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

Abstract

FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli. The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB. Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535-27540). We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA. Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA. We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric. TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage ⌽80.

Crystal Structure of a 92-Residue C-terminal Fragment of TonB from Escherichia coli Reveals Significant Conformational Changes Compared to Structures of Smaller TonB Fragments

“…5 of Ref. 21). In contrast, in the TonB-92 structure the ␤1-␤2 hairpin does not exchange with another molecule but rather takes up the same place that is filled in the tight dimer with the ␤1Ј-␤2Ј hairpin from the other molecule.…”

“…Protein Expression and Purification-TonB-92, a C-terminal fragment containing the last 92 amino acid residues of the TonB protein of E. coli, was overexpressed in BL21(DE3) cells containing the plasmid pTB92 and was subsequently purified to near homogeneity (21). Purification of the SeMet 1 -TonB-92 was performed according to the protocol for the native TonB-92 with the following exceptions: cells were grown in M63 minimal medium (27) to an A 600 of 0.6, and a selected set of amino acids was then added to a medium containing lysine, phenylalanine, and threonine at a concentration of 100 mg/liter; leucine, isoleucine, and valine at 50 mg/liter; and selenomethionine at 60 mg/liter.…”

“…At a rotor speed of 34,000 rpm, equilibrium was achieved after 20 h. Data were analyzed using the software SEGAL 2.1. 3 In vitro binding experiments of TonB-92 to ferrichrome-loaded FhuA were done as previously described for other C-terminal TonB-fragments (21).…”

“…Synthetic nonapeptides corresponding to the amino acid sequence of TonB between residues 155 and 166 were found to be able to inhibit FhuA-dependent transport of ferrichrome in vivo (20). Complex formation between the Cterminal domain of TonB and the outer membrane receptors FhuA or FepA, respectively, has also been demonstrated by co-purification (17,21), and disulfide cross-linking was demonstrated between cysteine substitutions in the Q160 region of TonB and the TonB box of BtuB (9) and FecA (22).…”

“…Other recent results have indicated that the situation may be more complicated, however. In a study of C-terminal TonB fragments of increasing length (from 77 to 126 residues), it was found that fragments of 85 residues and shorter formed homodimers in solution, whereas the longer fragments were monomeric (21). In addition, the shortest fragment with 77 residues (which lacked glutamines 160 and 162) was unable to bind to FhuA, whereas the longer ones (which contained the glutamines) formed a complex with it as determined by gel filtration studies and could inhibit the function of native TonB when produced in vivo.…”