title: The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.
abstract: The region of the surface of the histidine-containing protein (HPr) which interacts with the A domain of the mannitol-specific Enzyme II (II(Amt1)) has been mapped by titrating the A-domain into a solution of 15N-labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty-five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of II(Amt1) while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of II(Amt1) resulted in chemical shift changes in a sub-set of the above residues; these were located more in the vicinity of the active site phospho-histidine. Phosphorylation of the HPr/II(Amt1) complex resulted in a HSQC spectrum which was indistinguishable from the P-HPr spectrum in the absence of II(Amt1) indicating that, as expected, the complex P-HPr/P-II(Amt1) does not exist even at the high concentrations necessary for NMR.
authors.affiliation: BIOSON Research Institute, University of Groningen, The Netherlands.
authors.forename: N A
authors.initials: NA
authors.lastname: van Nuland
authors.forename: G J
authors.initials: GJ
authors.lastname: Kroon
authors.forename: K
authors.initials: K
authors.lastname: Dijkstra
authors.forename: G K
authors.initials: GK
authors.lastname: Wolters
authors.forename: R M
authors.initials: RM
authors.lastname: Scheek
authors.forename: G T
authors.initials: GT
authors.lastname: Robillard
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D046911:Macromolecular Substances; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr