In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG.
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We have characterized in vivo and in vitro the recently identified DsbG from Escherichia coli. In addition to sharing sequence homology with the thiol disulfide exchange protein DsbC, DsbG likewise was shown to form a stable periplasmic dimer, and it displays an equilibrium constant with glutathione comparable with DsbA and DsbC. DsbG was found to be expressed at approximately 25% the level of DsbC. In contrast to earlier results (Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) Mol. Microbiol. 26, 121-132), we showed that dsbG is not essential for growth and that dsbG null mutants display no defect in folding of multiple disulfide-containing heterologous proteins. Overexpression of DsbG, however, was able to restore the ability of dsbC mutants to express heterologous multidisulfide proteins, namely bovine pancreatic trypsin inhibitor, a protein with three disulfides, and to a lesser extent, mouse urokinase (12 disulfides). As in DsbC, the putative active site thiols in DsbG are completely reduced in vivo in a dsbD-dependent fashion, as would be expected if DsbG is acting as a disulfide isomerase or reductase. However, the latter is not likely because DsbG could not catalyze insulin reduction in vitro. Overall, our results indicate that DsbG functions primarily as a periplasmic disulfide isomerase with a narrower substrate specificity than DsbC. (+info)
Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.
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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. (+info)
Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1.
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The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution. (+info)
A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein.
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Misfolding or unfolding of polypeptides can occur as a consequence of environmental stress and spontaneous mutation. The abundance of general chaperones and proteases suggests that cells distinguish between proteins that can be refolded and "hopeless" cases fated to enter the proteolytic pathway. The mechanisms controlling this key metabolic decision are not well understood. We show here that the widely conserved heat shock protein DegP (HtrA) has both general molecular chaperone and proteolytic activities. The chaperone function dominates at low temperatures, while the proteolytic activity is present at elevated temperatures. These results show that a single cellular factor can switch between two key pathways, controlling protein stability and turnover. Implications of this finding for intracellular protein metabolism are discussed. (+info)
Structural basis of chaperone self-capping in P pilus biogenesis.
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PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of Escherichia coli. It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm. We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface. Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2-D2 and F1-G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface. An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits. (+info)
The Escherichia coli pldC gene encoding lysophospholipase L(1) is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively.
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We deduced the amino acid sequence of Escherichia coli lysophospholipase L(1) by determining the nucleotide sequence of the pldC gene encoding this enzyme. The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues. The molecular weight of the purified enzyme (20,500) was in good agreement with the predicted size (20,399) of the processed protein. A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively. Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E. coli overexpressing the pldC gene showed both protease I and thioesterase I activities. (+info)
Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.
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TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus. (+info)
Structural basis of chaperone function and pilus biogenesis.
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Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange. (+info)