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Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles

¹ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
² Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

Reprint requests to: Guangshun Wang, Eppley Cancer Institute, Room ECI3018, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA; e-mail: gwang{at}unmc.edu; fax: (402) 559-4651.

The N-terminal domain of enzyme IIA^Glc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system confers amphitropism to the protein, allowing IIA^Glc to shuttle between the cytoplasm and the membrane. To further understand this amphitropic protein, we have elucidated, by NMR spectroscopy, the solution structure of a synthetic peptide corresponding to the N-terminal domain of IIA^Glc. In water, this peptide is predominantly disordered, consistent with previous data obtained in the absence of membranes. In detergent micelles of dihexanoylphosphatidylglycerol (DHPG) or sodium dodecylsulfate (SDS), however, residues Phe 3–Val 10 of the peptide adopt a helical conformation in the ensemble of structures calculated on the basis of NOE-derived distance restraints. The root mean square deviations for superimposing the backbone atoms of the helical region are 0.18 Å in DHPG and 0.22 Å in SDS. The structure, chemical shifts, and spin–spin coupling constants all indicate that, of the four lysines in the N-terminal domain of IIA^Glc, only Lys 5 and Lys 7 in the amphipathic helical region interact with DHPG. In addition, the peptide-detergent interactions were investigated using intermolecular NOESY experiments. The aliphatic chains of anionic detergents DHPG, SDS, and 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) all showed intermolecular NOE cross-peaks to the peptide, providing direct evidence for the putative membrane anchor of IIA^Glc in binding to the membrane-mimicking micelles.

Keywords: Amphitropism; dihexanoylphosphatidylglycerol; IIA^Glc; lipid binding; NMR; membrane proteins; signal transduction

Abbreviations: IIA^Glc, glucose-specific enzyme IIA (older abbreviation III^Glc) • PTS, phosphoenolpyruvate:sugar phosphotransferase system • SDS, sodium dodecylsulfate • DHPC, dihexanoylphosphatidylcholine • DHPG, dihexanoylphosphatidylglycerol • DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser enhancement spectroscopy • TOCSY, total correlation spectroscopy • DQF-COSY, double-quantum filtered correlation spectroscopy • HPLC, high-pressure liquid chromatography • CD, circular dichroism • rms, root mean square

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