Vladimir P. Zav'yalov

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.

Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

Donor strand complementation mechanism in the biogenesis of non-pilus systems.

The F1 antigen of Yersinia pestis belongs to a class of non‐pilus adhesins assembled via a classical chaperone–usher pathway. Such pathways consist of PapD‐like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone–subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three inde‐pendent methods showed that these complexes are rod‐ or coil‐shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N‐terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone–subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule‐like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone–usher pathways.

A new gene of the ƒ1 operon of Y. pestis involved in the capsule biogenesis

The DNA sequence determination or the ƒ1 operon between the genes encoding the F1 subunit (caf1) and chaperone‐like protein (caf1M) revealed a large open reading frame that codes for a polypeptide similar to some E. coli proteins involved in the biogenesis of fimbria. The deletion and in trans complementation analyses showed that this gene is not necessary for extracellular transport or the F1 subunit but plays a role in the capsule assembly.

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.

Caf1R gene and its role in the regulation of capsule formation of Y. pestis

A new transcription unit of the f1 gene cluster was found. The DNA sequencing revealed one long open reading frame. Deletion and frame shift mutation analyses have demonstrated the importance of a corresponding gene product for the F1 antigen biosynthesis. A homology of the deduced amino acid sequence with that of AraC family DNA‐binding regulators was shown. A potential regulatory DNA region is discussed.

Specific high affinity binding of human interleukin 1β by Caf1A usher protein of Yersinia pestis

Understanding the interaction of Yersinia pestis with the key components of the immune system is important for elucidation of the pathogenesis of bubonic plague, one of the most severe and acute bacterial diseases. Here we report the specific, high affinity binding (K d = 1.40 × 10−10 M ± 0.14 × 10−10) of radiolabelled human interleukin 1β (hIL‐1β) to E. coli cells carrying the capsular fl operon of Y. pestis. Caf1A outer membrane usher protein was isolated to greater than 98% purity. Competition studies with purified Caf1, together with immunoblotting studies, identified Caf1A as the hIL‐1β receptor. Competition between Caf1 subunit and hIL‐1β for the same or an overlapping binding site on CaflA was demonstrated. Relevance of these results to the pathogenesis of Y. pestis and other Gram negative bacterial pathogens with homologous outer membrane usher proteins is discussed.

An extended hydrophobic interactive surface of Yersinia pestis Caf1M chaperone is essential for subunit binding and F1 capsule assembly

A single polypeptide subunit, Caf1, polymerizes to form a dense, poorly defined structure (F1 capsule) on the surface of Yersinia pestis. The caf‐encoded assembly components belong to the chaperone–usher protein family involved in the assembly of composite adhesive pili, but the Caf1M chaperone itself belongs to a distinct subfamily. One unique feature of this subfamily is the possession of a long, variable sequence between the F1 β‐strand and the G1 subunit binding β‐strand (FGL; F1 β‐strand to G1 β‐strand long). Deletion and insertion mutations confirmed that the FGL sequence was not essential for folding of the protein but was absolutely essential for function. Site‐specific mutagenesis of individual residues identified Val‐126, in particular, together with Val‐128 as critical residues for the formation of a stable subunit–chaperone complex and the promotion of surface assembly. Differential effects on periplasmic polymerization of the subunit were also observed with different mutants. Together with the G1 strand, the FGL sequence has the potential to form an interactive surface of five alternating hydrophobic residues on Caf1M chaperone as well as in seven of the 10 other members of the FGL subfamily. Mutation of the absolutely conserved Arg‐20 to Ser led to drastic reduction in Caf1 binding and surface assembled polymer. Thus, although Caf1M–Caf1 subunit binding almost certainly involves the basic principle of donor strand complementation elucidated for the PapD–PapK complex, a key feature unique to the chaperones of this subfamily would appear to be capping via high‐affinity binding of an extended hydrophobic surface on the respective single subunits.

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)

Receptor-binding properties of the peptides corresponding to the β-endorphin-like sequence of human immunoglobulin G

The decapeptide H2N-Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-COOH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of the human immunoglobulin G1 Eu heavy chain and displaying a 43% identity with the antigenic determinant of beta-endorphin was synthesized. Immunorphin was found to compete with 125I-beta-endorphin for high-affinity receptors on murine peritoneal macrophages (K = 2.5 +/- 0.9 x 10(-9) M) and with 3H-morphin for receptors on murine thymocytes (Ki = 2.7 +/- 0.6 x 10(-9) M) and murine macrophages (Ki = 5.9 +/- 0.7 x 10(-9) M). In particular two types of receptors to 125I-beta-endorphin with Kd1 = 6.1 +/- 0.6 x 10(-9) M and Kd2 = 3.1 +/- 0.2 x 10(-8) M were revealed on macrophages. The second type of receptors interacted with 125I-beta-endorphin, 3H-Met-enkephalin, 3H-Leu-enkephalin and 3H-morphin; the first displayed reactivity with 125I-beta-endorphin, 3H-morphin and immunorphin. The first type receptors are not present on murine brain cells nor are inhibited by naloxone. A minimum fragment of immunorphin practically completely retaining its inhibitory activity in the competition tests with 125I-beta-endorphin for common receptors on thymocytes was found to correspond to the tetrapeptide H2N-Lys-Gly-Phe-Tyr-COOH (Ki = 5.6 +/- 0.5 x 10(-9) M).