The active site of the SGNH hydrolase-like fold proteins: Nucleophile-oxyanion (Nuc-Oxy) and Acid-Base zones.

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.

Canonical structural-binding modes in the calmodulin-target protein complexes.

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.

Structural and functional significance of the amino acid differences Val35Thr, Ser46Ala, Asn65Ser, and Ala94Ser in 3C-like proteinases from SARS-CoV-2 and SARS-CoV.

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.

Structural leitmotif and functional variations of the structural catalytic core in (chymo)trypsin-like serine/cysteine fold proteinases.

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, Salo-Ahen OMH, Uversky VN, Denessiouk K. Int J Biol Macromol. 2020;153:399-411), we described a universal three-dimensional (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 ß-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 ß-barrel) with the "supplementary" core (S-Core, C-terminal ß-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.

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.

Papain-like cysteine proteinase zone (PCP-zone) and PCP structural catalytic core (PCP-SCC) of enzymes with cysteine proteinase fold.

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.

System Approach for Building of Calcium-Binding Sites in Proteins.

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.

NBCZone: Universal three-dimensional construction of eleven amino acids near the catalytic nucleophile and base in the superfamily of (chymo)trypsin-like serine fold proteases.

(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 acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements.

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.

Highly specific interaction of monomeric S100P protein with interferon beta.

S100 proteins are EF-hand calcium-binding proteins of vertebrates exerting numerous intra- and extracellular actions and involved into multiple diseases. Some of S100 proteins serve as extracellular damage signals via interaction with receptors. Although several S100 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-ß) depending on calcium level and oligomeric state of S100P. Dimeric Ca2+-loaded S100P binds IFN-ß with equilibrium dissociation constants, Kd, of 0.05-0.6 µM. S100P monomerization favors this interaction decreasing Kd values down to 0.3-2 nM. Calcium depletion drastically lowers S100P affinity to IFN-ß. Other related EF-hand proteins studied (calmodulin, α-parvalbumin and S100G) do not bind IFN-ß, thereby confirming selectivity of the S100P - IFN-ß interaction. Crystal violet assay reveals that the S100P binding suppresses IFN-ß cytotoxicity to MCF-7 breast cancer cells. Since several cancers (breast, colon, lung, liver, etc.) exhibit dysregulated functioning of S100P and IFN-ß, their interaction could be relevant to the cancer progression and directed therapeutic interventions.

Experimental Insight into the Structural and Functional Roles of the 'Black' and 'Gray' Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs.

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.

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.

Distinctive structural motifs co-ordinate the catalytic nucleophile and the residues of the oxyanion hole in the alpha/beta-hydrolase fold enzymes.

The alpha/beta-hydrolases (ABH) are among the largest structural families of proteins that are found in nature. Although they vary in their sequence and function, the ABH enzymes use a similar acid-base-nucleophile catalytic mechanism to catalyze reactions on different substrates. Because ABH enzymes are biocatalysts with a wide range of potential applications, protein engineering has taken advantage of their catalytic versatility to develop enzymes with industrial applications. This study is a comprehensive analysis of 40 ABH enzyme families focusing on two identified substructures: the nucleophile zone and the oxyanion zone, which co-ordinate the catalytic nucleophile and the residues of the oxyanion hole, and independently reported as critical for the enzymatic activity. We also frequently observed an aromatic cluster near the nucleophile and oxyanion zones, and opposite the ligand-binding site. The nucleophile zone, the oxyanion zone and the residue cluster enriched in aromatic side chains comprise a three-dimensional structural organization that shapes the active site of ABH enzymes and plays an important role in the enzymatic function by structurally stabilizing the catalytic nucleophile and the residues of the oxyanion hole. The structural data support the notion that the aromatic cluster can participate in co-ordination of the catalytic histidine loop, and properly place the catalytic histidine next to the catalytic nucleophile.

On the relationship between the conserved 'black' and 'gray' structural clusters and intrinsic disorder in parvalbumins.

Recently we found two highly conserved structural motifs in the members of the EF-hand protein family, which provide a supporting scaffold for their Ca2+ binding loops. Each structural motif is formed by a cluster of three amino acids. These clusters were called 'black' cluster (cluster I) and 'gray' cluster (cluster II). In the present work, we studied the relationship between the location of the 'black' and 'gray' structural clusters in parvalbumins and the location of the amino acid sequence regions with a tendency for intrinsic disorder. This analysis revealed that in parvalbumins, the residues in the vicinity of the conserved structural clusters constitute parts of the conserved motifs enriched in the disorder-promoting residues. Therefore, the clusters found in parvalbumins are characterized not only by the presence of conserved amino acid residues, but also by the conserved distribution of the intrinsic disorder predisposition within their sequences, suggesting the presence of conserved structural dynamics in the apo-forms of parvalbumins, where the black cluster appears to have greater mobility than the gray cluster.

Comprehensive analysis of the roles of 'black' and 'gray' clusters in structure and function of rat ß-parvalbumin.

Recently we found two highly conserved structural motifs in the proteins of the EF-hand calcium binding protein family. These motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif forms a cluster of three amino acids called cluster I ('black' cluster) and cluster II ('grey' cluster). Cluster I is much more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II includes a mix of aromatic, hydrophobic, and polar amino acids. The 'black' and 'gray' clusters in rat ß-parvalbumin consist of F48, A100, F103 and G61, L64, M87, respectively. In the present work, we sequentially substituted these amino acids residues by Ala, except Ala100, which was substituted by Val. Physical properties of the mutants were studied by circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probe methods. The Ca2+ and Mg2+ binding affinities of these mutants were evaluated by intrinsic fluorescence and equilibrium dialysis methods. In spite of a rather complicated pattern of contributions of separate amino acid residues of the 'black' and 'gray' clusters into maintenance of rat ß-parvalbumin structural and functional status, the alanine substitutions in the cluster I cause noticeably more pronounced changes in various structural parameters of proteins, such as hydrodynamic radius of apo-form, thermal stability of Ca2+/Mg2+-loaded forms, and total energy of Ca2+ binding in comparison with the changes caused by amino acid substitutions in the cluster II. These findings were further supported by the outputs of computational analysis of the effects of these mutations on the intrinsic disorder predisposition of rat ß-parvalbumin, which also indicated that local intrinsic disorder propensities and the overall levels of predicted disorder were strongly affected by mutations in the cluster I, whereas mutations in cluster II had less pronounced effects. These results demonstrate that amino acids of the cluster I provide more essential contribution to the maintenance of structuraland functional properties of the protein in comparison with the residues of the cluster II.

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like ß-propeller structures.

An integrin-like ß-propeller domain contains seven repeats of a four-stranded antiparallel ß-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type ß-propeller. Here, we show unique structural links that join different blades of the ß-propeller structure, which together with the structural motif for a single blade are repeated in a ß-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type ß-propeller domain and two non-integrin type ß-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5.

Building kit for metal cation binding sites in proteins.

Starting with conformations of calcium-binding sites in parvalbumin and integrin (representative structures of EF-hand and calcium blade zones, respectively) we introduce four new different local Ca2+-recognition units in proteins: a one-residue unit type I (ORI); a three-residue unit type I (TRI); a one-residue unit type II (ORII) and a three-residue unit type II (TRII). Based on the amount and nature of variable atoms, the type I and II units theoretically can have four and twelve variants, respectively. Analysis of known "Ca2+-bound functional niches" in proteins revealed presence of almost all possible variants of Ca2+-recognition units in actual structures. Parvalbumin, integrin alpha-IIb and sixteen other proteins with different Ca2+-bound functional niches contain various consecutively joined combinations of OR(I/II) and TR(I/II) units. Such a OR(I/II)+TR(I/II) joint unit forms a tripeptide, which uses three main-chain atoms for metal binding: nitrogenn (Donor), oxygenn (Acceptor) and nitrogenn+2 (Donor). Thus, taken together, the described ORI, TRI, ORII and TRII units can serve as elementary blocks to construct more complex calcium recognizing substructures in a variety of calcium binding sites of unrelated proteins.

Alpha/beta-hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families.

The alpha/beta-hydrolases are a family of acid-base-nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn. We show that binding of an external ligand does not change the structure of the catalytic acid zone and both the ligand-free and ligand-bound forms of the protein belong to the same catalytic acid zone subgroup. We also show that the catalytic acid zone coordinates the position of the catalytic histidine loop directly above its plane, and consequently, fixes the catalytic histidine in a proper position near the catalytic acid. Finally, we demonstrate that the catalytic acid zone plays a key role in multi-subunit complex formation in ABH enzymes, and is involved in interactions with other proteins. As a result, we speculate that each of the catalytic triad residues has its own supporting structural scaffold, similar to the catalytic acid zone, described above, which together form the extended catalytic triad motif. Each scaffold coordinates the function of its respective catalytic residue, and can even compensate for the loss of protein function, if the catalytic amino acid is mutated.

Novel calcium recognition constructions in proteins: Calcium blade and EF-hand zone.

Metal ions can regulate various cell processes being first, second or third messengers, and some of them, especially transition metal ions, take part in catalysis in many enzymes. As an intracellular ion, Ca2+ is involved in many cellular functions from fertilization and contraction, cell differentiation and proliferation, to apoptosis and cancer. Here, we have identified and described two novel calcium recognition environments in proteins: the calcium blade zone and the EF-hand zone, common to 12 and 8 different protein families, respectively. Each of the two environments contains three distinct structural elements: (a) the well-known characteristic Dx[DN]xDG motif; (b) an adjacent structurally identical segment, which binds metal ion in the same way between the calcium blade zone and the EF-hand zone; and (c) the following structurally variable segment, which distinguishes the calcium blade zone from the EF-hand zone. Both zones have sequence insertions between the last residue of the zone and calcium-binding residues in positions V or VI. The long insertion often connects the active and the calcium-binding sites in proteins. Using the structurally identical segments as an anchor, we were able to construct the classical calmodulin type EF-hand calcium-binding site out of two different calcium-binding motifs from two unrelated proteins.