X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr

Abstract

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-A 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.

Fig 1.

The HPr–HprK/P complex. The top half of the L. casei HprK/P hexamer is viewed along its threefold axis. The three subunits colored in red, green, and blue bind three B. subtilis HPr molecules, drawn with their molecular surface in gold.

Fig 2.

The two interface regions of the HPr–HprK/P contact. Enzyme subunits are in red and green as described for Fig. 1. HPr (in gold) is phosphorylated on Ser-46. (A) Stereoview of the binding site shared by the red and green subunits. (B) The Ser-46 region of HPr interacts with the active site of the green subunit and with Arg-245 of the red subunit. The 236–258 loop bearing the arginine is ordered in the phosphorylated complex. (C) The His-15 region of HPr interacts with helix α4 of the red subunit.

Fig 3.

Stereoview of the HprK/P active site in the HPr and P-Ser-HPr complexes. (A) The complex with HPr. Dashes indicate possible hydrogen bonds. A Ca²⁺ ion and a water molecule are seen in the P loop formed by residues 157–162 of the enzyme. (B) The complex with Ser-46-phosphorylated HPr. The electron density around the phosphoserine and the phosphate ion is from an Fo-Fc omit map contoured at 2σ. The gray sphere is Ca²⁺, and Arg-245 is from a neighboring subunit.