Evidence for dynamic clustering of carboxy-terminal aromatic amino acids in TonB-dependent energy transduction

Escherichia coli uses the proton motive force of the cytoplasmic membrane and TonB protein to energize the active transport of iron-siderophores and vitamin B12 across the outer membrane. TonB shuttles between the cytoplasmic and outer membranes, presumably during the course of energy transduction. Previous results indicated that the carboxy-terminal 65 amino acids of TonB are essential for both its outer membrane association and activity. A highly conserved region (residues 199-216) within this domain, predicted to be an amphipathic alpha-helix, was the initial focus of this study. Scanning mutagenesis indicated that only the aromatic residues F202, W213 and Y215 were individually important for activity. When the crystal structure of a dimeric TonB carboxy-terminus subsequently became available, we observed that two additional aromatic residues outside that region, F180 and F230, were potentially engaged in end-on hydrophobic interactions with the three residues identified previously. Changing these five aromatic residues individually to alanine reduced TonB activity. Surprisingly, however, each substitution exhibited a unique phenotypic profile with respect to ability to support 55Fe]-ferrichrome transport, sensitivity to colicins B, D, Ia and M or sensitivity to bacteriophage phi80. The phenotypic results suggested that the carboxy-terminus of TonB was a flexible and dynamic domain that could interact specifically with different ligands or transporters, perhaps through the aromatic residues. The possibility of interactions among all the aromatic residues was tested using double-mutant cycle analysis. All possible combinations of alanine substitutions were constructed, with the result that TonB containing any double-alanine substitution was inactive in the phenotypic assays, while retaining the ability to associate with the outer membrane. This synergistic, rather than additive, effect of the double mutants suggested that, consistent with the flexibility suggested by analysis of the single substitutions, all the aromatic residues might be capable of interacting with one another. A means of reconciling these results with the crystal structure is presented.