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Papers In Press, published online ahead of print October 23, 2000
J. Biol. Chem, 10.1074/jbc.C000709200
Submitted on October 9, 2000
Revised on October 23, 2000
Accepted on October 21, 2000
Lab of Chemical Physics, NIDDK/National Institutes of Health, Bethesda, MD 20892-0510
Corresponding Author: clore{at}speck.niddk.nih.gov
Enzyme IIAGlucose (IIAGlc) is a signal transducing protein in the phosphoenolpyruvate:glucose phosphotransferase system (PTS) of E. coli. Previous structural studies of both free IIAGlc (X-ray and NMR) and the HPr-IIAGlc complex (NMR) have shown that IIAGlc comprises a globular b-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 IIAGlc to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIAGlc to the membrane bound IIBCGlc. The amino acid sequence of the N-terminal tail suggests that it has the potential to form an amphiphatic helix. Using circular dichroism, we demonstrate that a synthetic peptide, corresponding to the N-terminal 18 residues of IIAGlc, 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, remains in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine. These results indicate that electrostatic interactions play an essential role in the binding of the lipid to the peptide. In addition, we show that intact IIAGlc 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 IIAGlc comprises two functional domains: a folded domain (residues 19-168) containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBCGlc; and the N-terminal tail which interacts with the negatively charged E. coli membrane, thereby stabilizing the complex of IIAGlc with IIBCGlc. This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway.
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