Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1–19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, ‘black’ and ‘gray’ clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr²⁺ interference with Ca²⁺ binding to proteins of the EF-hand family, we studied Sr²⁺/Ca²⁺ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca²⁺ binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 10⁹ M⁻¹ and 4 × 10⁹ M⁻¹ for Ca²⁺, and 2 × 10⁷ M⁻¹ and 2 × 10⁶ M⁻¹ for Sr²⁺. Inactivation of the EF site by homologous substitution of the Ca²⁺-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca²⁺/Sr²⁺ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca²⁺/Sr²⁺ affinity. These results suggest that Sr²⁺ and Ca²⁺ bind to CD/EF sites of α-PA and the Ca²⁺/Sr²⁺ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr²⁺ titration of the Ca²⁺-loaded α-PA revealed presence of secondary Sr²⁺ binding site(s) with an apparent equilibrium association constant of 4 × 10⁵ M⁻¹. Fourier-transform infrared spectroscopy data evidence that Ca²⁺/Sr²⁺-loaded forms of α-PA exhibit similar states of their COO⁻ groups. Near-UV circular dichroism (CD) data show that Ca²⁺/Sr²⁺ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca²⁺/Sr²⁺ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr²⁺-induced increase in stability of tertiary structure of α-PA, compared to the Ca²⁺-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca²⁺-binding sites of α-PA are well protected against exchange of Ca²⁺ for Sr²⁺ regardless of coordination number of Sr²⁺, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca²⁺/Sr²⁺ selectivity. Overall, despite lowered affinity of α-PA to Sr²⁺, the latter competes with Ca²⁺ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr²⁺ binding site(s) could be a factor contributing to Sr²⁺ impact on the functional activity of proteins.

Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here, we report that among eighteen representatives of the multifunctional S100 protein family, only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants (K_d) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent K_d values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1–0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatics analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect the efficacy of anti-TNF treatment in various diseases.

An imbalance between production and excretion of amyloid β peptide (Aβ) in the brain tissues of Alzheimer’s disease (AD) patients leads to Aβ accumulation and the formation of noxious Aβ oligomers/plaques. A promising approach to AD prevention is the reduction of free Aβ levels by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aβ. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aβ. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA–Aβ40 interaction: prednisone favors HSA–Aβ interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.