Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles.
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 A in DHPG and 0.22 A 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.
Wang
Guangshun
G
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA. gwang@unmc.edu
Keifer
Paul A
PA
Peterkofsky
Alan
A
D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008567:Membranes, Artificial; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012967:Sodium Dodecyl Sulfate; D012996:Solutions
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D008823:Micelles; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; C032166:1,2-hexanoylphosphatidylcholine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase