THE NMR DETERMINATION OF THE IIA(MTL) BINDING-SITE ON HPR OF THE ESCHERICHIA-COLI PHOSPHOENOL PYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM

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 N-15-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 N-15 or H-1 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.