High-Resolution Structure of the Histidine-Containing Phosphocarrier Protein (HPr) fromStaphylococcus aureusand Characterization of Its Interaction with the Bifunctional HPr Kinase/Phosphorylase

Abstract

A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant K d was determined to be 0.10 ؎ 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopyheteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L. casei in crystals.The histidine-containing phosphocarrier protein (HPr) plays a central role in the uptake of carbohydrates by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and in the regulation of carbohydrate metabolism in bacteria (for a review, see reference 54). In the transport system, it is part of a phosphate shuttle, which transfers a phosphate group from phosphoenolpyruvate to the carbohydrate transported through the cell membrane. As a second function, HPr is involved in gene regulation of the PTS carbon catabolite repression system. In that system, it acts as an activator of gene repression. Both of these mechanistically very different processes are controlled by HPr through phosphorylation-dephosphorylation reactions. In the phosphate shuttle, the amino acid that participates in phosphorylation-dephosphorylation reactions in HPr is a histidine residue at position 15. It is phosphorylated by enzyme I (EI) at N ␦1 and transfers this group to N ε2 of a histidine residue of the enzyme...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Vice versa, phosphorylation of HPr by EI at His-15 slowed its phosphorylation at Ser-46 by the ATP-dependent HPr kinase (177). In the HPr:HPr kinase complex, helix ␣4 of HPr kinase interacts with the region around His-15 (228,533). Phosphorylation at His-15 probably diminishes the affinity between the kinase and its protein substrate.…”

Ca2+-dependent Conformational Changes in a C-terminal Cytosolic Domain of Polycystin-2

“…In case of slow exchange conditions in onedimensional spectroscopy Equation 2 also holds but the intensity of the line under consideration in the absence of ligand must be zero. In case of two-dimensional HSQC spectra the situation is more complex, because relaxation effects during the mixing time may also influence the result (24).…”

Examination of the Structure, Stability, and Catalytic Potential in the Engineered Phosphoryl Carrier Domain of Pyruvate Phosphate Dikinase,

“…The histidine-containing phosphocarrier protein (HPr) is an independent protein having a function analogous to that of the central domain of PPDK (35,36). However, its fold with segregated R and regions (37,38) is different from that of PPDK. By checking the surface of HPr protein, it is also found to contain a large nonpolar face with a few hydrophilic residues surrounding its periphery.…”