Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and phi 80 and colicin M bind to the gating loop of FhuA

Abstract

Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli. Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and 80 and to colicin M. In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M. Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues. Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M. Two of these regions were common among all four FhuA ligands. Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head. These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and 80 and colicin M.Phages T1, 80, and T5 bind to the FhuA (formerly TonA) outer membrane protein of Escherichia coli. FhuA is also required for the uptake of ferrichrome, the structurally analogous antibiotic albomycin, and colicin M. For transport of the latter compounds through the outer membrane, the proteins TonB, ExbB, and ExbD are required (6). After deletion of residues 322 to 355 of FhuA (FhuA ⌬322-355), uptake of ferrichrome and albomycin no longer required TonB and ExbBD (12). The uptake kinetics were typical of a diffusion process, in contrast to the TonB-and ExbBD-dependent uptake, which showed saturation kinetics and required energy provided by the electrochemical potential of the cytoplasmic membrane (2, 7). Cells expressing FhuA ⌬322-355 became sensitive to sodium dodecyl sulfate and bacitracin, in contrast to the resistance of cells expressing wild-type FhuA, indicating the formation of open channels by FhuA ⌬322-355. In black lipid membranes FhuA ⌬322-355 formed stable channels three times as large as the porin OmpF, while wild-type FhuA did not increase the conductance of KCl through artificial membranes (12). In a model predicting the transmembrane arrangement of the FhuA protein, residues 316 to 356 formed the largest loop at the cell surface (14). This segment of FhuA carrying an inserted C3 epitope of poliovirus reacted with anti-C3 antibodies, indicating exposure of the epitope at the E. coli cell surface (17). Since removal of this loop converted the FhuA transport protein into a diffusion channel, we proposed that the basic structure of FhuA is a channel which is closed by the loop formed by residues 316 to 356 (gating loop). Through interaction with the TonB-ExbBD complex, FhuA undergoes a conformational change in which the gating loop moves and opens the channel (3, 12).Cells expressing FhuA ⌬322-355 were also resistant to phages T1, 80, and T5 and were not killed by colicin M. Deletion of Asp-348 in the gating loop reduced T5 sensitiv...

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies

“…This result refutes the idea that a MAb bound to its surface-located determinant would sterically inhibit the binding of all ligands. Because the determinant recognized by Fhu4.1 is proposed to be linear, it follows that there exists some linear sequence within amino acids 316 to 356 which acts as the determinant for Fhu4.1, despite the requirement for a phage binding region with secondary structure (24). It is possible that the binding of Fhu4.1 to its determinant prevents the inactivation of T5 phage by inducing or preventing a conformational change in FhuA.…”

“…It was proposed that the transmembrane strands of FhuA assume the conformation of a large beta barrel, with the predicted loop of amino acids 316 to 356 forming a ''gate'' that somehow regulates ferrichrome transport through the channel (4,22). Recently, Killmann et al (24) identified amino acids within the proposed gating loop of FhuA which contribute to the binding of phages T1, T5, and 80 and of colicin M. Preincubation of phages or colicin M with selected synthetic hexapeptides representing sequences between amino acids 316 and 356 of FhuA reduced the efficiency of plating of the lethal agents by up to 7 orders of magnitude. It was concluded that three regions of the gating loop were involved in the binding of T1, T5, 80, and colicin M and that the regions probably formed a single binding region for the ligands.…”

Colicin Biology

“…T1 and 80 phages interact with the FhuA receptor, and mutational studies have delineated the fixation site on a large external gating loop (residues 322 to 355) (70,341,342). Contrary to Tol-dependent bacteriophages, the viral protein responsible for host (or TonBdependent receptor) recognition has yet to be identified.…”

Using experimental evolution and next-generation sequencing to teach bench and bioinformatic skills

“…It has a large 121,752 bp 80 genome with composition that is typical of members of this genus (Wang et al 2005). T5 infects E. coli by binding to the bacterial ferrichrome transporter, encoded by the FhuA gene, and specifically to its gating loop (Killmann et al 1995). In the course of several terms using …”