In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence

Abstract

Transport of iron (III) hydroxamates across the inner membrane of Escherichia coli depends on a binding protein-dependent transport system composed of the FhuB, C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in a fhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.

FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus

“…Nevertheless, that such a muted conformational change can result in a protein form that is more resistant to proteolytic digestion is interesting and suggests that the ligand may protect a protease accessible site or loop structure within the binding pocket. This protection has now been demonstrated for three FhuD family members, E. coli FhuD (35) and S. aureus FhuD2 (14) and FhuD1 (this study).…”

A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B 12 utilization

“…As previously performed with other ABC importers (36,37), BtuF and the radiotracer are added to the spheroplast suspension and samples are removed at intervals. Bulk solution is removed by rapid filtration followed by several washes.…”

X-ray Crystallographic Structures of the Escherichia coli Periplasmic Protein FhuD Bound to Hydroxamate-type Siderophores and the Antibiotic Albomycin

“…His-tag FhuD was purified by metal chelate chromatography (POROS 20MC) charged with nickel, similarly as previously described (21) except that the BioCad high performance liquid chromatography system (PerSeptive Biosystems) was used. After cell lysis in 50 mM HEPES, pH 7.6, 0.5 M NaCl, the protein was bound to the column and washed with 10 volumes of 50 mM HEPES, pH 7.6, 0.5 M NaCl to remove unbound proteins and eluted with a gradient of 0 -0.5 M imidazole.…”

“…The protein was then dialyzed extensively against 10 mM Tris, pH 7.5 at 4°C. The His-tag of the protein was not cleaved off by enterokinase, because previous studies showed that the modified protein was functional (21).…”

“…Crystallization and Data Collection-Albomycin ␦ 2 and coprogen were purified as previously described (10,21). Desferal (desferrioxamine mesylate) was obtained from Ciba-Geigy Canada.…”