Siderophore Transport through Escherichia coli Outer Membrane Receptor FhuA with Disulfide-tethered Cork and Barrel Domains

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded β-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced. The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded β-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced. Iron is required by most living cells because of its diverse roles in numerous metabolic processes, including glycolysis, energy generation by electron transport, and DNA synthesis (1Klebba P.E. Front. Biosci. 2003; 8: s1422-s1436Crossref PubMed Scopus (35) Google Scholar). Under physiological conditions, however, iron forms highly insoluble ferric hydroxide complexes, thereby severely limiting the bioavailability of iron. The outer membrane receptor FhuA of Escherichia coli transports Fe3+ chelated to hydroxamate siderophores such as ferrichrome and its structural analog ferricrocin. FhuA also serves as a primary receptor for the antibiotics albomycin and rifamycin CGP 4832; the antibiotic peptide microcin J25; the bacterial toxin colicin M; and the bacteriophages T5, T1, UC-1, and Φ80. Structural determination of FhuA identified a monomeric C-terminal 22-stranded β-barrel domain (residues 161-714) and an N-terminal globular cork domain (residues 1-160) that resides within the barrel lumen (2Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar, 3Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The position of the cork domain within the barrel occludes the barrel lumen and prevents diffusion of ligand into the periplasm. The mechanism by which this occlusion is modulated to facilitate siderophore transport remains unclear. Ferric siderophore transport requires a pathway with a minimal diameter of ∼15 Å, which is not apparent in either apo-FhuA (Protein Data Bank code 2FCP) or the ferrichrome-bound crystal structure (Protein Data Bank code 1FCP) (1Klebba P.E. Front. Biosci. 2003; 8: s1422-s1436Crossref PubMed Scopus (35) Google Scholar, 4Scott D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M.C. Klebba P.E. J. Biol. Chem. 2001; 276: 13025-13033Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Comparison of these two FhuA structures revealed distinct conformations; most differences are localized to the cork domain, thereby providing direct evidence for conformational changes that occur within FhuA upon ligand binding. In the ferrichrome-bound state, apices in the cork domain are translated 1.7 Å toward the ferrichrome-binding site, forming multiple hydrogen bonds with the ligand (2Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar, 3Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The most conspicuous structural change takes place in an N-terminal region referred to as the switch helix. Upon ferrichrome binding to FhuA, the switch helix is entirely unwound, resulting in a 17.3-Å translocation of the N terminus (2Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar). Despite these changes in the ferrichrome-bound state, a transport pathway sufficient to accommodate a siderophore remains undetected. Therefore, after siderophore binding, the additional structural alterations that FhuA must undergo to allow passage of ligand though the barrel lumen are as yet undisclosed by x-ray crystallographic structures. Proposed models of siderophore transport through FhuA involve subtle rearrangement(s) of the cork domain; alternatively, complete or partial removal of the cork domain from the barrel has been postulated (1Klebba P.E. Front. Biosci. 2003; 8: s1422-s1436Crossref PubMed Scopus (35) Google Scholar, 4Scott D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M.C. Klebba P.E. J. Biol. Chem. 2001; 276: 13025-13033Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Postle K. Kadner R.J. Mol. Microbiol. 2003; 49: 869-882Crossref PubMed Scopus (249) Google Scholar, 6Ferguson A.D. Deisenhofer J. Cell. 2004; 116: 15-24Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Transport into the periplasm requires reduced stability of ferrichrome for its initial binding site, eventually facilitating dissociation from the outer membrane receptor. In ferrichrome-bound FhuA, the N-terminal region of the receptor interacts with the energy-transducing complex TonB-ExbB-ExbD anchored in the cytoplasmic membrane. Selected residues of the Ton box, a sequence of amino acids highly conserved among TonB-dependent outer membrane receptors, make direct interactions with TonB as demonstrated by disulfide cross-linking (7Cadieux N. Kadner R.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10673-10678Crossref PubMed Scopus (145) Google Scholar). These interactions are not only required for transport, but are likely coupled to conformational changes in the cork and barrel during transport (8Endriss F. Braun M. Killmann H. Braun V. J. Bacteriol. 2003; 185: 4683-4692Crossref PubMed Scopus (41) Google Scholar). Such conformational changes have been proposed to destabilize ferrichrome contacts with extracellular loops of the barrel domain and apices of the cork domain (9Braun V. Braun M. FEBS Lett. 2002; 529: 78-85Crossref PubMed Scopus (120) Google Scholar). Disruption of siderophore binding to the receptor may result from TonB interactions, shifting the cork apices toward the periplasm. When viewed along the barrel axis, a previously identified 10-Å solvent-accessible channel (2Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar), now referred to as the putative siderophore transport pathway, connects the extracellular environment to the periplasm. Conformational changes in loops of the cork domain could result in sufficient widening of this pathway to accommodate ferrichrome. Highly conserved residues along the inner barrel wall of the putative siderophore transport pathway may facilitate the diffusion of ferrichrome through FhuA by a series of low affinity binding interactions. Such a mechanism was shown for diffusion of maltose and maltodextrins along the greasy slide of LamB porin (10Van Gelder P. Dumas F. Bartoldus I. Saint N. Prilipov A. Winterhalter M. Wang Y. Philippsen A. Rosenbusch J.P. Schirmer T. J. Bacteriol. 2002; 184: 2994-2999Crossref PubMed Scopus (35) Google Scholar). Conversely, complete or partial removal of the cork domain could expose the lumen of the barrel and allow diffusion of siderophores (11Braun M. Killmann H. Braun V. Mol. Microbiol. 1999; 33: 1037-1049Crossref PubMed Scopus (80) Google Scholar). Experiments comparing the structure and function of wild-type FhuA with FhuA containing N-terminal deletions (FhuAΔ21-128) indicate that the cork domain contributes to overall protein stability (12Bonhivers M. Desmadril M. Moeck G.S. Boulanger P. Colomer-Pallas A. Letellier L. Biochemistry. 2001; 40: 2606-2613Crossref PubMed Scopus (49) Google Scholar). The cork domain is stabilized within the barrel by an extensive network of hydrogen bonds and salt bridges, rendering complete removal of the cork during ferrichrome transport energetically (1Klebba P.E. Front. Biosci. 2003; 8: s1422-s1436Crossref PubMed Scopus (35) Google Scholar, 6Ferguson A.D. Deisenhofer J. Cell. 2004; 116: 15-24Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Kadner R.J. Biol. 2003; PubMed Scopus Google Scholar, L. D. L. M. R. D. Deisenhofer J. Biol. 1999; PubMed Scopus Google Scholar). that of interactions within this network the energy for cork removal J.D. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of the cork from the outer membrane FhuA and result in affinity for but of TonB-dependent D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M.C. Klebba P.E. J. Biol. Chem. 2001; 276: 13025-13033Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, M. Desmadril M. Moeck G.S. Boulanger P. Colomer-Pallas A. Letellier L. Biochemistry. 2001; 40: 2606-2613Crossref PubMed Scopus (49) Google Scholar). has been postulated that of the siderophore from its initial binding an of the extracellular resulting in removal of the cork from the barrel lumen D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M.C. Klebba P.E. J. Biol. Chem. 2001; 276: 13025-13033Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In this the siderophore could through a pathway by has been that the cork domain may through the barrel during its translocation into the periplasm A.D. Deisenhofer J. Cell. 2004; 116: 15-24Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, E. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus (54) Google Scholar). Despite and crystal structures in the mechanism of siderophore transport through FhuA is In this site-directed disulfide cross-linking was to the role of the cork domain during transport of the ferrichrome analog ferricrocin. Selected amino acids along the putative siderophore transport pathway and in the cork domain and and in the barrel were to cysteine residues to allow disulfide bond formation in the of an oxidative catalyst. tethering of barrel and cork was predicted to siderophore either by blocking the putative siderophore transport pathway or by the removal of the cork domain. of ferricrocin with that does not of the cork domain and that siderophores the FhuA receptor through a than the putative siderophore transport pathway. and antibiotics and were from as were and were Oregon Green Oregon Green outer membrane outer membrane from Molecular and and DNA from and from and coli which FhuA a was for thiol-specific G.S. P. H. Coulton J.W. Mol. Microbiol. PubMed Scopus Google Scholar). E. coli G.S. Coulton J.W. J. Bacteriol. PubMed Google J.W. P. J. Bacteriol. PubMed Google and the was for iron are of the E. coli were in with and E. coli were in and with of FhuA within the putative siderophore transport pathway were for cysteine by the modeling B. PubMed Scopus Google Scholar). with a Data Bank the for disulfide formation by possible and cysteine and were created by of the site-directed changes were by into E. coli were for cysteine was with the and and into the of the that been with the These were into E. coli for iron with were in containing and to Cells were and to and were into Cells were and in and K. Mol. Microbiol. 2003; 49: PubMed Scopus Google Scholar), by the of in After a was by the of Cells were with and outer membrane were as previously K. Mol. PubMed Scopus Google Scholar). cells were in by the of and and After formation of were with and and outer membrane were with to SDS-PAGE with was also and outer membrane that been from by with was with FhuA and under the conditions as with or with were denatured in nonreducing by of FhuA was also after the of ferricrocin and of thiol-specific probe to FhuA was for Transport and cells were in and to of was Cells were with iron and and in of iron with was with ferricrocin in and for were for in a to or to oxidative the of were and with were and in a cells containing double cysteine FhuA disulfide bond formation was enhanced by the of an oxidative catalyst. cells were for with or with either or after of uptake. and were by SDS-PAGE containing were for a cross-linking and were from to in a by in and were in of and containing for The blocking was to the primary G.S. Coulton J.W. J. Bacteriol. PubMed Google and the coupled to with a of for were for with containing and in a with a of After were in and The was by the of were with a by to the of disulfide bond formation in the double cysteine FhuA within the Transport evaluate the role of amino acids within the putative siderophore transport pathway of FhuA, site-directed was were to disulfide bonds that the cork and barrel and to a within the putative siderophore transport pathway. the FhuA crystal structure (Protein Data Bank code 2FCP) as the modeling B. PubMed Scopus Google identified amino acids for to cysteine the and of residues in the crystal predicted to form disulfide cysteine mutants (L109C and Q112C) were created in the cork domain, and two were created in and of the barrel domain these predicted disulfide formation for the double cysteine mutants L109C/S356C and Q112C/M383C of Transport of cysteine mutants and M383C) within the putative siderophore transport pathway was by of sulfhydryl with the thiol-specific probe The additional FhuA mutants in and in were created as to probe for and E. coli was with wild-type FhuA, cysteine FhuA mutants and or double cysteine FhuA mutants and E. coli were for with alternatively, and were labeled with were to the of sulfhydryl with In cells and only in was with and mutants in the putative siderophore transport pathway were not that not have to the barrel lumen of of disulfide bond formation in the double cysteine mutants L109C/S356C and Q112C/M383C could not be by was not to in cells or FhuA to were in with was predicted to with In FhuA with of of also with with or were during ferricrocin were for and with FhuA not ligand not of FhuA by cells double cysteine FhuA mutants are to form predicted disulfide bonds within the putative siderophore transport pathway, FhuA was denatured under nonreducing conditions, by with Upon of FhuA, cysteine residues and were to wild-type FhuA of cells containing double cysteine FhuA mutants by SDS-PAGE under nonreducing conditions a of two distinct species: FhuA in which the disulfide bond and FhuA containing free sulfhydryl and The and by the was by free sulfhydryl groups. The and to the and could not be fluorescently the of disulfide When wild-type FhuA was denatured in the or of the resulting cysteine FhuA mutants under conditions were resulting in with to that of wild-type FhuA labeled with and by nonreducing SDS-PAGE revealed that the and putative siderophore transport pathway because was not of FhuA disulfide bond formation during ferricrocin transport an oxidative that of cells with an oxidative has been shown to disulfide bond formation cysteine K. Mol. Microbiol. 2003; 49: PubMed Scopus Google Scholar). the sulfhydryl of the double cysteine residues be with such as and The oxidative has been to disulfide bond formation of membrane J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To disulfide formation in double cysteine FhuA cells in iron were with an oxidative or To the of FhuA containing disulfide bonds under these conditions, the were by nonreducing SDS-PAGE and by the cork and barrel in double cysteine FhuA mutants was apparent from the reduced of with protein containing free sulfhydryl groups. protein was not in wild-type FhuA or in cysteine not with and for L109C/S356C and with as the cross-linking in equivalent of disulfide formation in cells L109C/S356C and Q112C/M383C in Cells FhuA and double cysteine FhuA mutants were for to transport the FhuA and the required for transport were in the E. coli The synthesis of which in transport Iron by wild-type FhuA was as of cells a of cysteine mutants and exhibited of wild-type FhuA and and with wild-type double cysteine FhuA mutants also of L109C/S356C of the wild-type Q112C/M383C These results demonstrate reduced transport of into cells double cysteine FhuA under these conditions, cells containing L109C/S356C and Q112C/M383C only and in FhuA cross-linking of the cork to the barrel would siderophore by FhuA, cells were with an oxidative to disulfide formation the of the was of cross-linking double cysteine residues to an siderophore wild-type transport by not has been shown that Fe3+ with for binding to in H. J. PubMed Google Scholar). In this the in transport may have been to for binding ferricrocin and resulting in the of the of double and were with to disulfide bond of cells were and the of into the cells was by by wild-type FhuA in the of the oxidative transport cysteine FhuA mutants within the putative siderophore transport pathway also to transport ferricrocin after and Despite complete disulfide L109C/S356C and Q112C/M383C and of Therefore, these double cysteine mutants were for transport of ferricrocin of and with double cysteine FhuA mutants disulfide bonds the putative siderophore transport pathway not ferricrocin transport through in FhuA during siderophore transport with cross-linking of L109C/S356C and Q112C/M383C within the putative siderophore transport pathway. transport of cells L109C/S356C not the of disulfide bond formation In cells Q112C/M383C exhibited disulfide bond formation during ferricrocin transport After of ferricrocin transport, the of cross-linking in Q112C/M383C was in the and of and cysteine FhuA mutants with the of an oxidative not reduced disulfide formation upon oxidation the previously after a with to of During ferricrocin transport, disulfide formation by in L109C/S356C from to Q112C/M383C from to Despite of the cork to the barrel and of the putative siderophore transport pathway, was in ferricrocin transport, with of and of cells These results that, during ferricrocin transport, of the cork domain residues in to the inner barrel wall along the putative siderophore transport pathway. The mechanism for siderophore transport through TonB-dependent is Proposed models have partial or complete removal of the cork domain from the barrel during siderophore passage (1Klebba P.E. Front. Biosci. 2003; 8: s1422-s1436Crossref PubMed Scopus (35) Google Scholar, 4Scott D.C. Cao Z. Qi Z. Bauler M. Igo J.D. Newton S.M.C. Klebba P.E. J. Biol. Chem. 2001; 276: 13025-13033Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Postle K. Kadner R.J. Mol. Microbiol. 2003; 49: 869-882Crossref PubMed Scopus (249) Google Scholar, 6Ferguson A.D. Deisenhofer J. Cell. 2004; 116: 15-24Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). FhuA crystal structures in the and of siderophore not completely conformational changes during energy and siderophore Therefore, in and in are required to possible of the cork domain during After residues in the FhuA crystal structure (2Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar), FhuA in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were The two models of siderophore transport, of the putative siderophore transport pathway and of the cork domain, could be by forming disulfide the cork and barrel within this To formation of disulfide bonds predicted by modeling and of the thiol-specific with was conformational changes during ferricrocin transport were also of FhuA was labeled with the cysteine residues within the putative siderophore transport pathway and and a cysteine in a with the thiol-specific probe results to with not to the periplasm by the outer membrane through demonstrated of of the cytoplasmic TonB K. Mol. Microbiol. 2003; 49: PubMed Scopus Google and D. K. E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Z. T. J. Bacteriol. 2001; PubMed Scopus Google Scholar). was not to in cells or of After from to with Conversely, the was of with in FhuA Therefore, the of the of with the thiol-specific acids to the have that with low by D. K. E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Z. T. J. Bacteriol. 2001; PubMed Scopus Google Scholar). These were by of required to in the of to is in with a by and Braun Braun V. Microbiol. Lett. PubMed Scopus Google Scholar), that the of with the thiol-specific is upon ferrichrome binding, structural changes in In were changes in the of FhuA or cysteine of FhuA to during ferricrocin that structural changes during transport not the of these cysteine of the cork domain during transport was predicted to cysteine mutants in the barrel lumen and cork cysteine mutants in the periplasm to because cells containing the cysteine FhuA mutants within the putative siderophore pathway were not labeled by during ferricrocin transport, these results a mechanism the cork remains within the barrel. To the of disulfide bond double cysteine FhuA mutants were denatured under nonreducing disulfide bonds to would not be to of the x-ray crystallographic structure of FhuA, the two disulfide bonds in FhuA were identified a thiol-specific Braun V. Microbiol. Lett. PubMed Scopus Google Scholar). After nonreducing wild-type FhuA was not labeled with cysteine FhuA mutants were to and The of two distinct FhuA in double cysteine mutants under nonreducing conditions partial disulfide bond The FhuA containing were by free sulfhydryl also SDS-PAGE of FhuA may be to the residues in L109C/S356C and residues in forming the disulfide bonds, the protein to a partial Conversely, the two disulfide bonds in FhuA, in loops and not with during to the residues in and residues in Braun V. Microbiol. Lett. PubMed Scopus Google Scholar). FhuA disulfide bonds and FhuA containing free sulfhydryl was in the of oxidative containing mutants were required to probe of the cork domain or of the putative siderophore transport pathway during siderophore uptake. oxidative that the cork domain is not from the barrel during ferricrocin cysteine FhuA mutants disulfide bonds the oxidative and has been that the the for cross-linking cysteine residues oxidation is Å and is and Å J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). L109C/S356C and Q112C/M383C have of and Å, tethering of the cork and barrel was to probe the of the conformational of the cork domain to the barrel. transport of in cells double cysteine FhuA mutants was not oxidative under these conditions, L109C/S356C and Q112C/M383C only and The cysteine mutants and transport, that these residues may with the barrel domain during ferricrocin Therefore, the reduced in double cysteine FhuA mutants may be to the transport by and in of the double cysteine mutants of cells with was to ferricrocin transport, of were cells with the oxidative with siderophore by cells not with FhuA containing double cysteine mutants within the putative siderophore transport pathway Therefore, tethering of the cork domain to the barrel does not conformational changes in the receptor that may ferricrocin transport into the periplasm. The of to form disulfide bonds with during transport additional into of the cork domain. through Q112C/M383C not disulfide formation in the of enhanced of the two putative pathway sulfhydryl and during Therefore, is that the cork from the barrel wall in this region to allow siderophore When cells ferricrocin were with the of Q112C/M383C disulfide formation to untreated however, L109C/S356C disulfide formation ferricrocin transport was not by disulfide formation during its passage through FhuA tethering could not occur the cork domain were displaced into the periplasm. disulfide bonds within the putative siderophore transport pathway not ferricrocin transport through FhuA, is likely that, upon with conformational changes may an pathway. tethering of the switch helix of the cork to of the barrel domain and the transport of and microcin through FhuA (8Endriss F. Braun M. Killmann H. Braun V. J. Bacteriol. 2003; 185: 4683-4692Crossref PubMed Scopus (41) Google Scholar). of the cork to the barrel with but does not allow in the cork to a transport Therefore, this double cysteine FhuA may indicate an siderophore transport pathway. cross-linking to be an to probe conformational changes during ferricrocin tethering of the cork to the barrel domain within the putative siderophore transport pathway was not sufficient to ferricrocin uptake. enhanced Q112C/M383C disulfide and upon oxidative double cysteine FhuA mutants disulfide formation through the putative siderophore transport pathway. that the cork domain remains within to the inner barrel wall during siderophore the siderophore likely the of the receptor through a series of low affinity interactions the cork domain and residues along the inner wall of the barrel domain. tethering of the cork and barrel in of FhuA to of the siderophore transport pathway. of this pathway and the mechanism for siderophore transport through FhuA into transport of by TonB-dependent outer membrane We B. M. A. and H. for the and J. A. for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.