PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA (original) (raw)
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Proceedings of the National Academy of Sciences, 1991
Molecular chaperones are found in the cytoplasm of bacteria and in various cellular compartments in eukaryotes to maintain proteins in nonnative conformations that permit their secretion across membranes or assembly into oligomerc structures. Virtually nothing, however, has been reported *mt a similar requirement for molecular chaperones in the periplasm of Gram-negative bacteria. We used the well-characterized P pilus biogenesis system in Escherichia coli as a model to elucidate the mechanism ofaction of a periplasmic chap PapD, which is specifically required for P pilus bions. PapD probably associates with at least six P pilus subunits after their secretion across the cytoplasmic membrane, but PapD is not incorporated into the pilus. We used
Structural basis of chaperone self-capping in P pilus biogenesis
Proceedings of the National Academy of Sciences, 1999
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.
Journal of Bacteriology, 2003
The assembly of type 1 pili on the surface of uropathogenic Escherichia coli proceeds via the chaperone-usher pathway. Chaperone-subunit complexes interact with one another via a process termed donor strand complementation whereby the G1 strand of the chaperone completes the immunoglobulin (Ig) fold of the pilus subunit. Chaperone-subunit complexes are targeted to the usher, which forms a channel across the outer membrane through which pilus subunits are translocated and assembled into pili via a mechanism known as donor strand exchange. This is a mechanism whereby chaperone uncapping from a subunit is coupled with the simultaneous assembly of the subunit into the pilus fiber. Thus, in the pilus fiber, the N-terminal extension of every subunit completes the Ig fold of its neighboring subunit by occupying the same site previously occupied by the chaperone. Here, we investigated details of the donor strand exchange assembly mechanism. We discovered that the information necessary for targeting the FimC-FimH complex to the usher resides mainly in the FimH protein. This interaction is an initiating event in pilus biogenesis. We discovered that the ability of an incoming subunit (in a chaperone-subunit complex) to participate in donor strand exchange with the growing pilus depended on a previously unrecognized function of the chaperone. Furthermore, the donor strand exchange assembly mechanism between subunits was found to be necessary for subunit translocation across the outer membrane usher.
Structural Basis of Chaperone Function and Pilus Biogenesis
Science, 1999
Many Cram-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 C, P 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.
Molecular Microbiology, 2010
Attachment to host cells via adhesive surface structures is a prerequisite for the pathogenesis of many bacteria. Uropathogenic E. coli assemble P and type 1 pili for attachment to the host urothelium. Assembly of these pili requires the conserved chaperone/usher pathway, in which a periplasmic chaperone controls the folding of pilus subunits and an outer membrane usher provides a platform for pilus assembly and secretion. The usher has differential affinity for pilus subunits, with highest affinity for the tip-localized adhesin. Here, we identify residues F21 and R652 of the P pilus usher PapC as functioning in the differential affinity of the usher. R652 is important for high affinity binding to the adhesin whereas F21 is important for limiting affinity for the PapA major rod subunit. PapC mutants in these residues are specifically defective for pilus assembly in the presence of PapA, demonstrating that differential affinity of the usher is required for assembly of complete pili. Analysis of PapG deletion mutants demonstrated that the adhesin is not required to initiate P pilus biogenesis. Thus, the differential affinity of the usher may be critical to ensure assembly of functional pilus fibers.
Proceedings of The National Academy of Sciences, 1996
The major subassemblies of virulenceassociated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperoneprotected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.
Chaperone Priming of Pilus Subunits Facilitates a Topological Transition that Drives Fiber Formation
Cell, 2002
bacterial attachment to host tissues, an essential early 2 Department of Biochemistry and Molecular step in UTI pathogenesis (Hultgren et al., 1996). Pilus-Biophysics mediated attachment facilitates bacterial colonization Washington University Medical School and triggers a complex web of events, including signal-660 South Euclid Avenue ing in both the bacterium and host, that then influences St. Louis, Missouri 63105 the course and outcome of the infection (Mulvey et al., 3 Birkbeck College 1998; Martinez et al., 2000; Hung et al., 2001). Department of Crystallography P pili are expressed by many strains of uropathogenic Malet Street E. coli. These pilus fibers are encoded by the pap gene London cluster (papA-K) and bind to the globoseries of glycolip-WC1E 7HX, UK ids present in the human kidney (Hull et al., 1981; Lund 4 The Ludwig Institute for Cancer Research et al., 1987). P pili have been shown to be required University College London for the establishment of pyelonephritis (Roberts et al., 91 Riding House Street 1994). Each P pilus consists of a thick, rigid rod with a London thinner, more flexible tip fibrillum at its distal end. The W1W 7BS, UK rod contains PapA subunits arranged to form a tightly wound, hollow, right-handed helical structure (Gong and Makowski, 1992; Bullitt and Makowski, 1995). The tip Summary fibrillum contains PapE subunits arranged in a more open helical conformation (Kuehn et al., 1992). The PapG Periplasmic chaperones direct the assembly of adheadhesin, which binds the glycolipid receptor, is at the sive, multi-subunit pilus fibers that play critical roles distal end of the tip fibrillum (Kuehn et al., 1992; Dodson in bacterial pathogenesis. Pilus assembly occurs via et al., 2001). The PapF and PapK pilus subunits are a donor strand exchange mechanism in which the N-terthought to link the PapG adhesin to the tip fibrillum and minal extension of one subunit replaces the chaperone the tip fibrillum to the rod, respectively (Jacob-Dubuis-G 1 strand that transiently occupies a groove in the son et al., 1993). neighboring subunit. Here, we show that the chaper-P pili are members of a large family of bacterial surface one primes the subunit for assembly by holding the fibers that are assembled by a conserved secretion and groove in an open, activated conformation. During doassembly system termed the chaperone-usher pathway nor strand exchange, the subunit undergoes a topo-(Thanassi et al., 1998a). This chaperone-usher pathway logical transition that triggers the closure of the groove participates in the assembly of surface organelles in and seals the N-terminal extension in place. It is this many pathogenic bacteria, including uropathogenic and topological transition, made possible only by the primenterotoxigenic E. coli, Haemophilus influenzae, Klebing action of the chaperone that drives subunit assemsiella pneumoniae, Proteus mirabilis, Bordetella pertusbly into the fiber. sis, and Salmonella and Yersinia species, including Yersinia pestis, the causative agent of bubonic plague Introduction (Hung et al.,
PapD-like chaperones provide the missing information for folding of pilin proteins
Proceedings of the National Academy of Sciences, 2000
A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperoneassisted folding.