X-Ray Structure of a Bifunctional Protein Kinase in Complex with Its Protein Substrate HPr

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 99; no. 21; pp. 13437 - 13441
• Main Authors: Fieulaine, Sonia, Morera, Solange, Poncet, Sandrine, Mijakovic, Ivan, Galinier, Anne, Janin, Joël, Deutscher, Josef, Nessler, Sylvie
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 15.10.2002; National Acad Sciences
• Subjects: Active sites; Bacillus subtilis - chemistry; Bacillus subtilis - genetics; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biochemistry, Molecular Biology; Biological Sciences; Calcium - chemistry; Carbon; Catalytic Domain; Crystallography, X-Ray; Data collection; Electron density; Enzymes; Hydrogen bonds; Lactobacillus casei - enzymology; Lactobacillus casei - genetics; Life Sciences; Macromolecular Substances; Models, Molecular; Molecules; Phosphates; Phosphoenolpyruvate Carboxykinase (ATP) - chemistry; Phosphoenolpyruvate Carboxykinase (ATP) - metabolism; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System - genetics; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Phosphorylation; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Static Electricity; Substrate Specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.192368699

HPr kinase/phosphorylase (HprK/P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK/P with Bacillus subtilis HPr, both at 2.8-Å resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK/P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions.