Abstract
The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.
Highlights
In Gram-positive bacteria, HPr kinase/phosphorylase (HPrK/P)6 is the central regulator of carbon catabolite repression, the paradigm of signal transduction (1)
In the Gram-negative Neisseria meningitidis devoid of CcpA-mediated carbon catabolite repression, Ser(P)-46-HPr is involved in the cell adhesion process (5), suggesting that HPrK/P might constitute a potential target for new antimicrobial agents in pathogenic bacteria
Structure of L. casei ⌬HPrK/P-V267F and Comparison with the Wild Type Enzyme—As for the wild type enzyme, ⌬HPrK/P-V267F forms a hexamer composed of two trimers (9)
Summary
The structure of the complex between L. casei ⌬HPrK/P and the protein substrate of the ATP-dependent kinase activity, unphosphorylated HPr, was similar and has been obtained in the absence of a nucleotide (15). These two complexes adopt the phosphorylase conformation and prevent a modeling of ATP in the conserved nucleotide-binding site. Kinetic measurements of ATP-dependent HPr phosphorylation provided sigmoid curves and suggested that ATP binds to Bacillus subtilis HPrK/P with strong positive cooperativity (16) This result was confirmed by fluorescence studies using the unique tryptophan of B. subtilis HPrK/P in either intrinsic fluorescence measurements or fluorescence resonance energy transfer (FRET) experiments with the fluorescent nucleotide analogue MantADP (2Ј-/3Ј-O-(NЈ-methylanthraniloyl) ADP (16). Fluorescence experiments aimed at studying the ATP binding mode of the mutant protein allowed us to gain insights into the allosteric behavior of the enzyme
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