In Escherichia coli, FhuA, together with the energy-transducing TonB-ExbB-ExbD complex, mediates the uptake of hydroxamate-type siderophores across the outer membrane. In addition to transporting ferrichrome, FhuA functions as the primary receptor for several bacteriophages, antibiotics, and colicin M. To comprehend how FhuA binds its cognate ligands, and to establish the molecular basis of active transport, the three-dimensional structure must be determined. In pursuit of this goal, an affinity tag was inserted into the fhuA gene at a known surface-exposed location. The recombinant FhuA protein was overexpressed and purified by metal-chelate chromatography. Sparse matrix screening established parameters for the growth of well-ordered crystals of FhuA. The crystallographic structure of FhuA and its complex with ferricrocin were solved at 2.50 and 2.70 A by multiwavelength anomalous dispersion. The crystallographic structure reveals that FhuA is a monomeric integral outer membrane protein that is composed of two domains. The C-terminal domain, a 22-stranded antiparallel beta-barrel, spans the outer membrane. Positioned within the beta-barrel is the N-terminal 'cork' domain, which is formed by a mixed four-stranded beta-sheet with four interspersed alpha-helices. Upon binding of ferricrocin, conformational changes are propagated from the extracellular pocket to the periplasmic pocket of FhuA; a transmembrane signal indicating the liganded status of the receptor. Structural analysis combined with sequence homologies and mutagenesis data are used to propose a mechanism for TonB-dependent siderophore-mediated iron acquisition. / Noncovalently associated with the membrane-embedded surface of FhuA is a single lipopolysaccharide molecule. Following the examination of electrostatic protein-lipid contacts, a structure-based algorithm revealed a conserved four-residue LPS-binding motif that is present among known LPS-binding proteins. / An expansion of these structural studies was the solution of the crystallographic structures of additional liganded complexes. The three-dimensional structures of FhuA in complex with the antibiotics albomycin and rifamycin CGP 4832 were determined at 3.10 and 2.90 A, respectively. In addition to probing the plasticity and accessibility of the ligand-binding site, these results establish a structural platform for the design of novel antibiotics. Moreover, insights into the physical association of FhuA with TonB can be derived from these data.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37739 |
| Date | January 2000 |
| Creators | Ferguson, Andrew D. |
| Contributors | Ward, Brian J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001802135, proquestno: NQ70183, Theses scanned by UMI/ProQuest. |
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