Evidence of Ball-and-chain Transport of Ferric Enterobactin through FepA

The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane β-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy- and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the β-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal β-barrel domain confirmed the feasibility of this mechanism. The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane β-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy- and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the β-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal β-barrel domain confirmed the feasibility of this mechanism. FepA is a Gram-negative bacterial outer membrane (OM) 6The abbreviations used are: OM, outer membrane; IM, inner membrane; FeEnt, ferric enterobactin; FM, fluorescein maleimide; MOPS, 4-morpholinepropanesulfonic acid; TBS, Tris-buffered saline; LGP, ligand-gated porin. protein that transports ferric enterobactin (FeEnt) (1Pugsley A.P. Reeves P. J. Bacteriol. 1976; 126: 1052-1062Crossref PubMed Google Scholar, 2McIntosh M.A. Earhart C.F. Biochem. Biophys. Res. Commun. 1976; 70: 315-322Crossref PubMed Scopus (30) Google Scholar, 3Wayne R. Frick K. Neilands J.B. J. Bacteriol. 1976; 126: 7-12Crossref PubMed Google Scholar). The crystal structures of FepA (4Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (492) Google Scholar) and other bacterial metal transporters (FhuA, BtuB, and FpvA (15Locher 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 (458) Google Scholar, 16Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar, 17Chimento D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. 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Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, 8Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2900) Google Scholar), because they selectively adsorb metal chelates with high affinity (3Wayne R. Frick K. Neilands J.B. J. Bacteriol. 1976; 126: 7-12Crossref PubMed Google Scholar, 9Di Masi D.R. White J.C. Schnaitman C.A. Bradbeer C. J Bacteriol. 1973; 115: 506-513Crossref PubMed Google Scholar, 10Luckey M. Neilands J.B. J. Bacteriol. 1976; 127: 1036-1037Crossref PubMed Google Scholar, 11Luckey M. Pollack J.R. Wayne R. Ames B.N. Neilands J.B. J. Bacteriol. 1972; 111: 731-738Crossref PubMed Google Scholar, 12Bradbeer C. Woodrow M.L. Khalifah L.I. J. Bacteriol. 1976; 125: 1032-1039Crossref PubMed Google Scholar, 13Bradbeer C. Kenley J.S. Di Masi D.R. Leighton M. J. Biol. Chem. 1978; 253: 1347-1352Abstract Full Text PDF PubMed Google Scholar, 14Braun V. Wolff H. FEBS Lett. 1973; 34: 77-80Crossref PubMed Scopus (39) Google Scholar). Ligand binding causes small conformational changes that activate them to transport competency (15Locher 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 (458) Google Scholar, 16Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar, 17Chimento D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. Biol. 2003; 10: 394-401Crossref PubMed Scopus (237) Google Scholar), hence their designation “ligand-gated porin” (LGP). The requirements for metabolic energy (18Wang C.C. Newton A. J Bacteriol. 1969; 98: 1142-1150Crossref PubMed Google Scholar, 19Pugsley A.P. Reeves P. J Bacteriol. 1977; 130: 26-36Crossref PubMed Google Scholar, 20Bradbeer C. J Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (148) Google Scholar) and another cell envelope protein, TonB (21Wang C.C. Newton A. J. Biol. Chem. 1971; 246: 2147-2151Abstract Full Text PDF PubMed Google Scholar, 22Guterman S.K. Dann L. J. Bacteriol. 1973; 114: 1225-1230Crossref PubMed Google Scholar, 23Reynolds P.R. Mottur G.P. Bradbeer C. J. Biol. Chem. 1980; 255: 4313-4319Abstract Full Text PDF PubMed Google Scholar, 24Postle K. J. Bioenerg. Biomembr. 1993; 25: 591-601PubMed Google Scholar), in LGP mediated transport are well known but unaccounted for: the OM has no source of energy and cannot sustain an ion gradient because of its open porin channels (7Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar); TonB is a minor cell envelope protein whose functions are not yet understood. In live cells, FepA binds and transports FeEnt via sub-reactions with different dependences on energy and TonB. (i) In the absence of ligand the receptor opens, and its flexible surface loops extend outward (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar). FeEnt binds to FepA in a M.A. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that with to in the C. Newton Mol. Microbiol. 2000; PubMed Scopus Google Scholar, R. B. Newton J. Bacteriol. PubMed Scopus Google Scholar). in (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar), on the iron a that the iron with and residues in the C. Newton Mol. Microbiol. 2000; PubMed Scopus Google Scholar, J.S. C. M.A. A. PubMed Scopus Google Scholar, D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar). binding FeEnt the for transport the β-barrel. In the binding a the from the β-barrel a of receptor from the cell surface to the (15Locher 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 (458) Google Scholar, 16Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar). data on the FepA binds FeEnt, but conformational motion in the of binds N. Kadner R.J. Nat. Struct. Biol. 2000; PubMed Scopus Google Scholar). These of ligand uptake are energy- and because they with affinity and in and cells. In FepA FeEnt its transmembrane the not in cells. The of the N-domain in transport is E. Deisenhofer J. A. 98: PubMed Scopus Google Scholar). has affinity for the ligand E. Deisenhofer J. A. 98: PubMed Scopus Google Scholar), and the FepA a to open a to the In the ball-and-chain the N-domain the periplasm, a globule that on the of its to the β-barrel residue by of its in the channel. N-domain expulsion ligand the metal with the N-domain of the N-domain to the transport FeEnt the channel. The on the other conformational motion in the N-domain is in the β-barrel, a for of the metal The of FeEnt that a a of to this the ligand the protein by between binding with affinity for its natural the FeEnt, this by binding to residues that the of the FepA N-domain and its membrane channel. In this for the between N-domain the periplasm, by the susceptibility of engineered Cys residues to by fluorescein maleimide during FeEnt and at with in in Scholar) and and labeling the from Smith J. Bacteriol. PubMed Google Scholar) M.A. Earhart C.F. Biochem. Biophys. Res. Commun. 1976; 70: 315-322Crossref PubMed Scopus (30) Google Scholar) the but the from iron the of the inner membrane and and periplasmic of the transport system and transport of FeEnt the OM is the in its uptake D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar, C. D.C. M.A. N. J. Newton J. Bacteriol. 2000; PubMed Scopus Google Scholar). and formed iron of enterobactin R. B. Newton J. Bacteriol. PubMed Scopus Google Scholar) and from van der by the in them with an of in in at the to with the ferric in and their from their at We and from of Escherichia coli M.A. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and engineered in E. coli M.A. Neilands J.B. J. Bacteriol. PubMed Google Scholar) by A. 2000; PubMed Scopus Google Scholar). of and and by from and of their cell and with of and confirmed their by of FeEnt and transport, and and susceptibility D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar). We by of the in M. F. H. V. J. Bacteriol. 2003; PubMed Scopus Google Scholar), was a from V. Cys engineered mutations on (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar, D.C. M. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a of the A. PubMed Scopus (138) Google Scholar) its natural Cys substitution mutations in on for the and by the and to transport FeEnt, and the of the FepA D.C. Mol. Microbiol. 1999; PubMed Scopus Google their from FepA. cells, to in by in on and by a cell at The OM and inner membrane formed by J. H. J. Bacteriol. PubMed Google Scholar) by gradient H. J. Bacteriol. PubMed Google Scholar) to with J. Bacteriol. PubMed Google Scholar). and in for and D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar) at to the was with with J. Bacteriol. PubMed Google in for and with D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar). of protein by in of for to and to and on an was on the of Cys residues in live cells, in the in and at they The by by in and and in with FeEnt the was by in its was was to for at in the The by the of cysteine and the with of TBS, in TBS, by at and by in to and the by of cell by the with and to a and for and the by FepA was the protein that labeled in the outer but other in the The of FepA Cys fluoresceination was to the labeling that was for other in the cell between the N-domain and the in and in at the of by and by and with to coli a of the was with the N-domain and β-barrel of FepA and with We the of the by and the of the protein expressed by susceptibility to and and by FeEnt of OM of complementation between of ferric receptor and receptor on K. J. Bacteriol. PubMed Scopus Google Scholar), engineered A. 2000; PubMed Scopus Google Scholar) an of bacterial of and in of with D.C. M. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar)), and with D.C. M. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar)), the for their susceptibility to and and L. F. B. W. B. M. M. and P. E. for and for their to and transport ferric enterobactin and The of the to the and the absence of FepA and and the complementation of the engineered to by and on this and to transport ferric enterobactin and the and of and to of FepA and in the and in the from the (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar, C. Newton Mol. Microbiol. 2000; PubMed Scopus Google Scholar, R. B. Newton J. Bacteriol. PubMed Scopus Google Scholar, D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar, D.C. M. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of FeEnt uptake in 1) from in P. Newton J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that the and with of receptor to previously and J. J. S.K. M.A. J.B. Science. PubMed Scopus Google Scholar). the of receptor in and Fig. a with an of another that in and E. H. J. Biol. Chem. Full Text PDF PubMed Google and of mutations of Cys by 25 Cys confirmed the of the FepA in the Cys of FepA FeEnt the protein in and susceptibility to B. The of the FepA expressed at in the of a cysteine not the functions of the outer membrane protein. We the sulfhydryl side chains for their to for at with FeEnt, residues The labeled engineered Cys residues in surface loops and at with in the and and modified a Cys residue in the protein on the surface of the N-domain The of labeling at of in to the binding and transport of FeEnt and the to with its was of that labeled residue buried in the on the surface of the but not residues on the of the β-barrel and The of was that of surface residues and of live E. coli was to FM, the Cys substitution of FepA other modified by the and labeled at The of labeling of other on the of and the of the labeling on the and of fluoresceination not to at for at in TBS, This labeling of the FepA Cys of and labeling of other at The of Cys reactivity at different in FepA was high of labeling residue of P. Newton J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), was labeled at a of of in the absence of FeEnt, and used the of fluoresceination a to the labeling at other was residue whose susceptibility to during the binding and transport of FeEnt, and the of its in FepA with this substitution of the of the in the OM and a small of in the In the other that with in cell Fig. in the and the not between the the from of the cell from the and the OM side residue in FepA, was of labeled that between and of and was the OM that of the of the OM the was for FepA in the OM but with other in the and inner of FeEnt on labeling of of to in the of the of FeEnt for at The by of by and the of the FepA was to the labeling of in the cell The fluoresceination of surface residue of at of cell labeling and was of and The fluoresceination of periplasmic of expressed of with absence of and and data from on the of on labeling of and the of FeEnt of and uptake with by the of of and open to and for The labeling of was to in the and a of in the The the for and with from the of data from on the of on labeling of in and In and on the data the of the of during FeEnt and the data Cys in different locations for and to at with FeEnt, in cells, in the absence of the at conformational changes that during FeEnt transport, not FepA but labeled in FeEnt was and the energy In cells, in the absence of FeEnt, with on the surface of the the OM The reactivity of this in the absence of FeEnt the that FepA an open in (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar), to from the In in the of FeEnt, not with but not transport FeEnt, and binding of the ferric FepA to a (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar) with to the from the the In cells, in the of FeEnt, was These and FeEnt, and the labeling of transport that during FeEnt uptake the N-domain surface its to was that with from the in the of FeEnt because the ligand the receptor and the to from the periplasmic FepA motion with transport. with this of energy by and hence FeEnt the fluoresceination of These data that the OM and labeled from the of from the during transport, other In cells, modified but in the of These data confirmed that the periplasm, was to at the of the FepA β-barrel. labeled and and of reside at the periplasmic FeEnt binding transport the susceptibility of and because ligand binding the from the β-barrel in other LGP (15Locher 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 (458) Google Scholar, 16Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar, 17Chimento D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. Biol. 2003; 10: 394-401Crossref PubMed Scopus (237) Google Scholar). the that the of FepA a to the formed during its reactivity with and on the surface of the β-barrel, in to In and not with that not to by the β-barrel. The labeling of but not the and the ball-and-chain in and labeled Cys at and was FepA was and the of its binding by FeEnt in from the metal FepA, to FM, and the labeling the and energy transport that modified FepA motion with transport. The fluoresceination of the of in the periplasm, to a to the the N-domain the the of reactivity with and to in the the is the of the mechanism. of a between and of a between and the transport of FeEnt and of the the of FepA to transport the the FepA was in transport, and with the FeEnt transport of labeled this at a reduced that was by the of of the N-domain the fluoresceination of the that was during transport. of FeEnt of the fluoresceination of in of the FepA transport the by the Cys that FeEnt and the receptor was that with from the during FeEnt transport, because the surface loops of the receptor conformational changes during ligand (25Scott D.C. Newton J. Bacteriol. PubMed Scopus Google Scholar, P. Newton J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. J.B. Newton Science. PubMed Scopus Google Scholar). the of of ligand the of uptake and by FeEnt the of the ferric to the receptor binding and other and adsorb to FepA with high affinity R. B. Newton J. Bacteriol. PubMed Scopus Google Scholar) and M.A. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but FeEnt uptake and of and at FeEnt the other from binding and transport with FepA, that the of to from the at of FeEnt in of solute with FepA was and the by the of in a We the of to the OM and in the periplasm, by of the cell and the in E. coli and and and with to an that of the and this was by FeEnt porin deficiency in high of the ferric to the fluoresceination of the resides in the of FeEnt and porin deficiency the labeling of previously and FeEnt binding and transport the of FeEnt was the for this this modified at a of that for the of of labeling not FeEnt was of solute uptake the by of the porins and a in the labeling of These data that of the by that the porin was the for of the the and that in the absence of and the by other to sustain a of the of FeEnt on the labeling of in and In of FeEnt and high fluoresceination of The at FeEnt a and the of labeling was These data and with the of FeEnt on and uptake that at FeEnt the receptor protein, its of other In cells, on the other the of FeEnt and the ferric In the of FM, the fluoresceination of a consistent but at the and not of the FeEnt, not this of with at a that was of that in the of FeEnt to fluoresceination from the of the labeling of in transport cells. The labeling for from the that was and from the that was to by FeEnt binding transport. with the confirmed this In the absence of FeEnt labeled at a that was of and porin deficiency a in labeling of in that of the fluoresceination from the In the of FeEnt, on the other porin deficiency a in labeling of that of the fluoresceination from the at FeEnt, the of the labeling of from the In residue the periplasmic in the of FeEnt, was labeled from the periplasmic side of the OM of by The data expulsion of the N-domain from the during transport, for transport to the N terminus within the β-barrel. We this by separately the and in The of in for the their in to to for in of the to out was to and during the but to the during the The data the of was to in by the of within the and of B. We not FeEnt transport by the of and E. coli was for an engineered of the and are of the and is a of residues of FepA, the of its natural residues by a of residues and with residues was in a a and of was to an previously with bacterial in The at The are the of the that of the bacterial was in a a and of was to an previously with bacterial in The at The are the of the that of the bacterial an engineered of the and are of the and is a of The residues of FepA, the of its natural residues by a of residues and with residues was in a a and of was to an previously with bacterial in The at The are the of the that of the bacterial in a The labeling of engineered Cys sulfhydryl by reflected their in These data in FepA during its transport of in FepA was energy- and the of the to that during transport. of in with the labeling with on the cell surface but porin channels and modified residues in the is the of porin but consistent with H. J. Bacteriol. PubMed Google Scholar, H. J. J. Bacteriol. PubMed Google Scholar). In an fluoresceination of the periplasmic residue This the uptake and demonstrated that to in the The of the of the labeling In the absence of FeEnt, between the and the β-barrel to but its susceptibility FepA These the of in the during FeEnt motion in (15Locher 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 (458) Google Scholar, 16Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (668) Google Scholar) and D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. Biol. 2003; 10: 394-401Crossref PubMed Scopus (237) Google Scholar, N. Kadner R.J. A. 2003; PubMed Scopus Google Scholar) but was not in the FepA crystal (4Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (492) Google Scholar) by other in between the N-domain and the the of with the that they are to in the absence of ligand 2003; PubMed Scopus Google Scholar). FeEnt this the for ligand and for reactivity with The reactivity of to the by in a not transport In the absence of FeEnt, labeled and the of FeEnt its that the from the and that FeEnt in the binding this labeling of during FeEnt transport from the periplasmic and of cell surface and periplasmic residues in and the in of in confirmed this In the absence of FeEnt, the of by that in with a OM, of labeling from the periplasmic and from the in a FeEnt of and porin deficiency of the during FeEnt transport of the from the periplasmic was labeled by periplasmic The of in the potentially between the and ball-and-chain modified during FeEnt transport, to and a on the of the β-barrel. This that the not the to with These labeling data are with the of the N-domain the The to this of evidence is the that of and from conformational within the β-barrel, their with the from of FepA to for the ball-and-chain mechanism. of transport and in labeling by between the and the β-barrel is consistent with the ball-and-chain with reduced but not but the engineered in that system formed Coulton J.W. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) in FepA. complementation of the by a separately expressed N terminus the that the globular domain the β-barrel. The of the FepA protein, at a of the feasibility of this The mechanism of a a bacterial the of FepA In the the E. coli OM FepA R. B. Newton J. Bacteriol. PubMed Scopus Google Scholar, D.C. Mol. Microbiol. 1999; PubMed Scopus Google Scholar, D.C. M. Newton J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar); the of from of the that of the N and C in We not see FeEnt uptake by the FepA protein, but this is not because of the of and of transport by a protein. its to the of the protein. was complementation was previously in the system M. F. H. V. J. Bacteriol. 2003; PubMed Scopus Google Scholar), the of LGP to their β-barrels. the of FepA in ball-and-chain the of its globular domain from the during solute an by and between the N-domain and β-barrel. are The surface of the N-domain is and the of to other residues to in the periplasmic the of to residues on the a of in the protein N-domain motion in and out of the the N terminus within the by changes in the protein a for its expulsion 2003; PubMed Scopus Google Scholar). the N-domain a to the and in by other them and the the ligand the with in its with and Wiener M.C. Science. PubMed Scopus Google Scholar, N. C. C.M. N. M. Coulton J.W. Science. PubMed Scopus Google Scholar), a to of the N-domain and ligand We and for the for providing and for providing