Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12

Molecular Microbiology
G S MoeckJ W Coulton

Abstract

Ferrichrome-iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA. To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of beta-sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrich...Continue Reading

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