Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop

Killmann, Helmut; Benz, Roland; Braun, Volkmar

doi:10.1128/jb.178.23.6913-6920.1996

Cited by 44 publications

(54 citation statements)

References 36 publications

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“…These results suggest a level of plasticity along the length of this well-conserved protein that allows TbpA to accommodate insertions with relatively little impact on structure or function. Results from a limited deletion analysis of TbpA (5) are in agreement with this assertion, and both studies support the theory that gonococcal TbpA, like other TonB-dependent receptors (3,7,33,34,39,44,47) is resilient to various types of mutations.…”

Section: Discussionsupporting

confidence: 74%

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Yost-Daljev

Cornelissen

2004

Infect Immun

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The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and ␤ strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative ␤ strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA.

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Section: Discussionsupporting

confidence: 74%

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Yost-Daljev

Cornelissen

2004

Infect Immun

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“…This is presumably reflective of a fundamental structural difference between the two barrels; the TolC barrel is mainly engaged in the removal of hydrophobic inhibitors from the cells, while the porin barrel lets small hydrophilic molecules diffuse through it. The conversion of FhuA and FepA, which are substrate-specific and TonB-dependent OMP transporters, to general diffusion OMPs has also been reported (17,31). These transporters do not allow general solute diffusion because of the existence of a large plug domain that blocks the inside of the barrels (4, 10).…”

Section: Discussionmentioning

confidence: 99%

“…These transporters do not allow general solute diffusion because of the existence of a large plug domain that blocks the inside of the barrels (4, 10). However, deletions that shorten the plug domain do allow the nonspecific diffusion of a variety of compounds, including novobiocin and rifampin (17,31).…”

Section: Discussionmentioning

confidence: 99%

Antibiotic-Sensitive TolC Mutants and Their Suppressors

Augustus¹,

Celaya

Husain

et al. 2004

J Bacteriol

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The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic ␣-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex ؉ phenotype. These novel phenotypes of TolC R367H and TolC R390C were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC R367H mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC R367H , mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.Escherichia coli cells lacking the outer membrane protein (OMP) TolC display hypersensitivity to a variety of inhibitors, including bile salts, detergents, and hydrophobic antibiotics (38). This was initially thought to be due to a defect in the outer membrane permeability barrier as a result of a defective lipopolysaccharide (LPS) (32). However, it was shown that tolC and rfa (LPS core) mutations produce an additive effect on hypersensitivity, thus suggesting that tolC mutations may confer hypersensitivity indepen...

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“…Uptake of microcin J25 and infection of Salmonella serovar Typhimurium by phage ES18 may require both the cork and the ␤-barrel. We have previously shown that the prominent loop of the FhuA ␤-barrel (18), which is loop 4 in the E. coli FhuA crystal structure (7,20) and lies above the cell surface, serves as the principal binding site of the phages and colicin M (13,14,15). This result implies that TonB, without the help of the cork, can change the conformation of loop 4 such that binding of phages T1 and 80 triggers DNA release from the phage head.…”

Section: Discussionmentioning

confidence: 99%

FhuA Barrel-Cork Hybrids Are Active Transporters and Receptors

et al. 2001

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The crystal structure of Escherichia coli FhuA reveals a ␤-barrel domain that is closed by a globular cork domain. It has been assumed that the proton motive force of the cytoplasmic membrane through the interaction of the TonB protein with the TonB box of the cork opens the FhuA channel. Yet, deletion of the cork results in an FhuA derivative, FhuA⌬5-160, that still displays TonB-dependent substrate transport and phage receptor activity. To investigate this unexpected finding further, we constructed FhuA⌬5-160 derivatives of FhuA proteins from Salmonella paratyphi B, Salmonella enterica serovar Typhimurium, and Pantoea agglomerans. The FhuA⌬5-160 proteins inserted correctly into the outer membrane, and with the exception of the P. agglomerans protein, transported ferrichrome and albomycin. FhuA hybrids consisting of the ␤-barrel of one strain and the cork of another strain were active and showed higher TonB-dependent ferrichrome transport rates than the corkless derivatives. Exceptions were the E. coli ␤-barrel/Salmonella serovar Typhimurium cork hybrid protein and the Salmonella serovar Typhimurium ␤-barrel/P. agglomerans cork hybrid protein, both of which were less active than the ␤-barrels alone. Each of the FhuA mutant proteins displayed activity for each of their ligands, except for phage T5, only when coupled to TonB. The hybrid FhuA proteins displayed a similar activity with the E. coli TonB protein as with their cognate TonB proteins. Sensitivity to phages T1, T5, and 80, rifamycin CGP 4832, and colicin M was determined by the ␤-barrel, whereas sensitivity to phage ES18 and microcin J25 required both the ␤-barrel and cork domains. These results demonstrate that the ␤-barrel domain of FhuA confers activity and specificity and responds to TonB and that the cork domains of various FhuA proteins can be interchanged and contribute to the activities of the FhuA hybrids.The FhuA outer membrane transport protein of Escherichia coli consists of 22 antiparallel ␤-sheets that form a ␤-barrel into which a globular domain is inserted from the periplasmic side. The globular domain seems to close the ␤-barrel channel and prevent entry of even small molecules and was for this reason designated the "cork" (7) or "plug" (20). Ferrichrome, the natural substrate of FhuA, binds in a cavity located well above the outer membrane lipid bilayer. The cork domain and the ␤-barrel domain contribute five and six amino acid side chains to the cavity, respectively, which are less than 4 Å away from the ferrichrome (7). It is thought that opening of the FhuA channel requires dislocation of the cork, resulting in a connection between the cavity exposed to the cell surface and the region exposed to the periplasm. Although binding of ferrichrome to FhuA moves the cork about 2 Å towards ferrichrome, this does not open the channel.Energy provided by the cytoplasmic membrane in the form of the proton motive force (3) and the TonB-ExbB-ExbD protein complex are required for active transport through FhuA. Binding of ferrichrome results in the ...

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Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop

Cited by 44 publications

References 36 publications

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Antibiotic-Sensitive TolC Mutants and Their Suppressors

FhuA Barrel-Cork Hybrids Are Active Transporters and Receptors

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