In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid ß Peptide.

An imbalance between production and excretion of amyloid ß peptide (Aß) in the brain tissues of Alzheimer's disease (AD) patients leads to Aß accumulation and the formation of noxious Aß oligomers/plaques. A promising approach to AD prevention is the reduction of free Aß levels by directed enhancement of Aß 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 Aß. 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 Aß. 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-Aß40 interaction: prednisone favors HSA-Aß 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.

Recognition of granulocyte-macrophage colony-stimulating factor by specific S100 proteins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic myelopoietic growth factor and proinflammatory cytokine, clinically used for multiple indications and serving as a promising target for treatment of many disorders, including cancer, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, COVID-19. We have previously shown that dimeric Ca2+-bound forms of S100A6 and S100P proteins, members of the multifunctional S100 protein family, are specific to GM-CSF. To probe selectivity of these interactions, the affinity of recombinant human GM-CSF to dimeric Ca2+-loaded forms of 18 recombinant human S100 proteins was studied by surface plasmon resonance spectroscopy. Of them, only S100A4 protein specifically binds to GM-CSF with equilibrium dissociation constant, Kd, values of 0.3-2 µM, as confirmed by intrinsic fluorescence and chemical crosslinking data. Calcium removal prevents S100A4 binding to GM-CSF, whereas monomerization of S100A4/A6/P proteins disrupts S100A4/A6 interaction with GM-CSF and induces a slight decrease in S100P affinity for GM-CSF. Structural modelling indicates the presence in the GM-CSF molecule of a conserved S100A4/A6/P-binding site, consisting of the residues from its termini, helices I and III, some of which are involved in the interaction with GM-CSF receptors. The predicted involvement of the 'hinge' region and F89 residue of S100P in GM-CSF recognition was confirmed by mutagenesis. Examination of S100A4/A6/P ability to affect GM-CSF signaling showed that S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. The ability of the S100 proteins to modulate GM-CSF activity is relevant to progression of various neoplasms and other diseases, according to bioinformatics analysis. The direct regulation of GM-CSF signaling by extracellular forms of the S100 proteins should be taken into account in the clinical use of GM-CSF and development of the therapeutic interventions targeting GM-CSF or its receptors.

Interaction of S100A6 Protein with the Four-Helical Cytokines.

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 have revealed the ability of specific S100 proteins to modulate cell signaling via direct interaction with cytokines. Previously, we revealed the binding of ca. 71% of the four-helical cytokines via the S100P protein, due to the presence in its molecule of a cytokine-binding site overlapping with the binding site for the S100P receptor. Here, we show that another S100 protein, S100A6 (that has a pairwise sequence identity with S100P of 35%), specifically binds numerous four-helical cytokines. We have studied the affinity of the recombinant forms of 35 human four-helical cytokines from 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 equilibrium dissociation constants from 0.3 nM to 12 µM. Overall, S100A6 interacts with ca. 73% of the four-helical cytokines studied to date, with a selectivity equivalent to that for the S100P protein, with the differences limited to the binding of interleukin-2 and oncostatin M. The molecular docking study evidences the presence in the 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 the S100 family of a site specific to a wide range of four-helical cytokines. This unique feature of the S100 proteins potentially allows them to modulate the activity of the numerous four-helical cytokines in the disorders accompanied by an excessive release of the cytokines.

Specific S100 Proteins Bind Tumor Necrosis Factor and Inhibit Its Activity.

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 (Kd) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent Kd 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.

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

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.

Ibuprofen Favors Binding of Amyloid-ß Peptide to Its Depot, Serum Albumin.

The deposition of amyloid-ß peptide (Aß) in the brain is a critical event in the progression of Alzheimer's disease (AD). This Aß deposition could be prevented by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aß. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aß40/Aß42 by a factor of 3-5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aß40 fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aß40-binding sites of HSA. The revealed enhancement of the HSA-Aß interaction by IBU and the strengthened inhibition of Aß fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD.

Interferon-ß Activity Is Affected by S100B Protein.

Interferon-ß (IFN-ß) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. Recently, we reported that IFN-ß activity is affected by interactions with S100A1, S100A4, S100A6, and S100P proteins, which are members of the S100 protein family of multifunctional Ca2+-binding proteins possessing cytokine-like activities (Int J Mol Sci. 2020;21(24):9473). Here we show that IFN-ß interacts with one more representative of the S100 protein family, the S100B protein, involved in numerous oncological and neurological diseases. The use of chemical crosslinking, intrinsic fluorescence, and surface plasmon resonance spectroscopy revealed IFN-ß binding to Ca2+-loaded dimeric and monomeric forms of the S100B protein. Calcium depletion blocks the S100B-IFN-ß interaction. S100B monomerization increases its affinity to IFN-ß by 2.7 orders of magnitude (equilibrium dissociation constant of the complex reaches 47 pM). Crystal violet assay demonstrated that combined application of IFN-ß and S100B (5-25 nM) eliminates their inhibitory effects on MCF-7 cell viability. Bioinformatics analysis showed that the direct modulation of IFN-ß activity by the S100B protein described here could be relevant to progression of multiple oncological and neurological diseases.

Erythropoietin Interacts with Specific S100 Proteins.

Erythropoietin (EPO) is a clinically significant four-helical cytokine, exhibiting erythropoietic, cytoprotective, immunomodulatory, and cancer-promoting activities. Despite vast knowledge on its signaling pathways and physiological effects, extracellular factors regulating EPO activity remain underexplored. Here we show by surface plasmon resonance spectroscopy, that among eighteen members of Ca2+-binding proteins of the S100 protein family studied, only S100A2, S100A6 and S100P proteins specifically recognize EPO with equilibrium dissociation constants ranging from 81 nM to 0.5 µM. The interactions occur exclusively under calcium excess. Bioinformatics analysis showed that the EPO-S100 interactions could be relevant to progression of neoplastic diseases, including cancer, and other diseases. The detailed knowledge of distinct physiological effects of the EPO-S100 interactions could favor development of more efficient clinical implications of EPO. Summing up our data with previous findings, we conclude that S100 proteins are potentially able to directly affect functional activities of specific members of all families of four-helical cytokines, and cytokines of other structural superfamilies.

Specific cytokines of interleukin-6 family interact with S100 proteins.

Cytokines of interleukin-6 (IL-6) family are important signaling proteins involved in various physiological and pathological processes. Earlier, we described interactions between IL-11 and S100P/B proteins from the family of S100 proteins engaged in the pathogenesis of numerous diseases. We probed here interactions between seven IL-6 family cytokines (IL-6, IL-11, OSM, LIF, CNTF, CT-1, and CLCF1) and fourteen S100 proteins (S100A1/A4/A6/A7/A8/A9/A10/A11/A12/A13/A14/A15/B/P). Surface plasmon resonance spectroscopy revealed formation of calcium-dependent complexes between IL-11, OSM, CNTF, CT-1, and CLCF1 and distinct subsets of S100A1/A6/B/P proteins with equilibrium dissociation constants of 19 nM - 12 µM. The existence of a network of interactions between Ca2+-loaded S100 proteins and IL-6 family cytokines suggest regulation of these cytokines by the extracellular forms of S100 proteins.

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid ß Peptide.

Prevention of amyloid ß peptide (Aß) deposition via facilitation of Aß binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer's disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aß by a factor of 3 (BBRC, 510(2), 248-253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aß monomer to HSA by a factor of 7/17 for Aß40/Aß42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA's affinity to monomeric Aß, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aß release from HSA in the central nervous system due to impairment of the SRO-mediated Aß trapping by HSA.

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins.

Interferon-ß (IFN-ß) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-ß and S100P lowering IFN-ß cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633-639). S100P is a member of large family of multifunctional Ca2+-binding proteins with cytokine-like activities. To probe selectivity of IFN-ß-S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca2+-dependent binding to IFN-ß with equilibrium dissociation constants, Kd, of 0.04-1.5 µM for their Ca2+-bound homodimeric forms. Calcium depletion abolishes the S100-IFN-ß interactions. Monomerization of S100A1/A4/A6 decreases Kd values down to 0.11-1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-ß-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-ß activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-ß.

Mouse S100G protein exhibits properties characteristic of a calcium sensor.

Bovine S100 G (calbindin D9k, small Ca2+-binding protein of the EF-hand superfamily) is considered as a calcium buffer protein; i.e., the binding of Ca2+ practically does not change its general conformation. A set of experimental approaches has been used to study structural properties of apo- and Ca2+-loaded forms of mouse S100 G (81.4% identity in amino acid sequence with bovine S100 G). This analysis revealed that, in contrast to bovine S100 G, the removal of calcium ions increases α-helices content of mouse S100 G protein and enhances its accessibility to digestion by α-chymotrypsin. Furthermore, mouse apo-S100 G is characterized by a decreased surface hydrophobicity and reduced tendency for oligomerization. Such behavior is typical of calcium sensor proteins. Apo-state of mouse S100 G still has rather compact structure, which can be cooperatively unfolded by temperature and GdnHCl. Computational analysis of amino acid sequences of S100 G proteins shows that these proteins could be in a disordered state upon a removal of the bound calcium ions. The experimental data show that, although mouse apo-S100 G is flexible compared to the Ca2+-loaded state, the apo-form is not completely disordered and preserves some cooperatively meting structure. The origin of the unexpectedly high stability of mouse S100 G can be rationalized by an exceptionally strong association of its N- and C-terminal parts containing the EF-hands I and II, respectively.

Monomeric state of S100P protein: Experimental and molecular dynamics study.

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca2+-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04-10 µM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 µM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.

Analyzing the structural and functional roles of residues from the 'black' and 'gray' clusters of human S100P protein.

Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I ('black' cluster) and cluster II ('grey' cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these 'black' and 'gray' clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1-2 strong calcium binding sites (K1 = 4 × 106 M-1) and two cooperative low affinity (K2 = 4 × 104 M-1) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II.

Calcium-dependent interaction of monomeric S100P protein with serum albumin.

S100 proteins are multifunctional (intra/extra)cellular mostly dimeric calcium-binding proteins engaged into numerous diseases. We have found that monomeric recombinant human S100P protein interacts with intact human serum albumin (HSA) in excess of calcium ions with equilibrium dissociation constant of 25-50nM, as evidenced by surface plasmon resonance spectroscopy and fluorescent titration by HSA of S100P labelled by fluorescein isothiocyanate. Calcium removal or S100P dimerization abolish the S100P-HSA interaction. The interaction is selective, since S100P does not bind bovine serum albumin and monomeric human S100B lacks interaction with HSA. In vitro glycation of HSA disables its binding to S100P. The revealed selective and highly specific conformation-dependent interaction between S100P and HSA shows that functional properties of monomeric and dimeric forms of S100 proteins are different, and raises concerns on validity of cell-based assays and animal models used for studies of (patho)physiological roles of extracellular S100 proteins.

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

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.

The impact of alpha-N-acetylation on structural and functional status of parvalbumin.

The effect of alpha-N-acetylation (Nt-acetylation) on the properties of parvalbumin (PA), a Ca2+-binding relaxing factor of skeletal muscles and major food allergen, has been explored. Intact PA contains an N-terminal acetyl group which is absent in the protein expressed in Escherichia coli (rWT), as confirmed by mass spectrometry. Compared to intact pike α-PA, its rWT form exhibits essentially altered profile of thermal unfolding, lowered α-helicity, and decreased affinities to Ca2+ and Mg2+. The structural destabilization of the rWT protein results in lowered resistance to chymotryptic digestion and increased propensity to oligomerization. The rate constants of Ca2+ dissociation from the rWT PA are markedly increased, which indicates that Nt-acetylation modifies functional status of the protein. Rat α-PA demonstrates similar properties for intact and rWT forms. The drastic difference in the effects induced by Nt-acetylation in the PA orthologs can be rationalized by higher disorder level of AB domain in pike PA. Though evolution of PA's genes resulted in the protein sequences with highly divergent properties, Nt-acetylation unifies their functional properties. The structural stability conferred to PA by Nt-acetylation may contribute to its allergenicity. Overall, Nt-acetylation is shown to be a prerequisite for maintenance of structural and functional status of some parvalbumins.

Metal-controlled interdomain cooperativity in parvalbumins.

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (alpha and beta isoforms), Baltic cod, and rat (alpha and beta isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike alpha-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 degrees C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca(2+) ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg(2+)- and Na(+)-loaded forms of pike alpha-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg(2+)- and Na(+)-loaded forms of pike alpha-PA), to the more complex mechanisms (Ca(2+)-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca(2+)-loaded pike alpha-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike alpha-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na(+)-bound state), thermophilic (Mg(2+)-form), or even of the hyperthermophilic origin (Ca(2+)-form).