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 phosp hotransferase system of Escherichia coli. Structural studies of free IIA(Gl c) and the HPr-IIA(Glc) complex have shown that IIA(Glc) comprises a globul ar P-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 requi red for IIA(Glc) to accept a phosphorus from the histidine phosphocarrier p rotein HPr, its presence is essential for effective phosphotransfer from II A(Glc) to the membrane-bound IIBCGlc. The sequence of the N-terminal tail s uggests that it has the potential to form an amphipathic helix. Using CD, w e demonstrate that a peptide, corresponding to the N-terminal 18 residues o f IIA(Glc), adopts a helical conformation in the presence of either the ani onic lipid phosphatidylglycerol or a mixture of anionic E. coli lipids phos phatidylglycerol (25%) and phosphatidylethanolamine (75%), The peptide, how ever, is in a random coil state in the presence of the zwitterionic lipid p hosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide. In addition, we show that intac t 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 fol ded domain containing the active site and capable of weakly interacting wit h the peripheral IIB domain of the membrane protein IIBCGlc; and the N-term inal tail, which interacts with the negatively charged E, coli membrane, th ereby stabilizing the complex of IIA(Glc) with IIBCGlc. This stabilization is essential for the final step of the phosphoryl transfer cascade in the g lucose transport pathway.