A novel membrane anchor function for the N-terminal amphipathic sequence of the signal-transducing protein IIA^Glucose of the Escherichia coli phosphotransferase system

Enzyme IIA^Glucose (IIA^Glc) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIA^Glc and the HPr-IIA^Glc complex have shown that IIA^Glc comprises a globular β-sheet sandwich core (residues 19-168) and a disordered N-terminal tail (residues 1-18). Although the presence of the N-terminal tail is not required for IIA^Glc to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIA^Glc to the membrane-bound IIBC ^Glc. The sequence of the N-terminal tail suggests that it has the potential to form an amphipathic helix. Using CD, we demonstrate that a peptide, corresponding to the N-terminal 18 residues of IIA^Glc, adopts a helical conformation in the presence of either the anionic lipid phosphatidylglycerol or a mixture of anionic E. coli lipids phosphatidylglycerol (25%) and phosphatidylethanolamine (75%). The peptide, however, is in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide. In addition, we show that intact IIA^Glc also interacts with anionic lipids, resulting in an increase in helicity, which can be directly attributed to the N-terminal segment. From these data we propose that IIA^Glc comprises two functional domains: a folded domain containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBC^Glc; and the N-terminal tail, which interacts with the negatively charged E. coli membrane, thereby stabilizing the complex of IIA^Glc with IIBC^Glc. This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway.