The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.

TitleThe NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.
Publication TypeJournal Article
Year of Publication1993
Authorsvan Nuland, N. A. J., G. J. Kroon, K. Dijkstra, G. K. Wolters, R. M. Scheek, and G. T. Robillard
JournalFEBS Lett
Volume315
Issue1
Pagination11-5
Date Published1993 Jan 2
ISSN0014-5793
KeywordsBacterial Proteins, Binding Sites, Escherichia coli, Macromolecular Substances, Magnetic Resonance Spectroscopy, Phosphoenolpyruvate Sugar Phosphotransferase System, Phosphorylation, Protein Structure, Tertiary
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.

Alternate JournalFEBS Lett.
PubMed ID8416803