Channel Properties of TpsB Transporter FhaC Point to Two Functional Domains with a C-terminal Protein-conducting Pore

Méli, Albano C.; Hodak, Hélène; Clantin, Bernard; Locht, Camille; Molle, Gérard; Jacob‐Dubuisson, Françoise; Saint, Nathalie

doi:10.1074/jbc.m508524200

Cited by 57 publications

(81 citation statements)

References 43 publications

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“…Its structural integrity is critical for FhaC function, as shown by the drastic effects of deletions. In support of this, we showed earlier that insertions in B5, B6 and L4 inactivated FhaC 35,37 . Notably, B5-B8 are generally long, and B9 is short in TpsB proteins (Supplementary Fig.…”

Section: Discussionsupporting

confidence: 70%

“…Note the presence of two FhaC-containing bands in some cases, the upper one corresponding to the complex and the other to an FhaC dimer that most likely formed upon cell lysis. Some FhaC variants with Cys in the barrel were devoid of H1 to facilitate crosslinker access (denoted as DH1), since H1 is not essential for activity 37 and moves to the periplasm for FHA translocation 23 . We checked for one such variant, FhaC C406 , that similar results were obtained with or without H1.…”

Section: Resultsmentioning

confidence: 99%

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Translocation path of a substrate protein through its Omp85 transporter

et al. 2014

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TpsB proteins are Omp85 superfamily members that mediate protein translocation across the outer membrane of Gram-negative bacteria. Omp85 transporters are composed of N-terminal POTRA domains and a C-terminal transmembrane b-barrel. In this work, we track the in vivo secretion path of the Bordetella pertussis filamentous haemagglutinin (FHA), the substrate of the model TpsB transporter FhaC, using site-specific crosslinking. The conserved secretion domain of FHA interacts with the POTRA domains, specific extracellular loops and strands of FhaC and the inner b-barrel surface. The interaction map indicates a funnel-like pathway, with conformationally flexible FHA entering the channel in a non-exclusive manner and exiting along a four-stranded b-sheet at the surface of the FhaC barrel. This sheet of FhaC guides the secretion domain of FHA along discrete steps of translocation and folding. This work demonstrates that the Omp85 barrel serves as a channel for translocation of substrate proteins.

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Section: Discussionsupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Translocation path of a substrate protein through its Omp85 transporter

et al. 2014

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“…Two POTRA domains are found in a periplasmic region between the N-terminal ␣-helix and the ␤-barrel. FhaC has also been characterized in electrophysiology and found to have a conductance of 1200 pS in 1 M KCl (17,18), a value comparable with a single OmpF porin monomer in these conditions (19). Deletion of the POTRA domains abrogated secretion but had no effect on the channel properties (13,18).…”

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confidence: 99%

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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“…Despite limited overall sequence conservation among the TpsA members, functional studies have established that TpsA proteins contain common features, including an atypical N-terminal signal peptide and an adjacent region of about 250 residues that forms the so-called secretion domain (1)(2)(3)(4). Although our knowledge of TpsB proteins remains relatively limited, recent studies have established that TpsB proteins have a modular structure with a C-terminal pore-forming domain (5,6).…”

mentioning

confidence: 99%

“…The functionally characterized TpsA/TpsB pairs in the TPS family include FHA/FhaC of B. pertussis, ShlA/ShlB of S. marcescens, and HMW1/HMW1B of H. influenzae (5,6,(15)(16)(17)(18)(19). In all of these examples, proper secretion requires that the secretion domain of TpsA and the periplasmic domain of TpsB recognize each other in the periplasm.…”

mentioning

confidence: 99%

The Structure of the Haemophilus influenzae HMW1 Pro-piece Reveals a Structural Domain Essential for Bacterial Two-partner Secretion

Yeo

Yokoyama

Walkiewicz

et al. 2007

Journal of Biological Chemistry

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In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and In Gram-negative bacteria, the two-partner secretion (TPS) 2 pathway serves as a common secretion mechanism for large protein virulence factors and is essential for the virulence of many human pathogens, including Haemophilus influenzae, Bordetella pertussis, Serratia marcescens, and Proteus mirabilis, among others. The TPS pathway consists of a secreted exoprotein (referred to as a TpsA protein) and a cognate outer membrane translocator (referred to as a TpsB protein) (1-3). TpsA proteins are synthesized as preproteins 100 -500 kDa in size that are processed in the course of secretion across the bacterial inner and outer membranes, yielding functional TpsA proteins (3, 4). Despite limited overall sequence conservation among the TpsA members, functional studies have established that TpsA proteins contain common features, including an atypical N-terminal signal peptide and an adjacent region of about 250 residues that forms the so-called secretion domain (1-4). Although our knowledge of TpsB proteins remains relatively limited, recent studies have established that TpsB proteins have a modular structure with a C-terminal pore-forming domain (5, 6).The H. influenzae HMW1 and HMW2 proteins are high molecular weight, non-pilus adhesins that were originally identified as major targets of the human serum antibody response during acute otitis media (7). These proteins are present in ϳ80% of nontypeable H. influenzae strains and mediate adherence to a variety of epithelial cell types (4, 8). HMW1 and HMW2 are encoded by separate chromosomal loci, with each locus consisting of three genes, designated hmwA, hmwB, and hmwC (8 -10). The hmwA genes encode the surface-exposed adhesins (HMW1 and HMW2), and the hmwB and hmwC genes encode accessory proteins required for proper processing and secretion of the adhesins (5,(11)(12)(13)(14). In H. influenzae strain 12, the HMW1 and HMW2 proteins are synthesized as preproproteins and undergo two discrete cleavage events during the process of maturation and surface localization, the first releasing the N-terminal signal peptide corresponding to residues 1-68 and the second releasing the pro-piece corresponding to residues 69 -441 (see Fig. 1). In H. influenzae strain 12, mature HMW1 corresponds to residues 442-1536 in the HMW1 preproprotein, and mature HMW2 corresponds to residues 442-1477 in the HMW2 preproprotein. In both HMW1 and HMW2, adhesive activity resides in a ϳ360-amino acid region at the N terminus of the mature species, and anchoring to the bacterial surface is mediated by a 20-amino acid region at the very C te...

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Channel Properties of TpsB Transporter FhaC Point to Two Functional Domains with a C-terminal Protein-conducting Pore

Cited by 57 publications

References 43 publications

Translocation path of a substrate protein through its Omp85 transporter

Translocation path of a substrate protein through its Omp85 transporter

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

The Structure of the Haemophilus influenzae HMW1 Pro-piece Reveals a Structural Domain Essential for Bacterial Two-partner Secretion

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