The Outer Membrane Usher Forms a Twin-pore Secretion Complex

Li, Huilin; Qian, Luping; Chen, Zhiqiang; Thibault, Danielle; Guang, Liu; Liu, Tianbo; Thanassi, David G.

doi:10.1016/j.jmb.2004.10.008

Cited by 68 publications

(64 citation statements)

References 62 publications

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“…The protein used for these studies was produced from a histidine affinity tag-tagged construct. More recently, similar experiments conducted with an untagged HMW1B construct suggest that HMW1B rather forms dimers (22) in a manner reminiscent of other polypeptide-transporting proteins, such as PapC (23) and the TIM and TOM complexes of mitochondrial inner and outer membranes, respectively (24,25).…”

mentioning

confidence: 85%

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

Duret

Szymanski

Choi

et al. 2008

Journal of Biological Chemistry

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The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane ␤-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.Secretion of proteins in the external medium from the cytosol of a Gram-negative bacterium is a complex mechanism. Because of the compartmented structure of the cell envelope, a periplasm limited by the inner membrane and the outer membrane, secreted proteins may need to undergo two translocation processes. Seven main types of mechanisms exist to translocate proteins through the cell envelope (1-6). Some of these pathways export the protein directly from the cytosol to the extracellular milieu, whereas others include a periplasmic intermediate step. In this case, independent translocation pathways exist through the inner membrane and comprise the Sec pathway and the TAT pathway (7). The type V secretion pathway is one of those two-step processes, where proteins are first exported to the periplasm in a Sec-dependent manner (5). The second step involves the translocation through the outer membrane of a passenger peptide through a transporter. In many cases, the passenger and the transporter domains belong to the same polypeptide, thus defining an auto-transporter type of mechanism. The two-partner secretion pathway is distinct in that the secreted exoprotein (TpsA) and its cognate transporter protein (TpsB) are translated as two separate proteins but are coded on the same operon or the same locus. Each TpsB transporter is devoted to the translocation of one specific TpsA. For example, the Haemophilus influenzae HMW1/HMW1B pair represents a prototype t...

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mentioning

confidence: 85%

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

Duret

Szymanski

Choi

et al. 2008

Journal of Biological Chemistry

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“…Interaction of the periplasmic chaperone/subunit complexes with the usher, an integral outer membrane protein, facilitates the release of fimbrial subunits and their secretion through the usher channel, with the most distal proteins being secreted first (26,50,113,185,195,233,234,292,328). The N terminus of subunits harbors a conserved ␤-strand motif similar to the G 1 donor ␤-strand motif of chaperones.…”

Section: Characteristics Of the Chaperone/usher Assembly Pathwaymentioning

confidence: 99%

Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek

Nuccio

Bäumler

2007

Microbiol Mol Biol Rev

293

456

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SUMMARY Many Proteobacteria use the chaperone/usher pathway to assemble proteinaceous filaments on the bacterial surface. These filaments can curl into fimbrial or nonfimbrial surface structures (e.g., a capsule or spore coat). This article reviews the phylogeny of operons belonging to the chaperone/usher assembly class to explore the utility of establishing a scheme for subdividing them into clades of phylogenetically related gene clusters. Based on usher amino acid sequence comparisons, our analysis shows that the chaperone/usher assembly class is subdivided into six major phylogenetic clades, which we have termed α-, β-, γ-, κ-, π-, and σ-fimbriae. Members of each clade share related operon structures and encode fimbrial subunits with similar protein domains. The proposed classification system offers a simple and convenient method for assigning newly discovered chaperone/usher systems to one of the six major phylogenetic groups.

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“…1C). In the presence of detergent, the apparent mass of a membrane protein can be ϳ200 kDa greater than the actual mass, reflecting the surrounding detergent micelle in aqueous solution (21,30). Taking the effect of detergent into account, we concluded that peak 2 corresponds to a dimer and that peak 1 corresponds to a dimer of dimers or a tetramer.…”

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confidence: 90%

“…To extend our observations, we examined the HMW1B preparation by negative staining electron microscopy, as described previously (21). As shown in Fig.…”

mentioning

confidence: 92%

“…Based on the 10-Å projection structure, there is a low-density area consistent with a narrow channel at the center of each subunit in the dimer. This infor- mation suggests that HMW1B is a twin-pore complex, analogous to a variety of other protein translocating channels, including the PapC outer membrane usher involved in the assembly of E. coli P pili, the Sec complex responsible for transport of proteins across the bacterial inner membrane and the eukaryotic endoplasmic reticulum, the TIM22 protein insertion complex in the mitochondrial inner membrane, and the TOM protein import complex in the mitochondrial outer membrane, among others (1,2,4,20,21,25).…”

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confidence: 99%

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Structure of the Haemophilus influenzae HMW1B Translocator Protein: Evidence for a Twin Pore

Grass

Wang

et al. 2007

J Bacteriol

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Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded ␤-barrel domain, the approximate size of the ␤-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 Å resolution. The native HMW1B structure is a dimer of ␤-barrels, with each ␤-barrel measuring 40 Å by 50 Å in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.Gram-negative bacteria have evolved a number of sophisticated pathways for secreting proteins (19). Among the simplest of these pathways is the two-partner secretion (TPS) system, which consists of an exoprotein termed TpsA and a poreforming outer membrane (OM) translocator protein termed TpsB (16). Based on experimental data and genome sequence analysis, over 100 TPS members have been identified (16). The TpsB proteins are large (60 to 80 kDa), pore-forming, outer membrane proteins that are predicted to form ␤-barrels and that translocate the cognate TpsA protein from the periplasm to the bacterial surface (16).Among the predicted TpsB proteins, the transmembrane topologies of the Serratia marcescens ShlB protein, the Bordetella pertussis FhaC protein, and the Haemophilus influenzae HMW1B protein have been studied. ShlB is involved in secretion and activation of the ShlA hemolysin, a pore-forming cytotoxin. Based on sequence alignments, secondary structure predictions, and characterization of M2 epitope insertions, ShlB is predicted to have 20 ␤ strands with a periplasmic N terminus and two large surface loops (18). FhaC is involved in secretion of filamentous hemagglutinin (FHA), a multifunctional adhesin. Initial work with FhaC suggested that this protein contains 19 transmembrane ␤ strands with a surface-exposed N terminus and forms two large loops on the bacterial surface (12,15,22). However, more recent studies suggest that FhaC has a maximum of 16 transmembrane ␤ strands and contains two functional domains, including an N-terminal domain that modulates pore properties and may participate in the recognition of FHA and a 350-amino-acid C-terminal domain that forms the pore (22). HMW1B facilitates surface localization of the HMW1 adhesin, a 125-kDa protein that forms fibers on the bacterial surface and mediates H. influenzae interaction with respi...

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The Outer Membrane Usher Forms a Twin-pore Secretion Complex

Cited by 68 publications

References 62 publications

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

The TpsB Translocator HMW1B of Haemophilus influenzae Forms a Large Conductance Channel

Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek

Structure of the Haemophilus influenzae HMW1B Translocator Protein: Evidence for a Twin Pore

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