Siderophore-mediated iron transport: Crystal structure of FhuA with bound lipopolysaccharide

Science

Volumn 282, Issue 5397, 1998, Pages 2215-2220

  Ferguson, Andrew D ^a,b   Hofmann, Eckhard ^b   Coulton, James W ^a   Diederichs, Kay ^b   Welte, Wolfram ^b  

^a MCGILL UNIVERSITY   (Canada)

^b UNIVERSITY OF KONSTANZ   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

IRON; LIPOPOLYSACCHARIDE; OUTER MEMBRANE PROTEIN; SIDEROPHORE;

ARTICLE; CELL MEMBRANE TRANSPORT; CONFORMATIONAL TRANSITION; ESCHERICHIA COLI; IRON TRANSPORT; MEMBRANE STRUCTURE; NONHUMAN; PRIORITY JOURNAL; PROTEIN BINDING; SIGNAL TRANSDUCTION; THREE DIMENSIONAL IMAGING;

BACTERIAL OUTER MEMBRANE PROTEINS; BACTERIAL PROTEINS; BINDING SITES; BIOLOGICAL TRANSPORT, ACTIVE; CELL MEMBRANE; CRYSTALLOGRAPHY, X-RAY; DIFFUSION; ESCHERICHIA COLI; ESCHERICHIA COLI PROTEINS; FERRIC COMPOUNDS; FERRICHROME; HYDROGEN BONDING; LIGANDS; LIPOPOLYSACCHARIDES; MEMBRANE PROTEINS; MODELS, MOLECULAR; PROTEIN CONFORMATION; PROTEIN STRUCTURE, SECONDARY; RECEPTORS, VIRUS; SIGNAL TRANSDUCTION;

ESCHERICHIA COLI; NEGIBACTERIA;

EID: 0032545324     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5397.2215     Document Type: Article

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13. Crystallization of FhuA is dependent on the presence of stoichiometric amounts of LPS. If LPS is completely removed from FhuA protein preparations or if an excess of LPS is present in such preparations, the growth of FhuA crystals is inhibited. We propose that LPS remained bound to FhuA throughout the process of purification and crystallization and that it did not adsorb to FhuA during isolation. Because it is known that LPS is localized to the outer leaflet of the outer membrane, the location of bound LPS marks its position relative to the upper aromatic girdle of FhuA and to the outer membrane.
14. 702 from β21.
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16. 244 cornes in close contact with the iron atom of the ferrichrome-iron molecule. This observation may explain the decreased affinity for apoferrichrome.
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18. 708 from β22.
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23. 128 results in a complete loss of all FhuA function [G. Carmel et al., J. Bacteriol. 172, 1861 (1990)].
24. The tryptophan emission spectra were measured for FhuA and the FhuA-ferrichrome-iron complex [K. Locher and J. Rosenbusch, Eur. J. Biochem. 247, 779 (1997)]. The tryptophan fluorescence was shown to decrease after the addition of the ferrichrome-iron, which suggests a change in accessibility.
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37. A more complicated mechanism could avoid the possible loss of the ferrichrome-iron into the external medium after the formation of the FhuA-TonB complex by the steric blockage of the surface-located pocket. However, the TonB-dependent binding and uptake of the FhuA-spedfic toxin colicin M through FhuA would require that the putative channel-forming region remain open from the external environment to the periplasm for an extended period of time and therefore would contradict such a mechanism [C. J. Lazdunski et al., J. Bacteriol. 180, 4993 (1998); R. M. Stroud et al., Curr. Opin. Struct. Biol. 8, 525 (1998)].
38. A more complicated mechanism could avoid the possible loss of the ferrichrome-iron into the external medium after the formation of the FhuA-TonB complex by the steric blockage of the surface-located pocket. However, the TonB-dependent binding and uptake of the FhuA-spedfic toxin colicin M through FhuA would require that the putative channel-forming region remain open from the external environment to the periplasm for an extended period of time and therefore would contradict such a mechanism [C. J. Lazdunski et al., J. Bacteriol. 180, 4993 (1998); R. M. Stroud et al., Curr. Opin. Struct. Biol. 8, 525 (1998)].
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41. Single-letter abbreviations for the amino acid residues 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.
42. - auxotrophic E. coli strain DL41 and grown as recommended [S. Doublié, Methods Enzymol. 276, 523 (1997)].
43. - auxotrophic E. coli strain DL41 and grown as recommended [S. Doublié, Methods Enzymol. 276, 523 (1997)].
44. - auxotrophic E. coli strain DL41 and grown as recommended [S. Doublié, Methods Enzymol. 276, 523 (1997)].
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46. 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.
47. 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.
48. 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.
49. 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.
50. 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.
51. 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.
52. 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.
53. 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.
54. 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.
55. All figures were prepared with the programs MOL-SCRIPT [P. Kraulis, J. Appl. Crystatlogr. 24, 946 (1991)] and Raster-30 [E. A. Merrit and D. J. Bacon, Methods Enzymol. 277, 505 (1997)], except for Fig. 3, which was prepared with the program O.
56. All figures were prepared with the programs MOL-SCRIPT [P. Kraulis, J. Appl. Crystatlogr. 24, 946 (1991)] and Raster-30 [E. A. Merrit and D. J. Bacon, Methods Enzymol. 277, 505 (1997)], except for Fig. 3, which was prepared with the program O.
57. We gratefully acknowledge A. Svensson at MAX-lab II and A. Thompson at the European Radiation Synchrotron Facility for their assistance and generous support during data collection; E. A. Meighen for providing E. coli strain DL41; P. A. Karplus for a critical reading of the manuscript; J. Wang for genetic constructs; V. Braun and H. Killmann for bacterial strains and discussions; A. Patel for his assistance with protein purification; J. Breed for crystallization trials and a critical reading of the manuscript; D. M. Allan and J. A. Kashul for editing, K. Hegetschweiler for providing cis-inositol; and B. Herrmann, A. Hirsch, C Peinelt, O. Seth, and J. Telioriclis, who made important contributions to the early phase of this project. This work was supported by the Deutsche Forschungsgemeinschaft (W.W.); by the Medical Research Council, Canada (grant MT-14133 to J.W.C.); and by NATO International Collaborative Research grant 960082. A.D.F. is the recipient of a Deutscher Akademischer Austauschdienst Grant for Study and Research.