LexA/signal peptidase-like superfamily proteins are serine proteases that use the Ser-Lys catalytic dyad to carry out their biological functions. Here, we investigate the two known families of LexA/signal peptidase-like superfamily proteins, the type I signal peptidase and LexA endopeptidase domain-like, and describe the structural catalytic cores that govern the catalytic residues in these proteins. We show that the structural catalytic core of these proteins is a combination of two subzones, the NucBaseOmega and Omega. While the NucBaseOmega subzone is a pattern observed in all proteins of the studied superfamily, the Omega subzone in the type I signal peptidase family differs from that of the LexA endopeptidase domain-like family. Thus, the amino acids and 3D characteristics of the Omega subzone are a structural marker of the proteins belonging to a specific family.

β-Lactamase/transpeptidase-like superfamily proteins are serine proteases that use the Ser–Lys catalytic dyad to carry out their biological functions. Here, we investigate the three known families of β-lactamase/transpeptidase-like superfamily proteins, β-lactamase/D-Ala carboxypeptidase, glutaminase, and Dac-like, and describe the structural catalytic cores that govern the catalytic residues in these proteins. We show that the structural catalytic core of these proteins is a combination of three zones, the mutual three-dimensional arrangement of which correspondingly determines their belonging to one of seven and twenty-four established groups and subgroups.

Receptor for Advanced Glycation Endproducts, RAGE, is a transmembrane multi-ligand receptor, which plays key role in amplifying immune response in numerous inflammatory processes and serious diseases. RAGE fulfills its function through binding of a variety of ligands. The structure of RAGE includes one transmembrane domain, one immunoglobulin V-like domain and two immunoglobulin C-like domains. Here, we have used combination of methods of domain structure analysis, fold classification, structure comparison and analysis of protein/ligand interactions with a chosen set of domains of RAGE and other immunoglobulin-like proteins from several different structural databases to describe structure-functional core and ligand recognition by the V-type domain of RAGE, and compare those to the other immunoglobulin-like proteins. We show that from one hand, similar to the other immunoglobulin-like proteins, RAGE contains a conserved substructure, the cross-β zone, with the cross-β motif at its core. The cross-β zone incorporates the hydrophobic core of RAGE and two multivalent surface ligand-binding sites for small molecules and proteins, one on the surface of the C-F-G β-sheet of the β-sheet sandwich, and the other formed by the CE-loop region. RAGE oligomerization allows RAGE to bind nucleic acids at a site different from the two mentioned above, leading to at least four distinct sites on the surface of RAGE: two for binding small molecules and proteins, one for binding nucleic acids, and one for RAGE dimerization. From the other hand, regardless of structural similarities with immunoglobulins, RAGE does not contain ligand-binding regions analogous to Complementary-Determining Regions in immunoglobulins, thus making RAGE and immunoglobulins functionally different.

Subtilisin-like proteins are serine proteases that use two types of catalytic triads: Ser-His-Asp and Ser-Glu-Asp. Here, we investigate the two known families of subtilisin-like proteins, the subtilases (Ser-His-Asp triad) and the serine-carboxyl proteinases (Ser-Glu-Asp triad), and describe the local structural arrangements (cores) that govern the catalytic residues in these proteins. We show the separation of the cores into conserved structural zones, which can be repeatedly found in different structures, and compare the structural cores in subtilisin-like proteins with those in trypsin-like serine proteases and alpha/beta-hydrolases.

The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering.

The nucleophile elbow is a well-known structural motif, which exists in proteins with catalytic triads and contains a catalytic nucleophile and the first node of an oxyanion hole. Here, we show that structural similarities of proteins with the nucleophile elbow extend beyond simple nucleophile elbow motifs. The motifs are incorporated into larger conserved structural organizations, the ElbowFlankOxy networks, incorporating motifs and flanking residues and networks of conserved interactions. A detailed structural analysis shows two major types of ElbowFlankOxy networks, depending on the formation of the oxyanion hole. Additionally, the ElbowFlankOxy networks show three classes: Class 1-2-3, 3-1-2, and 2-3-1, defined by the order in which the catalytic nucleophile and key interacting residues are located in the amino acid sequence, giving rise to six ElbowFlankOxy network variations. This makes it possible to properly position homologous non-catalytic, non-standard, and unusual catalytic triad active sites of proteins with the nucleophile elbow within the fold classification.

SGNH hydrolase-like fold proteins are serine proteases with the default Asp-His-Ser catalytic triad. Here, we show that these proteins share two unique conserved structural organizations around the active site: (1) the Nuc-Oxy Zone around the catalytic nucleophile and the oxyanion hole, and (2) the Acid-Base Zone around the catalytic acid and base. The Nuc-Oxy Zone consists of 14 amino acids cross-linked with eight conserved intra- and inter-block hydrogen bonds. The Acid–Base Zone is constructed from a single fragment of the polypeptide chain, which incorporates both the catalytic acid and base, and whose N- and C-terminal residues are linked together by a conserved hydrogen bond. The Nuc-Oxy and Acid-Base Zones are connected by an SHLink, a two-bond conserved interaction from amino acids, adjacent to the catalytic nucleophile and base.

Intracellular calcium sensor protein calmodulin (CaM) belongs to the large EF-hand protein superfamily. CaM shows a unique and not fully understood ability to bind to multiple targets, allows them to participate in a variety of regulatory processes. The protein has two approximately symmetrical globular domains (the N- and C-lobes). Analysis of the CaM-binding sites of target proteins showed that they have two hydrophobic 'anchor' amino acids separated by 10 to 17 residues. Consequently, several CaM-binding motifs: {1-10}, {1-11}, {1-13}, {1-14}, {1-16}, {1-17}, differing by the distance between the two anchor residues along the amino acid sequence, have been identified. Despite extensive structural information on the role of target-protein amino acid residues in the formation of complexes with CaM, much less is known about the role of amino acids from CaM contributing to these interactions. In this work, a quantitative analysis of the contact surfaces of CaM and target proteins has been carried out for 35 representative three-dimensional structures. It has been shown that, in addition to the two hydrophobic terminal residues of the target fragment, the interaction also involves residues that are 4 residues earlier in the sequence (binding mode {1-5}). It has also been found that the N- and C-lobes of CaM bind the {1-5} motif located at the ends of the target in a structurally identical manner. Methionine residues at positions 51 (corresponding to 124 in the C-lobe), 71 (144), and 72 (145) of the CaM amino acid sequence are key hydrophobic residues for this interaction. They are located at the N- and C-boundaries of the even EF-hand motifs. The hydrophobic core of CaM ('Ф-quatrefoil') consists of 10 amino acids in the N-lobe (and in the C-lobe): Phe16 (Phe89), Phe19 (Phe92), Ile27 (Ile100), Thr29 (Ala102), Leu32 (Leu105), Ile52 (Ile125), Val55 (Ala128), Ile63 (Val136), Phe65 (Tyr138), and Phe68 (Phe141) and do not intersect with the target-binding methionine residues. CaM belongs to the 'dynamic' group of EF-hand proteins, in which calcium and protein ligand binding causes only global conformational changes but does not alter the conservative 'black' and 'grey' clusters described in our earlier works (PLoS One. 2014; 9(10):e109287). The membership of CaM in the 'dynamic' group is determined by the triggering and protective methionine layer: Met51 (Met124), Met71 (Met144) and Met72 (Met145). HIGHLIGHTSInterchain interactions in the unique 35 CaM complex structures were analyzed.Methionine amino acids of the N- and C-lobes of CaM form triggering and protective layers.Interactions of the target terminal residues with these methionine layers are structurally identical.CaM belonging to the 'dynamic' group is determined by the triggering and protective methionine layer.Communicated by Ramaswamy H. Sarma.

Three dimensional structures of (chymo)trypsin-like proteinase (3CLpro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val86Leu, Lys88Arg, Phe134His, and Asn180Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CLpro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val35Thr, Ser46Ala, Asn65Ser, Ala94Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CLpro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CLpro(s) can use for functional regulation an autolytic loop and the unique Asp33-Asn95 region (the Asp33-Asn95 Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CLpro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.

Proteinases with the (chymo)trypsin-like serine/cysteine fold comprise a large superfamily performing their function through the Acid-Base-Nucleophile catalytic triad. In our previous work (Denesyuk AI, Johnson MS, SaloAhen OMH, Uversky VN, Denessiouk K. Int J Biol Macromol. 2020;153:399-411), we described a universal threedimensional (3D) structural motif, NBCZone, that contains eleven amino acids: dipeptide 42 T-43 T, pentapeptide 54 T-55 T-56 T-57 T(base)-58 T, tripeptide 195 T(nucleophile)-196 T-197 T and residue 213 T (T-numeration of amino acids in trypsin). The comparison of the NBCZones among the members of the (chymo)trypsin-like protease family suggested the existence of 15 distinct groups. Within each group, the NBCZones incorporate an identical set of conserved interactions and bonds. In the present work, the structural environment of the catalytic acid at the position 102 T and the fourth member of the "catalytic tetrad" at the position 214 T was analyzed in 169 3D structures of proteinases with the (chymo)trypsin-like serine/cysteine fold. We have identified a complete Structural Catalytic Core (SCC) consisting of two classes and four groups. The proteinases belonging to different classes and groups differ from each other by the nature of the interaction between their N-and C-terminal beta-barrels. Comparative analysis of the 3CLpro(s) from SARS-CoV-2 and SARS-CoV, used as an example, showed that the amino acids at positions 103 T and 179 T affect the nature of the interaction of the "catalytic acid" core (102 T-Core, N-terminal beta-barrel) with the "supplementary" core (S-Core, C-terminal beta-barrel), which ultimately results in the modulation of the enzymatic activity. The reported analysis represents an important standalone contribution to the analysis and systematization of the 3D structures of (chymo)trypsin-like serine/cysteine fold proteinases. The use of the developed approach for the comparison of 3D structures will allow, in the event of the appearance of new representatives of a given fold in the PDB, to quickly determine their structural homologues with the identification of possible differences. (c) 2021 Elsevier B.V. All rights reserved.

(Chymo)trypsin-like serine fold proteases belong to the serine/cysteine proteases found in eukaryotes, prokaryotes, and viruses. Their catalytic activity is carried out using a triad of amino acids, a nucleophile, a base, and an acid. For this superfamily of proteases, we propose the existence of a universal 3D structure comprising 11 amino acids near the catalytic nucleophile and base – Nucleophile-Base Catalytic Zone (NBCZone). The comparison of NBCZones among 169 eukaryotic, prokaryotic, and viral (chymo)trypsin-like proteases suggested the existence of 15 distinct groups determined by the combination of amino acids located at two “key” structure-functional positions 54T and 55T near the catalytic base His57T. Most eukaryotic and prokaryotic proteases fell into two major groups, [ST]A and TN. Usually, proteases of [ST]A group contain a disulfide bond between cysteines Cys42T and Cys58T of the NBCZone. In contrast, viral proteases were distributed among seven groups, and lack this disulfide bond. Furthermore, only the [ST]A group of eukaryotic proteases contains glycine at position 43T, which is instrumental for activation of these enzymes. In contrast, due to the side chains of residues at position 43T prokaryotic and viral proteases do not have the ability to carry out the structural transition of the eukaryotic zymogen-zyme type.

The alpha/beta-Hydrolases (ABH) are a structural class of proteins that are found widespread in nature and includes enzymes that can catalyze various reactions in different substrates. The catalytic versatility of the ABH fold enzymes, which has been a valuable property in protein engineering applications, is based on a similar acid-base-nucleophile catalytic mechanism. In our research, we are concerned with the structure that surrounds the key units of the catalytic machinery, and we have previously found conserved structural organizations that coordinate the catalytic acid, the catalytic nucleophile and the residues of the oxyanion hole. Here, we explore the architecture that surrounds the catalytic histidine at the active sites of enzymes from 40 ABH fold families, where we have identified six conserved interactions that coordinate the catalytic histidine next to the catalytic acid and the catalytic nucleophile. Specifically, the catalytic nucleophile is coordinated next to the catalytic histidine by two weak hydrogen bonds, while the catalytic acid is directly involved in the coordination of the catalytic histidine through by two weak hydrogen bonds. The imidazole ring of the catalytic histidine is coordinated by a CH-π contact and a hydrophobic interaction. Moreover, the catalytic triad residues are connected with a residue that is located at the core of the active site of ABH fold, which is suggested to be the fourth member of a "structural catalytic tetrad". Besides their role in the stability of the catalytic mechanism, the conserved elements of the catalytic site are actively involved in ligand binding and affect other properties of the catalytic activity, such as substrate specificity, enantioselectivity, pH optimum and thermostability of ABH fold enzymes. These properties are regularly targeted in protein engineering applications, and thus, the identified conserved structural elements can serve as potential modification sites in order to develop ABH fold enzymes with altered activities.

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2′,4,4′,6′-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p -coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p -coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.

We introduce five new local metal cation (first of all, Ca2+) recognition units in proteins: Clampn,(n−2), Clampn,(n−1), Clampn,n, Clampn,(n+1) and Clampn,(n+2). In these units, the backbone oxygen atom of a residue in position “n” of an amino acid sequence and side-chain oxygen atom of a residue in position “n + i” (i = −2 to +2) directly interact with a metal cation. An analysis of the known “Ca2+-bound niches” in proteins has shown that a system approach based on the simultaneous use of the Clamp units and earlier proposed One-Residue (OR)/Three-Residue (TR) units significantly improves the results of constructing metal cation-binding sites in proteins.

There are several families of cysteine proteinases with different folds – for example the (chymo)trypsin fold family and papain-like fold family – but in both families the hydrolase activity of cysteine proteinases requires a cysteine residue as the catalytic nucleophile. In this work, we have analyzed the topology of the active site regions in 146 three-dimensional structures of proteins belonging to the Papain-like Cysteine Proteinase (PCP) superfamily, which includes papain as a typical representative of this protein superfamily. All analyzed enzymes contain a unique structurally closed conformation – a “PCP-Zone” – which can be divided into two groups, Class A and Class B. Eight structurally conserved amino acids of the PCP-Zone form a common Structural Core. The Structural Core, catalytic nucleophile, catalytic base and residue Xaa – which stabilizes the side-chain conformation of the catalytic base – make up a PCP Structural Catalytic Core (PCP-SCC). The PCP-SCC of Class A and Class B are divided into 5 and 2 types, respectively. Seven variants of the mutual arrangement of the amino-acid side chains of the catalytic triad – nucleophile, base and residue Xaa – within the same fold clearly demonstrate how enzymes with the papain-like fold adapt to the need to perform diverse functions in spite of their limited structural diversity. The roles of both the PCP-Zone of SARS-CoV-2-PLpro described in this study and the NBCZone of SARS-CoV-2-3CLpro presented in our earlier article (Denesyuk AI, Johnson MS, Salo-Ahen OMH, Uversky VN, Denessiouk K. Int J Biol Macromol. 2020;153:399-411) that are in contacts with inhibitors are discussed.

Plants must precisely regulate their signalling pathways to respond to environmental changes promptly. Sucrose non fermenting1 (SNF1)-related protein kinases (SnRK) 2 are essential kinases in abiotic stress responses, including responses to abscisic acid. Although homologs of SnRKs in yeast require a γ-subunit for full activation, it has been unclear whether SnRK2s in higher plants are affected by γ-subunits. In this report, we aimed to show the effect of Arabidopsis KIN γ-subunit 1 (KING1), which is a potential γ-subunit, on the activity of SnRK2. A recombinant KING1 bound to SnRK2.6 and functionally inhibited its activity in vitro. On the other hand, KING1 facilitated the activity of SnRK2.2. Structural models suggest that significant structural changes occurred as a result of KING1 binding to the C-terminal tail of SnRK2s. Since KING1 inhibited the kinase activity of a chimeric protein consisting of the N-terminal domain of SnRK2.6 and the C-terminal domain of SnRK2.2, regulation by KING1 was determined by the N-terminal domain of SnRK2s. Together, these results show that KING1 can mediate activity of SnRK2s in vitro.

Recently, we have found that calcium binding proteins of the EF-hand superfamily (i.e., a large family of proteins containing helix-loop-helix calcium binding motif or EF-hand) contain two types of conserved clusters called cluster I ('black' cluster) and cluster II ('grey' cluster), which provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domains. Cluster I is more conservative and mostly incorporates aromatic amino acids, whereas cluster II includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. Recoverin is EF-hand Ca2+-binding protein containing two 'black' clusters comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172 (C-terminal domain) as well as two 'gray' clusters comprised of F70, Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain). To understand a role of these residues in structure and function of human recoverin, we sequentially substituted them for alanine and studied the resulting mutants by a set of biophysical methods. Under metal-free conditions, the 'black' clusters mutants (except for F35A and E169A) were characterized by an increase in the α-helical content, whereas the 'gray' cluster mutants (except for K119A) exhibited the opposite behavior. By contrast, in Ca2+-loaded mutants the α-helical content was always elevated. In the absence of calcium, the substitutions only slightly affected multimerization of recoverin regardless of their localization (except for K119A). Meanwhile, in the presence of calcium mutations in N-terminal domain of the protein significantly suppressed this process, indicating that surface properties of Ca2+-bound recoverin are highly affected by N-terminal cluster residues. The substitutions in C-terminal clusters generally reduced thermal stability of recoverin with F172A ('black' cluster) as well as W156A and K119A ('gray' cluster) being the most efficacious in this respect. In contrast, the mutations in the N-terminal clusters caused less pronounced differently directed changes in thermal stability of the protein. The substitutions of F172, W156, and K119 in C-terminal domain of recoverin together with substitution of Q46 in its N-terminal domain provoked significant but diverse changes in free energy associated with Ca2+ binding to the protein: the mutant K119A demonstrated significantly improved calcium binding, whereas F172A and W156A showed decrease in the calcium affinity and Q46A exhibited no ion coordination in one of the Ca2+-binding sites. The most of the N-terminal clusters mutations suppressed membrane binding of recoverin and its inhibitory activity towards rhodopsin kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated rhodopsin phosphorylation regardless of the presence of calcium. Taken together, these data confirm the scaffolding function of several cluster-forming residues and point to their critical role in supporting physiological activity of recoverin.

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 mu M. 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 degrees 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 SWOP protein is very low (0.5 mu M 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, S100P 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.

S100 proteins are EF-hand calcium-binding proteins of vertebrates exerting numerous intra- and extracellular actions and involved into multiple diseases. Some of 5100 proteins serve as extracellular damage signals via interaction with receptors. Although several 5100 proteins directly bind specific cytokines, this phenomenon remains underexplored. Using chemical crosslinking, intrinsic fluorescence and surface plasmon resonance spectroscopies, we show that S100P protein interacts with interferon beta (IFN-beta) depending on calcium level and oligomeric state of S100P. Dimeric Ca2+-loaded S100P binds IFN-beta with equilibrium dissociation constants, K-d, of 0.05-0.6 mu M. S100P monomerization favors this interaction decreasing K-d values down to 0.3-2 nM. Calcium depletion drastically lowers S100P affinity to IFN-beta. Other related EF-hand proteins studied (calmodulin, alpha-parvalbumin and S100G) do not bind IFN-beta, thereby confirming selectivity of the S100P - IFN-beta interaction. Crystal violet assay reveals that the S100P binding suppresses IFN-beta cytotoxicity to MCF-7 breast cancer cells. Since several cancers (breast, colon, lung, liver, etc.) exhibit dysregulated functioning of S100P and IFN-beta, their interaction could be relevant to the cancer progression and directed therapeutic interventions. (C) 2019 Elsevier B.V. All rights reserved.

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.