The haemolysin‐secreting ShlB protein of the outer membrane of <i>Serratia marcescens</i> : determination of surface‐exposed residues and formation of ion‐permeable pores by ShlB mutants in artificial lipid bilayer membranes

Könninger, Ulrich; Hobbie, Silke; Benz, Roland; Braun, Volkmar

doi:10.1046/j.1365-2958.1999.01433.x

Cited by 61 publications

(46 citation statements)

References 51 publications

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“…This pore size is in agreement with the reported dimensions of other bacterial and eukaryotic Omp85-like proteins, and it is large enough to allow passage of an unfolded or partially unfolded substrate protein (6,13,15,16,34). Both the relatively low specific activity of HMW1B for arabinose and the biphasic nature of the relative swelling data may be due to differences between the nature of HMW1B, which translocates a specific protein substrate, and bacterial porins, which are generally nonspecific diffusion channels (16,33,34).…”

Section: Discussionsupporting

confidence: 83%

“…However, previous studies performed with bacterial Omp85-like proteins have not supported this notion. Although several bacterial family members have been demonstrated to have pore activity, none had been found to be multimeric (7,15,16). Immunoblot analysis revealed that N. meningitidis Omp85 exists in a high-molecular weight complex (7); however, earlier work by Manning et al demonstrated that this protein interacts with several heterologous proteins in the OM (44), thus precluding any conclusion regarding the structural organization of N. meningitidis Omp85.…”

Section: Discussionmentioning

confidence: 99%

“…In bacteria, the only two family members that have been studied in detail are ShlB and FhaC, both of which have been demonstrated to have pore activity. However, no convincing evidence for multimer formation has been obtained for either ShlB or FhaC, thus contrasting with eukaryotic Omp85-like proteins (15,16). These findings have led others to suggest that the tetrameric Omp85 protein translocation system in eukaryotes may have evolved from more primitive, monomeric bacterial Omp85 homologs (3,5).…”

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confidence: 98%

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Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution

Surana

Grass

Hardy

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Omp85-like proteins represent a family of proteins involved in protein translocation, and they are present in all domains of life, except archaea. In eukaryotes, Omp85-like proteins have been demonstrated to form tetrameric pore-forming complexes that interact directly with their substrate proteins. Studies performed with bacterial Omp85-like proteins have demonstrated pore-forming activity but no evidence of multimerization. In this article, we characterize the Haemophilus influenzae HMW1B protein, an Omp85-like protein that has been demonstrated to be critical for secretion of the H. influenzae HMW1 adhesin. Analysis of purified protein by biochemical and electron microscopic techniques revealed that HMW1B forms a tetramer. Examination using liposomeswelling assays demonstrated that HMW1B has pore-forming activity, with a pore size of Ϸ2.7 nm. Far-Western blot analysis established that HMW1B interacts with the N terminus of HMW1. These results provide evidence that a bacterial Omp85-like protein forms a tetramer and interacts directly with a substrate protein, suggesting that the architecture and physical properties of Omp85-like proteins have been conserved throughout evolution. O mp85-like proteins represent a large family defined by phylogenetic relatedness, secondary-structure predictions, and a role in protein translocation (1-5). Members of this family are present in diverse organisms across the evolutionary spectrum, including bacteria, fungi, plants, and animals (1, 4). The widespread nature of Omp85-like proteins underscores their critical role in cellular processes, highlighted by the fact that many are required for cell viability (3,4,(6)(7)(8)(9). Although all Omp85-like proteins share sequence homology, they can be grouped into two classes based on the specific role that they play in protein translocation. One class is typified by Saccharomyces cerevisiae Sam50 and Neurospora crassa Tob55, which are chiefly involved in insertion of ␤-barrel proteins into the outer membrane (OM) of mitochondria (6, 9). The second class is epitomized by the chloroplast protein Toc75, which is involved in secretion of proteins across a membrane (10, 11). Interestingly, both classes of Omp85-like proteins are present in Gramnegative bacteria. Neisseria meningitidis Omp85, the namesake of this family of proteins, has been demonstrated recently to be critical for the insertion of proteins into the bacterial OM (7). Other Gram-negative bacterial Omp85-like proteins, including Serratia marcescens ShlB and Bordetella pertussis FhaC [and other members of the so-called two-partner secretion (TPS) pathway], have been demonstrated to function in protein export across the OM (12).The high level of amino acid homology among Omp85-like proteins from bacteria to humans raises the possibility that members of this family have similar structures. Although no crystal structures exist so far for Omp85-like proteins, several of these proteins have been subjected to biochemical studies of overall architecture. In eukaryotes, both classes...

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 98%

See 1 more Smart Citation

Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution

Surana

Grass

Hardy

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…2 In addition, they all bear a C-terminal motif with a terminal phenylalanine characteristic of the OmpF/C family of porins (13). Therefore, they are hypothesized to form ␤-barrel channels in the outer membrane to specifically let out their cognate exoproteins (14). In this paper we report on the purification and characterization of FhaC.…”

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confidence: 99%

Channel Formation by FhaC, the Outer Membrane Protein Involved in the Secretion of the Bordetella pertussis Filamentous Hemagglutinin

Jacob‐Dubuisson

El-Hamel²,

Saint³

et al. 1999

Journal of Biological Chemistry

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Many virulence factors of pathogenic microorganisms are presented at the cell surface. However, protein secretion across the outer membrane of Gram-negative bacteria remains poorly understood. Here we used the extremely efficient secretion of the Bordetella pertussis filamentous hemagglutinin (FHA) to decipher this process. FHA secretion requires a single specific accessory protein, FhaC, the prototype of a family of proteins necessary for the extracellular localization of various virulence proteins in Gram-negative bacteria. We show that FhaC is heat-modifiable and localized in the outer membrane. Circular dichroism spectra indicated that FhaC is rich in ␤-strands, in agreement with structural predictions for this protein. We further demonstrated that FhaC forms pores in artificial membranes, as evidenced by single-channel conductance measurements through planar lipid bilayers, as well as by liposome swelling assays and patch-clamp experiments using proteoliposomes. Single-channel conductance appeared to fluctuate very fast, suggesting that the FhaC channels frequently assume a closed conformation. We thus propose that FhaC forms a specific ␤-barrel channel in the outer membrane for the outward translocation of FHA.

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“…These recognition events are thought to trigger the translocation of the TpsA proteins, starting from their N terminus, across that membrane. The TpsB transporters appear to form rather small ␤-barrel channels in the outer membrane, through which their TpsA partners pass most likely in an extended conformation (6)(7)(8). Folding of the TpsA proteins is thus hypothesized to take place at the cell surface in a progressive fashion.…”

mentioning

confidence: 99%

The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway

Clantin

Hodak

Willery

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

151

184

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Filamentous hemagglutinin (FHA), the major 230-kDa adhesin of the whooping cough agent Bordetella pertussis, is one of the most efficiently secreted proteins in Gram-negative bacteria. FHA is secreted by means of the two-partner secretion (TPS) pathway. Several important human, animal, and plant pathogens also secrete adhesins and other virulence factors by using this mode of secretion. A TPS system is composed of two separate proteins, with TpsA the secreted protein and TpsB its associated specific outermembrane transporter. All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. We report here the 1.7-Å structure of a functionally secreted 30-kDa N-terminal fragment of FHA. It reveals that the TPS domain folds into a ␤-helix, with three extrahelical motifs, a ␤-hairpin, a fourstranded ␤-sheet, and an N-terminal capping, mostly formed by the nonconserved regions of the TPS domain. The structure thus explains why the TPS domain is able to initiate folding of the ␤-helical motifs that form the central domain of the adhesin, because it is itself a ␤-helical scaffold. It also contains less conserved extrahelical regions most likely involved in specific properties, such as the recognition of the outer-membrane transporter. This structure is representative of the TPS domains found so far in >100 secreted proteins from pathogenic bacteria. It also provides a mechanistic insight into how protein folding may be linked to secretion in the TPS pathway.

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The haemolysin‐secreting ShlB protein of the outer membrane of Serratia marcescens : determination of surface‐exposed residues and formation of ion‐permeable pores by ShlB mutants in artificial lipid bilayer membranes

Cited by 61 publications

References 51 publications

Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution

Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution

Channel Formation by FhaC, the Outer Membrane Protein Involved in the Secretion of the Bordetella pertussis Filamentous Hemagglutinin

The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway

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