical cytokines and the similar putative locations of their cytokine-binding sites, they could be recognized by the cytokines quite differently.

Since some of the predicted S100A6-binding residues of the specific four-helical cytokines (EPO, GM-CSF, IL-2, IL-3, IL-5, IL-13, IL-15, IL-21, GH, IFN-ω1, IL-20, IL-22 and IL-24) have previously been shown to participate in recognition of their respective receptors (Table S4), S100A6 binding is potentially able to affect the cytokine signaling, as exemplified by the inhibition of IFN-β signaling in MCF-7 cells by S100A1/A4/B/P [22,39,40], suppression of the cytotoxic activity of soluble TNF against Huh-7 cells by S100A12/A13 [37], inhibition of FGF2-induced increase in proliferation of MCF-7/MDA-MB468 cells by S100B [36], and S100A4 stimulation of the amphiregulin-mediated proliferation of embryonic fibroblasts [33].

Of note, the presented structural predictions based on the molecular docking suffer from an inability to take into consideration the structural flexibility inherent to the four-helical cytokines, as evidenced by the experimentally confirmed disorder in IL-15, THPO, G-CSF, LEP, IL-10 and the theoretical predictions described in [42].

3.3. Potential physiological significance of the S100A6 interactions with the four-helical cytokines

To get an insight into possible relative in vivo concentrations of S100A6 and the S100A6-specific cytokines (Table 1 and Table 3), we collected literature data on their concentrations in physiological fluids under normal and pathological conditions (Tables S3 and S5). Examination of the concentration ranges reported for S100A6 and the S100A6-specific cytokines in the blood serum/plasma (Figure 8) highlights several regularities. First, there is an overlap of the concentration ranges for the both interaction partners, which indicates a possibility of mutual regulation of activity of one of the partners due to an excess of the other partner. The binding of S100A6 could alter the cytokine signaling, as previously shown for IFN-β signaling in MCF-7 cells inhibited by S100A1/A4/B/P [22,39,40], and in other cases [33,36,37]. Alternatively, an excess of the S100A6-specific cytokine over S100A6 protein could affect its interaction with RAGE [56], integrin β1 [25] and/or other receptors. Second, with a few exceptions, there is a trend towards increased cytokine concentrations in pathological conditions, which should promote formation of the S100A6-cytokine complex. The most favorable conditions for that are expected for GH, GH-V, LEP, PRL and IL-26, which demonstrate the highest blood levels, exceeding 1 nM (Figure 8). As mentioned above, although the experimental estimates of the equilibrium dissociation constants (Table 3 and Figure 5), K_d, are low enough for efficient S100A6 binding only for THPO, S100A6 monomerization could decrease the K_d values by 1.4-2.2 orders of magnitude, which was earlier shown for S100P interaction with IL-11 and IFN-β [22,54,59]. In this case, many other cytokines could bind S100A6 in vivo at concentrations as high as several nM [60] (Table S3).

The binding of S100A6 to the cytokines may also promote their non-canonical secretion, as previously reported for S100A13 binding to IL-1α and FGF1 [34,35] and for CLCF1, which requires CRLF1 binding for efficient secretion [67]. Considering that sharing of interaction partners is inherent to many S100 proteins, including S100A6 and S100P [44,45], the revealed multiple interactions of the four-helical cytokines with S100A6/S100P and other members of S100 family [22,38,39,40,41,42,53,54] could serve as a common mechanism for non-canonical secretion of the cytokines.

4. Conclusions

In this work, we have extended the list of the four-helical cytokines studied with regard to their affinity to the promiscuous S100A6 protein [44,45] from 9 (see references to Table 1) to 44 (see also Table 2) cytokines, covering all structural families of this fold. Only 12 out of the 44 cytokines studied using SPR spectroscopy lack notable specificity to S100A6 (Table S2). The absence of detectable interactions implies that the respective equilibrium dissociation constants, K_d, exceed 10⁻⁴ M. Thus, about 73% of the cytokines are specific to Ca²⁺-loaded S100A6 dimer with the K_d values in the range from 0.3 nM to 12 µM (Table 3), which intersects with the K_d estimates for the complexes of Ca²⁺-bound S100A6 with extracellular RAGE fragments from 28 nM to 13 µM [55,56,57]. The fraction of the cytokines specific to S100A6 may be even higher, given the possibility that S100A6 monomerization promotes its interaction with the cytokines. The similarly high percentage of the four-helical cytokines specific to S100P protein of 71% [42] cannot be attributed to high homology between S100A6 and S100P, since their pairwise sequence identity is only 35%.

The promiscuous binding of S100A6 and S100P to wide range of the four-helical cytokines belonging to all structural families of this fold can be partly explained by the molecular docking analysis, which revealed that both S100 proteins have a nearly identical cytokine-binding site formed by helices α1, α3 and α4, and ‘hinge’ (Figure 6A, [42]). The involvement of ‘hinge’ and helix α4 in the cytokine recognition was previously confirmed by S100P mutagenesis [42]. This site overlaps with the RAGE-binding site of S100P, but differs markedly from that for S100A6. Meanwhile, the structural modeling shows that the S100-specific four-helical cytokines lack a conserved S100-binding site (Figure 7, [42]). Instead, the location of the putative S100A6/S100P-binding sites of the cytokines is variable and depends on the particular cytokine. Importantly, the modelling reveals distinct differences in the cytokine regions putatively involved in the recognition of S100A6 or S100P proteins, which explains some differences in their selectivity to the cytokines (limited to recognition of IL-2 and OSM). Nevertheless, similarly to the putative S100P-binding sites of the four-helical cytokines [42], some of their predicted S100A6-specific regions are involved in binding of the corresponding receptors. This fact raises the possibility that the interaction of some cytokines with extracellular S100A6/S100P may interfere with formation of the cytokine-receptor complexes, thereby inhibiting the proper signaling (see, for instance, [22,36,37,39,40]). The same effect is expected for other promiscuous members of S100 family, capable of interaction with multiple common binding partners [44,45]. In this case, the promiscuous S100 proteins are potentially able to serve as universal inhibitors of signaling of the four-helical cytokines. This unique feature of the S100 family could be used for reduction of severity of the disorders associated with excessive release of the cytokines, including cytokine storm. Another possibility is the cytokine binding-induced modulation of extracellular activity of the promiscuous S100 proteins in response to the pathological conditions accompanied by elevated levels of some four-helical cytokines. Finally, the binding of intracellular S100 proteins could facilitate the non-canonical secretion of certain cytokines [34,35,67].

Supplementary Materials

The following supporting information can be downloaded at: Preprints.org, Table S1: The PDB entries or AlphaFold2 predictions used for structural modelling of the complexes of Ca2+-loaded human S100A6 dimer (chains A, B of PDB entry 1K9K) with the four-helical cytokines using ClusPro docking server; Table S2: The samples of the four-helical cytokines lacking specificity to S100A6, as evidenced by SPR experiments using cytokine as a ligand; Table S3: Serum levels of S100A6 under normal and pathological conditions, according to the literature data; Table S4: The contact residues for the S100A6-cytokine complexes predicted using ClusPro docking server (numbering is according to the PDB entries); Table S5: The literature data on concentrations of the S100A6-specific four-helical cytokines in the physiological fluids under normal and pathological conditions; Table S6: The concentration ranges of the S100A6-specific cytokines in the physiological fluids under normal and pathological conditions, extracted from the literature data collected in Table S5.

Author Contributions

Conceptualization, S.E.P.; validation, A.S.K., E.I.D., V.A.R., V.N.U., E.A.P., S.E.P.; formal analysis, A.S.K., E.I.D., V.A.R., S.E.P.; investigation, A.S.K., E.I.D., V.A.R., A.S.S., M.E.P., E.A.L.; data curation, A.S.K., E.I.D., V.A.R., S.E.P.; writing—original draft preparation, A.S.K., E.I.D., V.A.R., S.E.P.; writing—review and editing, A.S.K., E.I.D., V.A.R., V.N.U., E.A.P., S.E.P.; supervision, E.A.P., S.E.P.; project administration, S.E.P.; funding acquisition, S.E.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Russian Science Foundation, grant number 19-14-00289-П to S.E.P.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data present in the current study are available from the corresponding authors on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

CHO, Chinese hamster ovary cells; CLCF1, cardiotrophin-like cytokine factor 1; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin-1; EDTA, ethylenediaminetetraacetic acid; EPO, erythropoietin; FGF, fibroblast growth factor; ER, endoplasmic reticulum; Flt3L, Fms-related tyrosine kinase 3 ligand; G-CSF, granulocyte colony-stimulating factor; GH, growth hormone; GH-V, growth hormone variant; GM-CSF, granulocyte-macrophage colony-stimulating factor; HEK293, Human embryonic kidney 293 cells; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; IFN, interferon; IL, interleukin; LEP, leptin; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor 1; NMR, nuclear magnetic resonance; OSM, oncostatin-M; PDB, Protein Data Bank [50]; PL, chorionic somatomammotropin hormone 1; PRL, prolactin; RAGE, receptor for advanced glycation end products; RU, resonance unit; SCF, stem cell factor, soluble form; SCOP, Structural Classification of Proteins [43]; SPR, surface plasmon resonance; THPO, thrombopoietin; TNF, Tumor necrosis factor; TSLP, thymic stromal lymphopoietin.

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Figure 1. SPR spectroscopy data on kinetics of association/dissociation of the complexes of Ca²⁺-bound S100A6 with the short-chain four-helical cytokines (see Table 3) immobilized on the sensor chip surface by amine coupling, 25°C (10 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 0.05% TWEEN 20, pH 7.4). The association phase is marked by the vertical dotted lines. Micromolar concentrations of S100A6 are indicated for the sensograms. The experimental curves (grey) are described by the heterogeneous ligand model (1) or one-site binding model (black curves) (see Table 3).

Figure 2. SPR spectroscopy data on kinetics of association/dissociation of the complexes of Ca²⁺-bound S100A6 with the short-chain four-helical cytokines (see Table 3) immobilized on the sensor chip surface by amine coupling, 25°C (10 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 0.05% TWEEN 20, pH 7.4). The experimental curves (grey) are described by the heterogeneous ligand model (1) (black curves) (see Table 3). For other designations, refer to the caption to Figure 1.

Figure 3. SPR spectroscopy data on kinetics of association/dissociation of the complexes of Ca²⁺-loaded S100A6 with the long-chain four-helical cytokines (see Table 3) immobilized on the sensor chip surface by amine coupling, 25°C (10 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 0.05% TWEEN 20, pH 7.4). The experimental curves (grey) are described by the heterogeneous ligand model (1) (black curves) (see Table 3). For other designations, refer to the caption to Figure 1.

Figure 4. SPR spectroscopy data on kinetics of association/dissociation of the complexes of Ca²⁺-bound S100A6 with the interferons/IL-10 four-helical cytokines (see Table 3) immobilized on the sensor chip surface by amine coupling, 25°C (10 mM HEPES, 150 mM NaCl, 1 mM CaCl₂, 0.05% TWEEN 20, pH 7.4). The experimental curves (grey) are described by the heterogeneous ligand model (1) (black curves) (see Table 3). For other designations, refer to the caption to Figure 1.

Figure 5. The free energy changes upon the binding of Ca²⁺-loaded S100A6 to the four-helical cytokines of short-chain (panel A), long-chain (B) or interferons/IL-10 (C) families at 25°C, estimated from the SPR data shown in Table 3. The scale of K_d values is indicated.

Figure 6. A, the cytokine-binding site predicted for the Ca²⁺-loaded S100A6 dimer (PDB entry 1K9K) using ClusPro docking server [49] and the tertiary structures listed in Table S1. The chains A and B are highlighted in cyan and grey, respectively. The α-helices are labeled as α1-α4, and the Ca²⁺-binding loops are yellow-colored. The S100A6 residues predicted to recognize at least 10 out of the 30 four-helical cytokines (I44, E52, R55, D59, I83, Y84, E86, A87 and K89 of chain A, and A2 and D6 of chain B) are marked as an orange mesh surface, whereas the residues predicted to interact with at least half of the cytokines (I44, R55, D59, I83 and Y84) are shown as red balls and sticks. B, distributions of the predicted contact residues of S100A6 over its amino acid sequence within the docking models of S100A6 complexes with the four-helical cytokines shown in Table S1 (10 models per each S100A6-cytokine pair). The boundaries of the α-helices α1-α4 are extracted from PDB entry 1K9K; the residues I44, R55, D59, I83, and Y84 of chain A are marked in red.

Figure 7. The S100A6-binding sites (orange-colored; see Table S4) predicted for the four-helical cytokines of short-chain (panel A), long-chain (B) and interferons/IL-10 (C) families using ClusPro docking server [49] and the tertiary structures listed in Table S1. Only one subunit of IL-5 is shown. N-terminus of each cytokine is located in the lower left corner.

Figure 8. The concentration ranges of the S100A6-specific cytokines in the blood serum or plasma under normal (blue bars) and pathological (red bars) conditions, extracted from the literature data (see Table S6). The blue and red dotted lines correspond to the upper limits of S100A6 serum levels in health and disease, respectively, according to the literature data (see Table S3).

Table 1. The literature data on equilibrium dissociation constants for the complexes between Ca²⁺-loaded S100A6 and four-helical cytokines at 25ºC. The SPR spectroscopy data (amine coupling of the cytokine on the SPR chip surface) are described using the heterogeneous ligand model (1).

Cytokine	K_d1, M	K_d2, M	Reference
Short-chain cytokines
EPO	(1.5 ± 0.3) × 10⁻⁷	(6.5 ± 2.6) × 10⁻⁷	[38]
Long-chain cytokines
CLCF1	(1.1 ± 0.8) × 10^-6	(3.7 ± 0.8) × 10^-6	[41]
CNTF	(1.1 ± 1.0) × 10^-7	(2.07 ± 0.08) × 10^-6	[41]
CT-1	(1.6 ± 0.5) × 10^-6	(1.2 ± 0.4) × 10^-5	[41]
IL-11	(8.3 ± 1.9) × 10^-6	(7.9 ± 2.1) × 10^-6	[41]
Interferons/IL-10
IFN-β	(8.2 ± 2.4) × 10^-8 (0.70 ± 0.03) × 10^-9 *	(2.67 ± 0.57) × 10^-7 (2.81 ± 0.49) × 10^-7 *	[39]

* S100A6 serves as a ligand

Table 2. The cytokine samples studied with regard to affinity to Ca²⁺-loaded S100A6 in the present work.

Full name	Abbreviation	UniProt ID	Manufacturer	Cat. number	Source
Short-chain cytokines
Fms-related tyrosine kinase 3 ligand	Flt3L	P49771	PeproTech	300-19	E.coli
Granulocyte-macrophage colony-stimulating factor	GM-CSF	P04141	PeproTech	300-03	E.coli
Interleukin-2	IL-2	P60568	PeproTech	AF-200-02	E.coli
Interleukin-3	IL-3	P08700	SCI-Store (Russia)	PSG160-10	CHO
Interleukin-4	IL-4	P05112	PeproTech	AF-200-04	E.coli
Interleukin-5	IL-5	P05113	PeproTech	200-05	E.coli
Interleukin-7	IL-7	P13232	SCI-Store (Russia)	PSG240-10	CHO
Interleukin-9	IL-9	P15248	PeproTech	200-09	E.coli
Interleukin-13	IL-13	P35225	PeproTech	200-13	E.coli
Interleukin-15	IL-15	P40933	PeproTech	200-15	E.coli
Interleukin-21	IL-21	Q9HBE4	PeproTech	200-21	E.coli
Macrophage colony-stimulating factor 1	M-CSF	P09603	PeproTech	300-25	E.coli
Stem cell factor, soluble form	SCF	P21583	PeproTech	300-07	E.coli
Thrombopoietin	THPO	P40225	SCI-Store (Russia)	PSG090-10	CHO
Thymic stromal lymphopoietin	TSLP	Q969D9	PeproTech	300-62	E.coli
Long-chain cytokines
Chorionic somatomammotropin hormone 1	PL	P0DML2	R&D Systems	5757-PL/CF	CHO
Granulocyte colony-stimulating factor	G-CSF	P09919	Pharmstandard (Russia)	n/a	E.coli
Growth hormone	GH	P01241	PeproTech	AF-100-40	E.coli
Growth hormone variant	GH-V	P01242	R&D Systems	7668-GH/CF	E.coli
Interleukin-12	IL-12	P29459* & P29460	PeproTech	200-12H	HEK293
Interleukin-23	IL-23	Q9NPF7* & P29460	PeproTech	200-23	Hi-5
Interleukin-27	IL-27	Q8NEV9* & Q14213	PeproTech	200-38	HEK293
Interleukin-31	IL-31	Q6EBC2	PeproTech	200-31	E.coli
Interleukin-35	IL-35	P29459* & Q14213	PeproTech	200-37	HEK293
Leptin	LEP	P41159	PeproTech	AF-300-27	E.coli
Prolactin	PRL	P01236	PeproTech	100-07	E.coli
Interferons/IL-10
Interferon α-2	IFN-α2	P01563	Vector-Medica (Russia)	n/a	E.coli
Interferon γ	IFN-γ	P01579	Pharmaclon (Russia)	n/a	E.coli
Interferon ω-1	IFN-ω1	P05000	PeproTech	300-02J	E.coli
Interleukin-10	IL-10	P22301	PeproTech	AF-200-10	E.coli
Interleukin-19	IL-19	Q9UHD0	PeproTech	200-19	E.coli
Interleukin-20	IL-20	Q9NYY1	PeproTech	200-20	E.coli
Interleukin-22	IL-22	Q9GZX6	PeproTech	200-22	E.coli
Interleukin-24	IL-24	Q13007	PeproTech	200-35	CHO
Interleukin-26	IL-26	Q9NPH9	R&D Systems	1375-IL/CF	E.coli

* denotes the chain used for SCOP 2 [43] family assignment. n/a, not applicable

Table 3. Parameters of the interactions between Ca²⁺-bound S100A6 and specific four-helical cytokines shown in Table 2 at 25°C, derived from the SPR spectroscopy data (Figure 1, Figure 2, Figure 3 and Figure 4) using the heterogeneous ligand model (1).

Cytokine	k_d1, s^-1	K_d1, M	k_d2, s^-1	K_d2, M
Short-chain cytokines
Flt3L	(8.83 ± 2.54) × 10^-4	(1.22 ± 0.27) × 10^-7	(8.32 ± 4.05) × 10^-4	(1.25 ± 0.46) × 10^-7
GM-CSF	(7.92 ± 4.02) × 10^-4	(2.26 ± 1.18) × 10^-6	(1.76 ± 0.49) × 10^-2	(3.63 ± 1.14) × 10^-6
IL-2*	(4.97 ± 0.78) × 10^-3	(1.20 ± 0.04) × 10^-5	n/a	n/a
IL-3	(2.71 ± 0.78) × 10^-4	(6.11 ± 2.19) × 10^-9	(7.28 ± 0.65) × 10^-3	(4.02 ± 2.03) × 10^-7
IL-5	(2.47 ± 0.45) × 10^-4	(1.16 ± 0.52) × 10^-8	(5.07 ± 0.20) × 10^-3	(3.23 ± 0.73) × 10^-8
IL-9	(5.97 ± 2.79) × 10^-3	(1.69 ± 0.90) × 10^-7	(5.85 ± 2.72) × 10^-3	(2.71 ± 2.42) × 10^-7
IL-13	(2.64 ± 0.98) × 10^-4	(1.97 ± 1.50) × 10^-7	(1.36 ± 0.06) × 10^-2	(1.91 ± 1.07) × 10^-6
IL-15	(1.62 ± 0.24) × 10^-4	(5.70 ± 0.46) × 10^-8	(6.29 ± 0.26) × 10^-3	(2.15 ± 1.32) × 10^-7
IL-21	(1.88 ± 1.33) × 10^-4	(4.57 ± 1.30) × 10^-8	(5.68 ± 0.34) × 10^-3	(3.87 ± 0.86) × 10^-7
SCF	(2.57 ± 0.92) × 10^-4	(8.72 ± 4.95) × 10^-9	(4.51 ± 1.52) × 10^-3	(1.90 ± 1.45) × 10^-6
THPO	(6.00 ± 3.29) × 10^-5	(2.89 ± 1.83) × 10^-10	(4.47 ± 2.46) × 10^-3	(3.96 ± 2.65) × 10^-9
Long-chain cytokines
G-CSF	(7.72 ± 0.31) × 10^-4	(3.91 ± 2.06) × 10^-9	(1.04 ± 0.04) × 10^-2	(1.68 ± 1.29) × 10^-6
GH	(3.73 ± 2.36) × 10^-4	(3.42 ± 2.85) × 10^-7	(5.87 ± 1.16) × 10^-3	(4.16 ± 2.65) × 10^-7
GH-V	(7.92 ± 3.35) × 10^-5	(4.62 ± 2.41) × 10^-8	(3.06 ± 1.27) × 10^-3	(1.78 ± 0.92) × 10^-6
IL-27^#	(9.61 ± 4.18) × 10^-4	(1.16 ± 0.63) × 10^-6	(1.47 ± 0.62) × 10^-2	(2.80 ± 0.85) × 10^-6
IL-31	(4.64 ± 0.13) × 10^-4	(1.06 ± 0.96) × 10^-7	(1.11 ± 0.12) × 10^-2	(2.56 ± 1.95) × 10^-7
IL-35^#	(2.59 ± 0.81) × 10^-3	(2.34 ± 1.26) × 10^-6	(3.91 ± 1.55) × 10^-2	(4.65 ± 2.42) × 10^-6
LEP	(3.18 ± 2.17) × 10^-3	(3.25 ± 1.92) × 10^-7	(2.87 ± 1.00) × 10^-4	(3.85 ± 1.06) × 10^-7
PRL	(1.59 ± 0.23) × 10^-4	(8.40 ± 0.58) × 10^-8	(1.55 ± 0.82) × 10^-2	(6.35 ± 0.96) × 10^-7
Interferons/IL-10
IFN-ω1	(4.71 ± 0.22) × 10^-4	(2.28 ± 0.64) × 10^-7	(1.29 ± 0.55) × 10^-2	(1.11 ± 0.63) × 10^-6
IL-10	(3.98 ± 0.28) × 10^-4	(3.61 ± 1.09) × 10^-7	(1.50 ± 0.37) × 10^-2	(2.13 ± 0.67) × 10^-6
IL-19	(6.28 ± 0.86) × 10^-4	(1.47 ± 1.08) × 10^-7	(2.77 ± 1.69) × 10^-2	(4.96 ± 4.04) × 10^-7
IL-20	(3.08 ± 0.86) × 10^-4	(4.51 ± 1.12) × 10^-8	(9.14 ± 2.51) × 10^-3	(3.85 ± 0.59) × 10^-7
IL-22	(3.57 ± 2.56) × 10^-3	(5.93 ± 0.84) × 10^-7	(3.26 ± 2.67) × 10^-2	(1.31 ± 0.49) × 10^-6
IL-24	(5.73 ± 0.85) × 10^-4	(2.39 ± 0.94) × 10^-7	(7.79 ± 1.72) × 10^-3	(6.01 ± 1.57) × 10^-7
IL-26	(5.98 ± 2.92) × 10^-4	(2.78 ± 2.31) × 10^-7	(2.56 ± 1.39) × 10^-3	(6.00 ± 2.63) × 10^-7

* single site binding model is used. # heterodimeric cytokines. n/a, not applicable

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