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The NMR determination of the IIA mtl binding site on HPr of the Escherichia coli phosphoenol pyruvate‐dependent phosphotransferase system
Author(s) -
van Nuland Nico A.J.,
Kroon Gerard J.A.,
Dijkstra Klaas,
Wolters Gea K.,
Scheek Ruud M.,
Robillard George T.
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81122-g
Subject(s) - heteronuclear single quantum coherence spectroscopy , chemistry , heteronuclear molecule , histidine , pep group translocation , chemical shift , nuclear magnetic resonance spectroscopy , stereochemistry , active site , enzyme , biochemistry , phosphoenolpyruvate carboxykinase
The region of the surface of the histidine‐containing protein (HPr) which interacts with the A domain of the mannitol‐specific Enzyme II (IIA mtl ) has been mapped by titrating the A‐domain into a solution of 15 N‐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 15 N or 1 H chemical shifts or both as a result of the presence of IIA mtl 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 IIA mtl 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/IIA mtl complex resulted in a HSQC spectrum which was indistinguishable from the P‐HPr spectrum in the absence of IIA mtl indicating that, as expected, the complex P‐HPr/P‐IIA mtl does not exist even at the high concentrations necessary for NMR.