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Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane β barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner β-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the β barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle. [ABSTRACT FROM AUTHOR]

FhaC is an integral outer membrane protein of the whooping cough agent B ordetella pertussis that mediates the transport to the cell surface of a major virulence factor, the filamentous haemagglutinin adhesin FHA. The FHA/ FhaC pair is a prototypic TpsA/ TpsB system of the widespread 'Two-Partner Secretion' pathway, dedicated to the transport of long extracellular proteins in various pathogenic and environmental Gram-negative bacteria. FhaC belongs to the ubiquitous Omp85 superfamily of protein transporters. The X-ray structure of FhaC shows that the transmembrane β-barrel channel hypothesized to serve as the FHA-conducting pore is obstructed by two structural elements conserved among TpsB transporters, an N-terminal α helix and an extracellular loop. Here, we provide evidence for conformational dynamics of FhaC related to the secretion mechanism. Using paramagnetic electron resonance, electrophysiology and in vivo approaches, we showed that FhaC exchanges between open and closed conformations. The interaction with its secretory partner FHA alters this distribution of conformations. The open conformation of FhaC implies a large displacement from the channel of the N-terminal 'plug' helix, which remains in the periplasm during FHA secretion. The membrane environment favours the dynamics of the TpsB transporter. [ABSTRACT FROM AUTHOR]

Summary Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, β-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a β barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a β sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with β augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion. [ABSTRACT FROM AUTHOR]

The chaperone/protease DegP belongs to the HtrA superfamily and is involved in protein quality control in the periplasm of Gram-negative bacteria. In Escherichia coli, typical substrates are unfolded or misfolded globular proteins that trigger the rearrangement of inactive DegP hexamers into substrate-sequestering 12- or 24-mers 'cages' for refolding or degradation. In Bordetella pertussis, DegP facilitates, in addition, the secretion of FHA, a long β-helical adhesin that passes through the periplasm in an extended conformation. We show that DegP exists as soluble trimers and as a membrane-associated form. Different substrates interact differently with the distinct forms of DegP, and membrane-associated DegP has high affinity for non-native FHA. Unlike more globular substrates, FHA does not efficiently mediate rearrangement of trimers into proteolytically active, short-lived dodecamers. In contrast to these dodecamers, membrane-associated DegP is not committed to substrate degradation, although it is proteolytically competent. In B. pertussis, membrane-associated DegP thus represents a specific functional form serving as a holding chaperone for client proteins including FHA. If FHA secretion is impaired, membrane-associated DegP participates in its degradation. This form of DegP is appropriate to handle substrates unsuitable to be sequestered in cages or non-folded, secretory proteins that must not be degraded. [ABSTRACT FROM AUTHOR]

In Gram-negative bacteria, the two-partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their transport through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA)/FhaC pair represents a model two-partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to decipher the mechanism of action of these proteins. FhaC is composed of a β-barrel preceded by two periplasmic polypeptide-transport-associated (POTRA) domains in tandem. The barrel is occluded by an N-terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA-TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two-partner secretion machinery. This motif is conserved in a predicted loop between two β-barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport. [ABSTRACT FROM AUTHOR]

The Bug proteins form a large family of periplasmic solute-binding receptors present in a number of bacterial species. Here, the crystal structure of Bordetella pertussis BugE, a member of the Bug family coded by the gene BP0250, is reported. It adopts the Venus flytrap architecture of periplasmic binding proteins, with two domains separated by a deep cleft. BugE has a bound ligand, identified as a glutamate. The structure of B. pertussis BugD, which is an aspartic acid transporter, has recently been reported. These structures reveal high conservation of the Bug architecture, despite limited sequence identity. They share a common carboxylate-binding motif defined by two strand–β-turn–α-­helix motifs, also involving two water molecules to bridge the carboxylate O atoms to the protein. The two water molecules are hydrogen bonded to a common main-chain carbonyl group. Although the features of the carboxylate-binding motif are totally conserved, the ligand in BugE is bound by its side-chain carboxylate group rather than by its α-­carboxylate as in BugD. This specific ligand-binding motif is highly conserved in Bug proteins and the BugE structure suggests that the cavity of the Bug proteins might also accommodate carboxylated solutes other than amino acids. The vast expansion of the Bug family in several bacterial genera is likely to be explained by the possible diversity of ligands. No charged residues are involved in glutamate binding by BugE, unlike what has been described for all glutamate receptors reported so far. [ABSTRACT FROM AUTHOR]

The sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram-negative bacteria, the widespread two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-related proteins. The secreted TpsA proteins carry a characteristic 250-residue-long N-terminal ‘TPS domain’ essential for secretion, while their TpsB transporters are pore-forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. However, the nature of the secretion signal has not been elucidated yet. The whooping cough agent Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter FhaC. In this work, we show specific interactions between an N-terminal fragment of FHA containing the TPS domain and FhaC by using two different techniques, an overlay assay and a pull-down of the complex. FhaC recognizes only non-native conformations of the TPS domain, corroborating the model that in vivo, periplasmic FHA is not yet folded. By generating single amino acid substitutions, we have identified interaction determinants forming the secretion signal. They are found unexpectedly far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA–TpsB interaction. The N-terminal domain of FhaC is involved in the FHA–FhaC interaction, in agreement with its proposed function and periplasmic localization. [ABSTRACT FROM AUTHOR]

Summary Many extracytoplasmic proteins undergo proteolytic processing during secretion, which is essential to their maturation. These post-translational modifications are carried out by specific enzymes whose subcellular localization is important for function. We have described a maturation subtilisin in Gram-negative Bordetella pertussis , the autotransporter SphB1. SphB1 catalyses the maturation of the precursor of the adhesin filamentous haemagglutinin (FHA) at the bacterial surface, in addition to the processing of its own precursor. Here, we show that the outer membrane anchor of SphB1 is crucial to its function, as evidenced by the lack of FHA maturation in a strain releasing a variant of SphB1 into the milieu. In contrast, surface association is not required for automaturation of SphB1. The surface retention of mature SphB1 is mediated by lipidation of the protein. The tethered protease appears to be stabilized by unusual Gly- and Pro-rich motifs at the N-terminus of the protein. This represents a new mode of localization for a protease involved in protein secretion. [ABSTRACT FROM AUTHOR]

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence white preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its ∼130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells. [ABSTRACT FROM AUTHOR]

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA- E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis. [ABSTRACT FROM AUTHOR]

The major adhesin of Bordetella pertussis , filamentous haemagglutinin (FHA), is produced and secreted at high levels by the bacterium. Mature FHA derives from a large precursor, FhaB, that undergoes several post-translational maturations. In this work, we demonstrate by site-directed mutagenesis that the N-terminal signal peptide of FHA is composed of 71 amino acids, including a 22-residue-long ‘N-terminal extension’ sequence. This sequence, although highly conserved in various other secretory proteins, does not appear to play an essential part in FHA secretion, as shown by deletion mutagenesis. The entire N-terminal signal region of FhaB is removed in the course of secretion by proteolytic cleavage at a site that corresponds to a Lep signal peptidase recognition sequence. After this maturation, the N-terminal glutamine residue is modified to a pyroglutamate residue. This modification is not crucial for heparin binding, haemagglutination or secretion. Interestingly, however, the modification is absent from Escherichia coli secreted FHA derivatives. In addition, it is dependent in B. pertussis on the presence of all three cysteines contained in the signal peptide of FhaB. These observations suggest that it does not occur spontaneously but perhaps requires a specific enzymatic machinery. [ABSTRACT FROM AUTHOR]

The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same Mr as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates. [ABSTRACT FROM AUTHOR]

DctP6 and DctP7 are two Bordetella pertussis proteins which belong to the extracytoplasmic solute receptors (ESR) superfamily. ESRs are involved in the transport of substrates from the periplasm to the cytosol of Gram-negative bacteria. DctP6 and DctP7 have been crystallized and diffraction data were collected using a synchrotron-radiation source. DctP6 crystallized in space group P41212, with unit-cell parameters a = 108.39, b = 108.39, c = 63.09 Å, while selenomethionyl-derivatized DctP7 crystallized in space group P212121, with unit-cell parameters a = 64.87, b = 149.83, c = 170.65 Å. The three-dimensional structure of DctP7 will be determined by single-wavelength anomalous diffraction, while the DctP6 structure will be solved by molecular-replacement methods. [ABSTRACT FROM AUTHOR]

Proteins with both peptidylprolyl isomerase (PPIase) and chaperone activities play a crucial role in protein folding in the periplasm of Gram-negative bacteria. Few such proteins have been structurally characterized and to date only the crystal structure of SurA from Escherichia coli has been reported. Par27, the prototype of a new group of parvulins, has recently been identified. Par27 exhibits both chaperone and PPIase activities in vitro and is the first identified parvulin protein that forms dimers in solution. Par27 has been expressed in E. coli. The protein was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Form A, which belongs to space group P2 (unit-cell parameters a = 42.2, b = 142.8, c = 56.0 A, beta = 95.1 degrees ), diffracts to 2.8 A resolution, while form B, which belongs to space group C222 (unit-cell parameters a = 54.6, b = 214.1, c = 57.8 A), diffracts to 2.2 A resolution. Preliminary diffraction data analysis agreed with the presence of one monomer in the asymmetric unit of the orthorhombic crystal form and two in the monoclinic form.