Alexander I. Denesyuk

Nucleotide sequence of the Yersinia pestis gene encoding F1 antigen and the primary structure of the protein. Putative T and B cell epitopes.

The plasmid‐located gene cafl encoding the capsular antigen fraction 1 (F1) of Yersinia pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced M r of 17.6 kDa. The signal peptide sequence was highly homologous to the E. coli consensus signal sequence. The F1 was assumed to have β‐sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells.

Dimer structure as a minimum cooperative subunit of small heat-shock proteins

Recently, it has been shown that small heat-shock proteins (Hsp25, Hsp27) are molecular chaperones. They bind to thermally unfolded proteins and can also assist refolding of denatured proteins. Mammalian small Hsps can form oligomeric structures of about 32 subunits. Until now, no data about cooperativity and stability of the interactions between the subunits of sHsps are available. To analyze these interactions we studied mouse Hsp25 and human Hsp27 by difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD). Here we show that, according to DASM data, the minimum cooperatively melting structure is a sHsp-dimer. CD data indicate that Hsp25 major secondary structure, the beta-pleated conformation, is resistant to acidic influence up to pH 4.5 and, at neutral pH values, to heat treatment up to 60 degrees C. The melting pattern of Hsp25/27 bears resemblance to alpha-crystallins. CD data indicate similar secondary, tertiary and quaternary structures of the proteins compared. This finding is in agreement with the revealed homology of primary structure of these proteins and their common chaperone function.

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.

Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes.

A detailed comparison of the structures of aspartate aminotransferase, alanine racemase, the β subunit of tryptophan synthase, D‐amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)‐binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent α‐β‐α‐β supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous α‐β‐α‐β structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements. Proteins 1999;35:250–261.

Theoretical analysis of conformation and active sites of interferons

Seven methods for prediction of protein secondary structures [1, 2, 14, 21, 22, 30, 31] were used for analysis of conformation of alpha- and beta-interferons (IFN-alpha and -beta). The results of the analysis indicate that the dominant secondary structure of IFNs-alpha and -beta is an alpha-helix. Five segments of the helix predicted by all the methods used have been identified. Using the method of Vonderviszt and Simon in IFNs-alpha and -beta two domains are predicted, the border between them lying at the region of amino acid (aa) residues 100-110. Comparison of primary structures of IFNs-alpha, -beta and -omega of different origin revealed two peculiar regions of conservative positions. In the first region, restricted to the N-terminal domain, 9 of 13 conservative positions are occupied by hydrophilic residues. In the second region, restricted to the C-terminal domain, all 8 conservative positions are occupied by hydrophobic residues. The C-terminal domain of the IFNs-alpha (aa residues 116-165) has been found to have a statistically valid homology (36%, probability of random coincidence is 5 x 10(-4) with prothymosin-alpha 1 (residues 1-47). The results of the theoretical analysis as compared to the experimental data available suggest the existence of at least two active sites in IFNs-alpha, beta and omega.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular modeling of the steric structure of the envelope F1 antigen of Yersinia pestis

Steric structure of the envelope F1 protein of Yersinia pestis was reconstructed by computer modeling taking into account structural similarities between F1 and interleukins (IL)-1 alpha, -beta, -ra and by using the known atomic coordinates for huIL-1 beta obtained by the X-ray crystallography. Of 18 hydrophobic positions forming a hydrophobic core in all the proteins studied with the IL-1-like conformation, 15 positions are occupied by hydrophobic residues in F1 protein as well. Of 8 homologous positions occupied by the amino acid residues of similar charge in all huIL-1 alpha, -beta, -ra, 5 positions are conserved in F1 antigen. The B-cell epitope accessible to antibodies in polymeric F1 is exposed as an hydrophilic loop at the surface opposite to the C-terminal sequence, forming a conserved binding site with periplasmic molecular chaperones.

Two Structural Motifs within Canonical EF-Hand Calcium-Binding Domains Identify Five Different Classes of Calcium Buffers and Sensors

Proteins with EF-hand calcium-binding motifs are essential for many cellular processes, but are also associated with cancer, autism, cardiac arrhythmias, and Alzheimers, skeletal muscle and neuronal diseases. Functionally, all EF-hand proteins are divided into two groups: (1) calcium sensors, which function to translate the signal to various responses; and (2) calcium buffers, which control the level of free Ca2+ ions in the cytoplasm. The borderline between the two groups is not clear, and many proteins cannot be described as definitive buffers or sensors. Here, we describe two highly-conserved structural motifs found in all known different families of the EF-hand proteins. The two motifs provide a supporting scaffold for the DxDxDG calcium binding loop and contribute to the hydrophobic core of the EF hand domain. The motifs allow more precise identification of calcium buffers and calcium sensors. Based on the characteristics of the two motifs, we could classify individual EF-hand domains into five groups: (1) Open static; (2) Closed static; (3) Local dynamic; (4) Dynamic; and (5) Local static EF-hand domains.

Phosphate group binding "cup" of PLP-dependent and non-PLP-dependent enzymes: leitmotif and variations.

Pyridoxal-5-phosphate (PLP) is widely used by many enzymes in reactions where amino acids are interconverted. Whereas the role of the pyridoxal ring in catalysis is well understood, the functional role of the single phosphate group in PLP has been less studied. Here we construct unambiguous connection diagrams that describe the interactions among the three non-ester phosphate oxygen atoms of PLP and surrounding atoms from the protein binding site and from water molecules, the so-called phosphate group binding cup. These diagrams provide a simple means to identify common recognition motifs for the phosphate group in both similar and different protein folds. Diagrams were constructed and compared in the cases of five newly determined structures of PLP-dependent transferases (fold type I enzymes) and, additionally, two non-PLP protein complexes (indole-3-glycerol phosphate synthase (IGPS) with bound indole-3-glycerol phosphate (IGP) and old yellow enzyme (OYE) with bound flavin mononucleotide (FMN)). A detailed comparison of the diagrams shows that three positions out of ten in the structure of the phosphate group binding cup contain invariant atoms, while seven others are occupied by conserved atom types. This level of similarity was also observed in the fold type III (TIM beta/alpha-barrel) enzymes that bind three different ligands: PLP, IGP and FMN.

Alpha‐fetoprotein antagonizes X‐linked inhibitor of apoptosis protein anticaspase activity and disrupts XIAP–caspase interaction

Previous results have shown that the human oncoembryonic protein α‐fetoprotein (AFP) induces dose‐dependent targeting apoptosis in tumor cells, accompanied by cytochromeu2003c release and caspaseu20033 activation. AFP positively regulates cytochromeu2003c/dATP‐mediated apoptosome complex formation in a cell‐free system, stimulates release of the active caspasesu20039 and 3 and displaces cIAP‐2 from the apoptosome and from its complex with recombinant caspasesu20033 and 9 [Semenkova et al. (2003) Eur. J. Biochem. 270, 276–282]. We suggested that AFP might affect the X‐linked inhibitor of apoptosis protein (XIAP)–caspase interaction by blocking binding and activating the apoptotic machinery via abrogation of inhibitory signaling. We show here that AFP cancels XIAP‐mediated inhibition of endogenous active caspases in cytosolic lysates of tumor cells, as well as XIAP‐induced blockage of active recombinant caspaseu20033 in a reconstituted cell‐free system. A direct protein–protein interaction assay showed that AFP physically interacts with XIAP molecule, abolishes XIAP–caspase binding and rescues caspaseu20033 from inhibition. The data suggest that AFP is directly involved in targeting positive regulation of the apoptotic pathway dysfunction in cancer cells inhibiting the apoptosis protein function inhibitor, leading to triggering of apoptosis machinery.

Citrullination of collagen II affects integrin-mediated cell adhesion in a receptor-specific manner

Citrullinated collagen II (CII) is a well‐known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance‐based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid‐phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts ~50% (P<0.05) and mesenchymal stem cells ~40% (P<0.05) by specifically decreasing the binding of integrins α10β1 and α11β1 to arginine‐containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1β1 and α2β1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells.—Sipilä, K., Haag, S., Denessiouk, K., Käpylä, J., Peters, E. C., Denesyuk, A., Hansen, U., Konttinen, Y., Johnson, M. S., Holmdahl, R., Heino, J. Citrullination of collagen II affects integrin‐mediated cell adhesion in a receptor‐specific manner. FASEB J. 28, 3758–3768 (2014). www.fasebj.org