High-resolution Structure of the Phosphorylated Form of the Histidine-containing Phosphocarrier Protein HPr from Escherichia coliDetermined by Restrained Molecular Dynamics from NMR-NOE Data

Abstract

The solution structure of the phosphorylated form of the histidine-containing phosphocarrier protein, HPr, from Escherichia colihas ben determined by NMR in combination with retrained molecular dynamics simulations. The structure of phospho-HPr ( P-HPr) results from a molecular dynamics simulation in water, using time-dependent distance restraints to attain agreement with the measured NOEs. Experimental restraints were identified from both three-dimensional 1H- 1H- 15N HSQC-NOESY and two-dimensional 1H- 1HNOSEY spectra, and compared with those of the unphosphorylated form. Structural changes upon phosphorylation of HPr are limited to the active sit, as evidenced by chages in chemical shifts, in 3 J NHHα-coupling constants and NOE patterns. Chemical shift changs were obtained mainly for protons that were positioned close to the phosphoryl group attachd to the His15 imidazole ring. Differences could be detected in the intensity of the NOEs involving the side-chain protons of His15 and Pro18, resulting from a change in th relative position of the two rings. In addition, a small chang could be detected in the three-bond J-coupling between the amide proton and the H αproton of Thr16 and Arg17 upon phosphorylation, in agreement with the changes of the φ torsion angle of these two residues obtained from time-averaged restrained molcular dynamics simulations in water. The proposed rol of the torsion-angle strain at rsidue 16 in th mechanism of Streptococcus faecalisHPr is not supported by these results. In contrast, phosphorylation seems to introduce torsion angle strain at residue His15. This strain could facilitate the trasfer of the phosphoryl group to the A-domain at enzyme II. The phospho-histidine is not stabilised by hydrogen bonds to the side-chain group of Arg17; instad stable hydrogen bonds are formed between the phosphate group and the backbone amide protons of Thr16 and Arg17, which show the largest changes in chemical shift upon phosphorylation, and a hydrogen bond involving the side-chain O γproton of Thr16. HPr accepts the phosphoryl group from enzyme I and donats it subsequently to the A domain of various enzyme II species. The binding site for EI on HPr resembles that of the A domain of the mannitol-specific enzyme II, as can be concluded from the changs on the amideproton and nitrogen chemical shifts observed viaheteromolecular single-quantum coherence spectroscopy.