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 independent 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 posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3.

The mechanism by which peptide release factor RF3 recycles RF1 and RF2 has been clarified and incorporated in a complete scheme for translation termination. Free RF3 is in vivo stably bound to GDP, and ribosomes in complex with RF1 or RF2 act as guanine nucleotide exchange factors (GEF). Hydrolysis of peptidyl-tRNA by RF1 or RF2 allows GTP binding to RF3 on the ribosome. This induces an RF3 conformation with high affinity for ribosomes and leads to rapid dissociation of RF1 or RF2. Dissociation of RF3 from the ribosome requires GTP hydrolysis. Our data suggest that RF3 and its eukaryotic counterpart, eRF3, have mechanistic principles in common.

Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli.

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.

Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

Thiol/disulfide exchange between small heat shock protein 25 and glutathione.

Murine small heat shock protein 25 (Hsp25) carries a single Cys-residue at position 141 of its amino acid sequence. In glutathione redox buffers, Hsp25 equilibrates between reduced protein (PSH), mixed disulfide (PSSG) and protein dimer (PSSP) forms. At highly oxidative conditions, native Hsp25 predominantly forms PSSP while denatured Hsp25 forms PSSG. Conversion of PSSP to PSSG correlates with urea and temperature denaturation of tertiary and/or quaternary structure of Hsp25. At pH 7.5, 25 degreesC, the second-order rate constant for the formation of PSSP in the reaction of native PSH with GSSG is 20.1+/-1.4 M-1 min-1. This is approximately 3-fold lower than the reaction velocity of GSSG with a typical, unhindered thiol of pKa 8.6. At redox equilibrium, the fractions of PSSP, PSSG, and PSH depend on the concentration of GSH and less on the ratio [GSH]/[GSSG] (R). At a constant R, the fractions of PSSG and PSH species depend similarly on GSH concentration, being approximately equal in glutathione redox buffers with low R. It is concluded that in oligomeric complexes, Hsp25 subunits in vitro form stable dimers, in which the reacting -SH groups are in a proximity to form intersubunit disulfide bonds. Within a reaction of one of these -SH groups with GSSG, steric hindrances and electrostatic repulsion complicate penetration of another reduced or oxidized glutathione molecule to the reaction site.

Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.