The periplasmic FbpA (ferric-binding protein A) from Haemophilus influenzae plays a critical role in acquiring iron from host transferrin, shuttling iron from the outer-membrane receptor complex to the inner-membrane transport complex responsible for transporting iron into the cytoplasm. In the present study, we report on the properties of a series of site-directed mutants of two adjacent tyrosine residues involved in iron co-ordination, and demonstrate that, in contrast with mutation of equivalent residues in the N-lobe of human transferrin, the mutant FbpAs retain significant iron-binding affinity regardless of the nature of the replacement amino acid. The Y195A and Y196A FbpAs are not only capable of binding iron, but are proficient in mediating periplasm-to-cytoplasm iron transport in a reconstituted FbpABC pathway in a specialized Escherichia coli reporter strain. This indicates that their inability to mediate iron acquisition from transferrin is due to their inability to compete for iron with receptor-bound transferrin. Wild-type iron-loaded FbpA could be crystalized in a closed or open state depending upon the crystallization conditions. The synergistic phosphate anion was not present in the iron-loaded open form, suggesting that initial anchoring of iron was mediated by the adjacent tyrosine residues and that alternate pathways for iron and anion binding and release may be considered. Collectively, these results demonstrate that the presence of a twin-tyrosine motif common to many periplasmic iron-binding proteins is critical for initially capturing the ferric ion released by the outer-membrane receptor complex.
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Research Article|
October 25 2010
The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A
Husain K. Khambati;
Husain K. Khambati
*Department of Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary, Canada, T2N 4N1
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Trevor F. Moraes;
Trevor F. Moraes
†Department of Biochemistry, University of Toronto, 1 Kings College Circle, Toronto, Canada, M5S 1A8
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Jagroop Singh;
Jagroop Singh
*Department of Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary, Canada, T2N 4N1
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Stephen R. Shouldice;
Stephen R. Shouldice
‡Institute for Molecular Bioscience, The University of Queensland, 306 Camody Rd, Brisbane, QLD 4072, Australia
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Rong-hua Yu;
Rong-hua Yu
*Department of Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary, Canada, T2N 4N1
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Anthony B. Schryvers
Anthony B. Schryvers
1
*Department of Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary, Canada, T2N 4N1
1To whom corrspondence should be addressed (email schryver@ucalgary.ca)
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Publisher: Portland Press Ltd
Received:
July 14 2010
Revision Received:
August 19 2010
Accepted:
August 27 2010
Accepted Manuscript online:
August 27 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 432 (1): 57–67.
Article history
Received:
July 14 2010
Revision Received:
August 19 2010
Accepted:
August 27 2010
Accepted Manuscript online:
August 27 2010
Citation
Husain K. Khambati, Trevor F. Moraes, Jagroop Singh, Stephen R. Shouldice, Rong-hua Yu, Anthony B. Schryvers; The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A. Biochem J 15 November 2010; 432 (1): 57–67. doi: https://doi.org/10.1042/BJ20101043
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