The NMR determination of the IIAmtl binding site on HPr of the Escherichia coli phosphoenol pyruvate‐dependent phosphotransferase system

FEBS Letters

van Nuland, Nico A.J.¹  Wolters, Gea K.¹  Dijkstra, Klaas¹  Robillard, George T.¹  Kroon, Gerard J.A.¹  Scheek, Ruud M.¹ 

[1] The BIOSON Research Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

关键词: Nuclear magnetic resonance;    Transport system;    Enzyme II;    P-HPr;    P-IIAmtl;    HSQC-spectroscopy;    NMR;    nuclear magnetic resonance;    HMQC;    heteronuclear multiple quantum correlation;    HSCQ;    heteronuclear single quantum correlation;    HPr;    histidine-containing protein;    EI;    Enzyme I;    EI;    Enzyme II;    Mtl;    mannitol;    IIA;    IIB;    IIC;    the A;    B and C domains of Enzyme II;    DTT;    dithiothreitol;    TPPI;    time-proportional phase incrementation;    rf;    radio frequency;    NOE;    nuclear Overhauser enhancement;   

DOI  :  10.1016/0014-5793(93)81122-G

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 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 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.

【 授权许可】

Unknown