PURIFICATION AND CRYSTALLIZATION OF THE SIDEROPHORE RECEPTOR FhuA FROM Escherichia coli K-12

Andrew D. Ferguson^l,2, Jason Breed², Kay Diederichs², Wolfram Welte², and James W. Coulton²

^lDepartment of Microbiology and Immunulogy, McGill University, 3775 University Street, Montreal, QC, Canada H3A 2B4 ²Fakultät für Biologie, Universität Konstanz, Postfach 5560, Konstanz, D-78434 Germany

In order to scavenge ferric iron from their environment, bacteria synthesize and secrete iron chelating substances called ferric siderophores of molecular mass 700-1000 Da. Non-specific and specific porin channels do not function in the import of these complexes due to their apparent molecular mass exclusion limit of 600 Da. Bacteria have evolved high-affinity iron transport mechanisms, distinct from porin-mediated diffusion, and these mechanisms require proteins in the outer membrane, the periplasm and the cytoplasmic membrane. Outer membrane-localized receptors bind their cognate ferric siderophore with high affinity and in Escherichia coli, the outer membrane receptor for ferrichrome hydroxamate uptake is FhuA, 714 amino acids, M_r 78,992 Da. In addition to binding ferrichrome-iron, FhuA also acts as receptor for four bacterial phages, colicin M, and the antibiotics albomycin and microcin 25.

FhuA has been affinity-tagged, purified, and crystallized. A hexahistidine-tag was genetically inserted after amino acid 405 which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed and purified to homogeneity in large amounts using metal-chelate chromatography. FhuA crystals suitable for X-ray diffraction analysis were grown using dimethyldecylamine-N-oxide as the primary detergent by the hanging drop vapour diffusion technique with 14% PEG 2,000 MME and 100 mM sodium cacodylate (pH 6.4) as the reservoir solution. These crystals grow in 7 days to a final size of 500 x 500 x 500 mm and show diffraction up to 3.0 A resolution on a rotating anode X-ray source. The crystals belong to the space group P6₁ or P6₅ with unit cell dimensions of a = b =174 A, c = 88 A (a = b = 90^o, g =120^o).