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.

The plasmid-located gene cafl encoding the capsular antigen fraction I (Fl) of Yersiniu pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced M, of 17.6 kDa. The signal peptide sequence was highly homologous to the E. co/i consensus signal sequence. The Fl was assumed to have p-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. 1, INTRODUCTIONThe ultimate goal of infectious disease research is their prevention. Vaccination is one of the most effective ways in which that goal can be attained. It is necessary to know the gene structure and putative immunogenic surface structures of antigens to create recombinant vaccines.More than 10 antigens have been isolated from Yersinia pestis. The capsular antigen fraction 1 (Fl) was shown to be a highly protective antigen among such thermolabile antigens as D, Fl, T, V and W [I). Some properties of the Fl structure have been studied recently [2], but the nucleotide and amino acid sequences have been unknown so far. Here, we report the cloning and sequencing of the Y. pestis cafl gene coding for the Fl , and the predicted secondary structure with potential antigenic determinants. MATERIALS AND METHODS Bacterial strains, plasmids and DNA manipulafionsThe E. co/i strains LE392 and HBIOI were used as transistent hosts for cosmid pHC79 [3], and pUC18 or f9 [4], respectively. Y. pestisF1 positive vaccine strain EV was obtained from the Culture Collection, All-Union Antiplague Institute 'Microb', USSR. Cultures were grown overnight while shaking at 37°C in liquid LB or on solid medium supplemented with the relevant antibiotics for plasmid selection. Construction of a gene library, screening, and subcloningThe Y. pestis plasmid pFra DNA (about 110 kb in size) was partially digested with EcoRI, ligated with EeoRIdigested cosmid pHC79 and packaged in vitro. The library was amplified in E. coli LE392 and ApRTcR colonies selected were further screened for Fl production by enzyme immunoassay. The isolated cosmid ~153 containing a 40-kb fragment of pFra DNA was then digested with EcoRl and an 8.6kb fragment was cloned into pHC79. The resulting cosmid pFS2 was digested with SalI and Hind111 and a 4.5kb fragment was cloned into pUCI9. The plasmid pFS2-13 generated was used for gene sequencing. The 1 .O-kb Alul fragment of pFS2-I 3 was cloned into the SmaIdigested pUCl8 (plasmid pF18L) and sequenced. Protein sequencingThe Fl protein was isolated from culture medium and purified by polyacrylamide gel electrophoresis as described [2]. The N-terminus of the mature protein was identified by a PTH-amino acid analyzer (Model 120A, Applied Biosystems). Secondary structure and anrigenic determinant analysis

SummaryA 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 b-strand and the G1 subunit binding b-strand (FGL; F1 b-strand to G1 b-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.

Steric structure of CaflM, a periplasmic molecular chaperone of Yersiniu p&is, was reconstructed by computer modelling based on a statistically significant primary structure homology between CaflM and PapD protein from Escherichia coli, and u,sing the known atomic coordinates obtained by the X-ray crystallography for PapD. In the three-dimensional model of CaflM an accessory sequence between Fl and Gl &strands (as compared to PapD) can form a strain-specific part of the binding pocket of surface organell subunits. This accessory sequence decreases the depth of the binding pocket. The characteristic structural feature of the subfamily of periplasmic molecular chaperones with the accessory sequence (CaflM subfamily) is the existence of exposed to a solvent Cys residues in Fl and Gl P-strands which can form disulfide bond in the putative binding pocket. The characteristic functional feature of CaflM subfamily is the chaperoning of more simple compositions of virulence-associated surface organells (in the case of Y. pestis a capsule consists of only Fl protein). Highly conserved R,, and D,,, located at the domain surface remote from the putative subunit binding pocket, can participate in direct contacts with the conserved portion of molecular usher proteins.

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