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Cited by 787Bacterial Genetics and BiotechnologyEnzyme Structure and FunctionDrug Transport and Resistance Mechanisms

Siderophore-Mediated Iron Transport: Crystal Structure of FhuA with Bound Lipopolysaccharide

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Ferguson, Andrew D., Hofmann, Eckhard, Coulton, James W., Diederichs, Kay, Welte, Wolfram·1998·Science
DOI 10.1126/science.282.5397.2215
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Abstract

FhuA, the receptor for ferrichrome-iron in Escherichia coli, is a member of a family of integral outer membrane proteins, which, together with the energy-transducing protein TonB, mediate the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. The three-dimensional structure of FhuA is presented here in two conformations: with and without ferrichrome-iron at resolutions of 2.7 and 2.5 angstroms, respectively. FhuA is a beta barrel composed of 22 antiparallel beta strands. In contrast to the typical trimeric arrangement found in porins, FhuA is monomeric. Located within the beta barrel is a structurally distinct domain, the "cork," which mainly consists of a four-stranded beta sheet and four short alpha helices. A single lipopolysaccharide molecule is noncovalently associated with the membrane-embedded region of the protein. Upon binding of ferrichrome-iron, conformational changes are transduced to the periplasmic pocket of FhuA, signaling the ligand-loaded status of the receptor. Sequence homologies and mutagenesis data are used to propose a structural mechanism for TonB-dependent siderophore-mediated transport across the outer membrane.

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  • Given their overall structural and sequence commonalities, other LGPs likely internalize metal complexes, including Trojan horse antibiotics, by the same or similar mechanisms.

    “Given their overall structural ( Ferguson et al., 1998 ; Locher et al., 1998 ; Buchanan et al., 1999 ; Ferguson et al., 2002 ; Chimento et al., 2003 ; Cobessi et al., 2005a , b ; Buchanan et al., 2007 ; Cobessi et al., 2010 ; Grinter and Lithgow, 2019a , b ; Grinter and Lithgow, 2020 ) and sequence ( Klebba et al., 2021 ) commonalities, other LGPs likely internalize metal complexes, including Troj”

    From Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu (2024) · cites this paper as Ferguson et al., 1998

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  35. Single-letter abbreviations for the amino acid resi- dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 35. Because FhuA is difficult to purify to homogeneity in large amounts, one prerequisite for crystallization and subsequent structural analysis by x-ray crystallography was the development of an overexpression system and efficient protocols for the rapid purification of FhuA [A. D. Ferguson et al., Protein Sci. 7, 1636 (1998)]. A recombinant FhuA was constructed by splicing a hexa- histidine tag and three additional residues (SHHHHH- HGS) (34) at a previously identified surface location (amino acid 405) [
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  39. W. Kabsch, J. Appl. Crystallogr. 21, 916 (1988)]. The 10 selenium sites were located with SOLVE [T. C. Terwilliger and J. Berendzen, Acta Crystallogr. D53, 571 (1997)], and MAD phases were obtained with SOLVE and SHARP [E. de La Fortelle and G. Bricogone, Methods Enzymol. 276, 472 (1997)]. Initial phases, which were calculat- ed to a resolution of 3.05 Å, were improved by solvent flattening with the program DM [K. Cowtan, Acta Crystallogr. D50, 760 (1994)]. The resulting electron density maps were of sufficient quality to build a model with the program O [T. A. Jones et al., Acta Crystallogr. A47, 110 (1991)] and unambigu- ously place a ferrichrome-iron molecule and a LPS molecule. The model was refined with the programs X-PLOR [A. T. Bru ¨nger, X-PLOR 3.1 (Yale University, New Haven, CT, 1992)] and CNS [
  40. A. T. Bru ¨nger et al., Acta Crystallogr. D54, 905 (1998)] and was used to solve the structure of FhuA to a resolution of 2.5 Å by difference Fourier techniques. Standard protocols for simulated annealing and minimization as implement- ed in CNS were used for refinement. Individual re- strained B-factor refinement was justified as judged by a substantial drop in R free [A. T. Bru ¨nger, Nature 355, 472 (1992)]. The current FhuA model contains residues 19 to 723, 1 LPS, and 99 water molecules. The average B factors for main-chain and side-chain atoms and the LPS molecule are 65, 67, and 78 Å 2 , respectively. The FhuA model was used in the refine- ment of the FhuA-ferrichrome-iron complex to a resolution of 2.7 Å. The current model for the FhuA- ferrichrome-iron complex contains residues 19 to 723, 1 LPS, 1 ferrichrome-iron molecule, and 52 water molecules. The average B factors for main- chain and side-chain atoms, the LPS, and fer- richrome-iron molecules are 63, 66, 75, and 49 Å 2 , respectively. Refinement is not complete for both structures. For the FhuA model, the R cryst is 24.2% (48,359 reflections), and the R free is 28.3% (2309 reflections); for the FhuA-ferrichrome-iron complex, the R cryst is 23.2% (37,362 reflections), and the R free is 28.1% (1532 reflections). All residues lie in allowed regions of the Ramachrandran plot, and all residues that are explicitly mentioned in the text reside in good electron density. Protein Data Bank accession codes are 2fcp and 1fcp for FhuA and the FhuA- ferrichrome-iron complex, respectively.
  41. P. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and Raster-3D [
  42. E. A. Merrit and D. J. Bacon, Methods Enzymol. 277, 505 (1997)], except for Fig. 3, which was prepared with the program O.

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Authors
Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., & Welte, W.
Affiliations
University of Konstanz (DE)
Corresponding author
Welte, Wolfram
ORCID
Venue
Science
Publisher
American Association for the Advancement of Science (AAAS)
Published
1998-12-18
Volume
282
Issue
5397
Pages
2215-2220
ISSN
  • 0036-8075 (ISSN-L)
  • 1095-9203
DOI
10.1126/science.282.5397.2215
OpenAlex
W2084608838
Cited by
787
References
45
Citations by year
2026: 7 · 2025: 9 · 2024: 15 · 2023: 10 · 2022: 10 · 2021: 17 · 2020: 13 · 2019: 14 · 2018: 16 · 2017: 21 · 2016: 19 · 2015: 12 · 2014: 22 · 2013: 24 · 2012: 27
FWCI
22.45
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Science (publishedVersion)
  • PubMed (publishedVersion)
  • KOPS (University of Konstanz) (submittedVersion, cc-by-nc-nd)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
Keywords
FhuA, siderophore transport, TonB, outer membrane receptor, lipopolysaccharide binding, beta-barrel proteins, Escherichia coli, Gram-negative bacteria, iron acquisition, conformational change, TonB-dependent transport, protein crystal structure
MeSH terms
Bacterial Outer Membrane Proteins, Bacterial Proteins, Binding Sites, Biological Transport, Active, Cell Membrane, Diffusion, Escherichia coli, Ferric Compounds, Ferrichrome, Hydrogen Bonding, Ligands, Lipopolysaccharides, Membrane Proteins, Models, Molecular, Protein Conformation, Receptors, Virus, Signal Transduction, Protein Structure, Secondary, Crystallography, X-Ray, Escherichia coli Proteins

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