Abstract
Uptake of iron complexes into the Gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC)) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BP), integral membrane proteins, and membrane-associated ATP-hydrolyzing proteins. In Gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. To date, there is little structural information about the components of ABC transport systems involved in iron complex transport. The recently determined structure of the Escherichia coli periplasmic ferric siderophore binding protein FhuD is unique for an ABC transport system (Clarke et al. 2000). Unlike other BP's, FhuD has two domains connected by a long α-helix. The ligand binds in a shallow pocket between the two domains. In vivo and in vitro analysis of single amino acid mutants of FhuD identified several residues that are important for proper functioning of the protein. In this study, the mutated residues were mapped to the protein structure to define special areas and specific amino acid residues in E. coli FhuD that are vital for correct protein function. A number of these important residues were localized in conserved regions according to a multiple sequence alignment of E. coli FhuD with other BP's that transport siderophores, heme, and vitamin B12. The alignment and structure prediction of these polypeptides indicate that they form a distinct family of periplasmic binding proteins.
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Altschul SF, Madden T, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.
Ames GF. 1986 Bacterial periplasmic transport systems: structure, function and evolution. Annu Rev Biochem 55, 397-425.
Böhm B, Boschert H, Köster W. 1996 Conserved amino acids in the N-and C-terminal domains of integral membrane transporter FhuB define sites important for intra-and intermolecular interaction. Mol Microbiol 20, 223-232.
Braun V. 1998 Pumping iron through cell membranes. Science 282, 2202-2203.
Braun V, Hantke K, Köster W. 1998 Bacterial iron transport, mechanisms, genetics, and regulation. In: Sigel A & Sigel H, eds. Iron Transport and Storage in Microorganisms, Plants, and Animals. Vol. 35 of 'Metal Ions in Biological Systems'. New York: Marcel Dekker, Inc.; 67-145.
Braun V, Killmann H. 1999 Bacterial solutions to the iron-supply problem. Trends Biochem Sci 24, 104-109.
Buchanan SK, Smith BS, Venkatramani L, Xia D, Esser L, Palnitkar M, Chakraborty R, van der Helm D, Deisenhofer J. 1999 Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat Struct Biol 6, 56-63.
Byers BR, Arceneaux JE. 1998 Microbial iron transport: iron acquisition by pathogenic microorganisms. Met Ions Biol Syst 35, 37-66.
Clarke TE, Ku SY, Dougan DR, Vogel HJ, Tari LW. 2000 The structure of the ferric siderophore binding protein FhuD complexed with gallichrome. Nat Struct Biol 7, 287-291.
Clarke TE, Tari LW, Vogel HJ. 2001 Structural biology of iron uptake systems. Curr Top Med Chem 1, 7-30.
Cox CD. 1982 Effect of pyochelin on the virulence of Pseudomonas aeruginosa. Infect Immun 36, 17-23.
Evans SV. 1993 SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph 11, 134-138.
Ferguson AD, Braun V, Fiedler HP, Coulton JW, Diederichs K, Welte W. 2000. Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA. Protein Sci 9, 956-963.
Ferguson AD, Hofmann E, Coulton JW, Diederichs K, Welte W. 1998. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 282, 2215-2220.
Groeger W, Köster W. 1998 Transmembrane topology of the two FhuB domains representing the hydrophobic components of bacterial ABC-transporters involved in the uptake of siderophores, heme, and vitamin B12. Microbiol 144, 2759-2769.
Köster W. 1991 Iron(III)hydroxamate transport across the cytoplasmic membrane of Escherichia coli. Biol Metals 4, 23-32.
Köster W. 2001 ABC transporter-mediated uptake of iron, siderophores, heme and vitamin B12. Res Microbiol 152, 291-301.
Köster W, Braun V. 1990 Iron (III) hydroxamate transport into Escherichia coli. Substrate binding to the periplasmic FhuD protein. J Biol Chem 265, 21407-21410.
Linton KJ, Higgins CF. 1998 The Escherichia coli ATP-binding cassette (ABC) proteins. Mol Microbiol 28, 5-13.
Locher KP, Rees B, Koebnik R, Mitschler A, Moulinier L, Rosenbusch JP, Moras D. 1998 Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 95, 771-778.
Mademidis A, Killmann H, Kraas W, Flechsler I, Jung G, Braun V. 1997 ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping. Mol Microbiol 26, 1109-1123.
Mao B, Pear MR, McCammon JA, Quiocho FA. 1982 Hinge-bending in L-arabinose-binding protein. The 'Venus-flytrap' model. J Biol Chem 257, 1131-1133.
Payne SM. 1988 Iron and virulence in the family Enterobacteriaceae. Crit Rev Microbiol 16, 81-111.
Payne SM. 1993 Iron acquisition in microbial pathogenesis. Trends Microbiol 1, 66-69.
Quiocho FA. 1990 Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria. Philos Trans R Soc Lond B Biol Sci 326, 341-351.
Quiocho FA, Ledvina PS. 1996. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol Microbiol 20, 17-25.
Ratledge C, Dover LG. 2000 Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54, 881-941.
Rohrbach MR. Braun V, Köster W. 1995a Ferrichrome transport in Escherichia coli K-12: Altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB. J Bacteriol 177, 7186-7193.
Rohrbach MR, Paul S, Köster W. 1995b In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence. Mol Gen Genet 248, 33-42.
Saurin W, Hofnung M, Dassa E. 1999. Getting in or out:early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters. J Mol Evol 48, 22-41.
Schryvers AB, Stojiljkovic I. 1999 Iron acquisition systems in the pathogenic Neisseria. Mol Microbiol 32, 1117-1123.
Tam R, Saier MH. 1993 Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol Rev 57, 320-346
Vasil ML, Ochsner UA. 1999 The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 34, 399-413.
Wandersman C, Stojiljkovic I. 2000 Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores. Curr Opin Microbiol 3, 215-220.
Winkelmann G. (ed.) 2001 Microbial Transport Systems. Weinheim: Wiley-VCH.
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Clarke, T.E., Rohrbach, M.R., Tari, L.W. et al. Ferric hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved and non-conserved regions. Biometals 15, 121–131 (2002). https://doi.org/10.1023/A:1015249530156
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DOI: https://doi.org/10.1023/A:1015249530156