An internal affinity‐tag for purification and crystallization of the siderophore receptor fhua, integral outer membrane protein from escherichia coli K‐12

Abstract

FhuA ( M , 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quanties sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 40.5, which resides in a known surface-exposed loop. Recombinant Fh~A40.5.H~ was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 8, resolution.The crystals belong to space group P61 or P65 with unit cell dimensions of u = b = 174 A, c = 88 8, (a = p = 90°, y = 120").Keywords: crystallization; FhuA; membrane protein; outer membrane; protein crystals; siderophore; TonB-dependent receptor; X-ray crystallography Biological membranes serve to partition the cell interior from the external milieu, and they play a critical role in maintaining cell integrity and cell function. The cell envelope of Gram-negative bacteria consists of three distinct layers: the surface-located outer membrane (OM), the periplasm in which the peptidoglycan layer is found, and the cytoplasmic membrane (CM). Bacteria have a variety of transport systems available for the import of essential nutrients through these cell envelope layers (reviewed by Nikaido & Saier, 1992). The OM acts as a permeability barrier for the cell. It also functions as a molecular sieve, excluding deleterious subReprint requests to: James Coulton, Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada H3Y 2G1; e-mail: jwcoulton@microimm.mcgill.ca.Abbreviarionst PEG, polyethyleneglycol; CloDAO, dimethyldecylamine-N-oxide; CI2DAO, dimethyldodecylamine-N-oxide; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. stances and permitting passive diffusion of small solutes through porins into the periplasm (Nikaido, 1996). Some compounds essential for bacterial growth are found at exceedingly low concentrations; iron is one such nutrient. To scavenge ferric iron from their environment, bacteria synthesize and secrete iron-chelating substances called siderophores of molecular mass 700-1000. Nonspecific porin channels from Escherichia coli have an apparent molecular mass exclusion limit of 600 and, therefore, do not function in the import of these complexes. Hence, bacteria have evolved high-affinity iron transport mechanisms, distinct from porin-mediated diffusion, and these mechanisms require proteins in the OM, the periplasm and the CM. OM-localized receptors bind their cognate ferric siderophore with high affinity and in E. coli, the OM receptor for ferric hydroxamate uptake is FhuA, 714 amino acids, M , 78,992 (Coulton et al., 1986). In addition to binding femchrome-iron, FhuA also acts...

Enhanced Binding of TonB to a Ligand-loaded Outer Membrane Receptor

“…Strains-Escherichia coli AW740 harbors plasmid pHX405 and expresses recombinant FhuA with a hexahistidine tag at position 405 (22). E. coli ER2566, transformed with pET28 plasmid containing H6.ЈTonB, was similar to the construct described by Moeck and Letellier (15) and was corrected to reflect the wild-type sequences of residues 32-239 of TonB.…”

Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli

“…Strain AB2847⌬ara was created by P1 transduction of leu::Tn10 and ⌬ara714 from LMG194 (Invitrogen) into AB2847 (35 (37). The protein was purified as described in the literature (38) with the following changes: for binding experiments the purification was stopped before the detergent exchange from LDAO to DDAO. Fractions containing FhuA were concentrated to 10 mg/ml and dialyzed overnight against 50 mM ammonium acetate pH 8.0 with 0.05% LDAO (N,N-dimethydodecylamine-N-oxide/FLUKA).…”

Molecular cloning and characterization of the ferric hydroxamate uptake (fhu) operon in Actinobacillus pleuropneumoniae The GenBank accession number for the sequence of the fhuCDBA operon of Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 described in this study is AF351135.

“…There were two gaps in the model : the first occurred between residues 74 and 79 in the cork domain and the second occurred between residues 375 and 391 in the β-barrel domain. The second gap overlapped the hexahistidine tag and flanking linker regions (a total of 11 aa) that were inserted into the sequence to facilitate affinity purification of the E. coli recombinant FhuA (Ferguson et al, 1998b). It is reasonable to assume that the A. pleuropneumoniae model structure should not be threaded against this region, since it is not present in the wild-type A. pleuropneumoniae fhuA gene.…”