Two groups and three classes of the conserved structural organization of nucleophile and non-canonical ElbowFlankOxy networks in different superfamily proteins.

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.

Structural Catalytic Core of the Members of the Superfamily of Acid Proteases.

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.

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.

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).

Structural and functional significance of the amino acid differences ValThr, SerAla, AsnSer, and AlaSer in 3C-like proteinases from SARS-CoV-2 and SARS-CoV.

Three dimensional structures of (chymo)trypsin-like proteinase (3CL) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the ValLeu, LysArg, PheHis, and AsnLys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CL 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.

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.

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

Interferon-β (IFN-β) 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-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633-639).

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.

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

We introduce five new local metal cation (first of all, Ca) recognition units in proteins: Clamp, Clamp, Clamp, Clamp and Clamp. 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 "Ca-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 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.

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.

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 Ca binding loops and contribute to the hydrophobic core of the EF-hand domains.

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 Ca-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers.

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).

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.

Comprehensive analysis of the roles of 'black' and 'gray' clusters in structure and function of rat β-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 Ca 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).

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 Ca 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).

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

An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition.

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 Ca-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 "Ca-bound functional niches" in proteins revealed presence of almost all possible variants of Ca-recognition units in actual structures.

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.

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, Ca 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.

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.

Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use.