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

Guangshun Wang, Alan Peterkofsky, G. Marius Clore

Research output: Contribution to journalArticle

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Abstract

Enzyme IIAGlucose (IIAGlc) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIAGlc and the HPr-IIAGlc complex have shown that IIAGlc 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 IIAGlc to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIAGlc 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 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, 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 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 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.

Original languageEnglish (US)
Pages (from-to)39811-39814
Number of pages4
JournalJournal of Biological Chemistry
Volume275
Issue number51
DOIs
StatePublished - Dec 22 2000

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Escherichia coli Proteins
Protein Sorting Signals
Anchors
Phosphotransferases
Membranes
Lipids
Escherichia coli
Phosphatidylglycerols
Peptides
Coulomb interactions
Static Electricity
Phosphatidylcholines
Histidine
Phosphorus
Conformations
Catalytic Domain
Membrane Proteins
Stabilization
Glucose
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A novel membrane anchor function for the N-terminal amphipathic sequence of the signal-transducing protein IIAGlucose of the Escherichia coli phosphotransferase system. / Wang, Guangshun; Peterkofsky, Alan; Clore, G. Marius.

In: Journal of Biological Chemistry, Vol. 275, No. 51, 22.12.2000, p. 39811-39814.

Research output: Contribution to journalArticle

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abstract = "Enzyme IIAGlucose (IIAGlc) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIAGlc and the HPr-IIAGlc complex have shown that IIAGlc 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 IIAGlc to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIAGlc 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 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, 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 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 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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AB - Enzyme IIAGlucose (IIAGlc) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIAGlc and the HPr-IIAGlc complex have shown that IIAGlc 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 IIAGlc to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIAGlc 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 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, 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 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 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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