Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found

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 β-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 β-barrel) with the “supplementary” core (S-Core, C-terminal β-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

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

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

Intrinsic Disorder in S100 Proteins

Although the members of the largest subfamily of the EF-hand proteins, S100 proteins, are evolutionarily young, their functional diversity is extremely broad, partly due to their ability to adapt to various targets. This feature is a hallmark of intrinsically disordered proteins (IDPs), but none of the S100 proteins are recognized as IDPs. S100 are predicted to be enriched in intrinsic disorder, with 62% of them being predicted to be disordered by at least one of the predictors: 31% are recognized as ‘molten globules’ and 15% are shown to be in extended disordered form. The disorder level of predicted disordered S100 regions is conserved compared to that of more structured regions. The central disordered stretch corresponds to the major part of pseudo EF-hand loop, helix II, hinge region, and an initial part of helix III. It contains about half of known sites of enzymatic post-translational modifications (PTMs), confirming that this region can be flexible in vivo. Most of the internal residues missing in tertiary structures belong to the hinge. Both hinge and pseudo EF-hand loop correspond to the local maxima of the PONDR® VSL2 score and are shown to be evolutionary hotspots, leading to gain of new functional properties. The action of PTMs is shown to be destabilizing, in contrast with the effect of metal-binding or S100 dimerization. Formation of the S100 heterodimers relies on the interplay between the structural rigidity of one of the S100 monomers and the flexibility of another monomer. The ordered regions dominate in the S100 homodimerization sites. Target-binding sites generally consist of distant regions, drastically differing in their disorder level. The disordered region comprising most of the hinge and the N-terminal half of helix III is virtually not involved into dimerization, being intended solely for target recognition. The structural flexibility of this region is essential for recognition of diverse target proteins. At least 86% of multiple interactions of S100 proteins with binding partners are attributed to the S100 proteins predicted to be disordered. Overall, the intrinsic disorder is inherent to many S100 proteins and is vital for activity and functional diversity of the family

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

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 β-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 β-barrel) with the “supplementary” core (S-Core, C-terminal β-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

Structural and Functional Significance of the Amino Acid Differences Val\u3csub\u3e35\u3c/sub\u3eThr, Ser\u3csub\u3e46\u3c/sub\u3eAla, Asn\u3csub\u3e65\u3c/sub\u3eSer, and Ala\u3csub\u3e94\u3c/sub\u3eSer 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

Apo-parvalbumin as an Intrinsically Disordered Protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (α- and β-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in α-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge–charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-α-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20°C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10–15°C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (α-PA) or two (β-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data. Proteins 2008. © 2008 Wiley-Liss, Inc

Metal-controlled Interdomain Cooperativity in Parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β 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 α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca2+ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg2+- and Na+-loaded forms of pike α-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., Mg2+- and Na+-loaded forms of pike α-PA), to the more complex mechanisms (Ca2+-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 Ca2+-loaded pike α-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 α-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 (Mg2+-form), or even of the hyperthermophilic origin (Ca2+-form)