Sylvie Nessler

Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: A relic of early life?

In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes (≈10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the β phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho–phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations (≈6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.

The HPr Kinase from Bacillus subtilis Is a Homo-oligomeric Enzyme Which Exhibits Strong Positive Cooperativity for Nucleotide and Fructose 1,6-Bisphosphate Binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2′(3′)-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.

Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism.

The oligomeric bifunctional HPr kinase/P‐Ser‐HPr phosphatase (HprK/P) regulates many metabolic functions in Gram‐positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C‐terminal region in contact with the ATP‐binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl‐phosphorylated HPr (P‐Ser‐HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P‐Ser‐HPr. The hprK V267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non‐PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non‐PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.

X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain.

HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram‐ positive bacteria. It catalyses the ATP‐dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide‐binding proteins. We report here the X‐ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 Å resolution, solved by the multiwavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP‐binding domain similar to nucleoside/nucleotide kinases, and a putative HPr‐binding domain unrelated to the substrate‐binding domains of other kinases. The Walker motif A forms a typical P‐loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes.

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

Bacterial tyrosine kinases: evolution, biological function and structural insights

Reversible protein phosphorylation is a major mechanism in the regulation of fundamental signalling events in all living organisms. Bacteria have been shown to possess a versatile repertoire of protein kinases, including histidine and aspartic acid kinases, serine/threonine kinases, and more recently tyrosine and arginine kinases. Tyrosine phosphorylation is today recognized as a key regulatory device of bacterial physiology, linked to exopolysaccharide production, virulence, stress response and DNA metabolism. However, bacteria have evolved tyrosine kinases that share no resemblance with their eukaryotic counterparts and are unique in exploiting the ATP/GTP-binding Walker motif to catalyse autophosphorylation and substrate phosphorylation on tyrosine. These enzymes, named BY-kinases (for Bacterial tYrosine kinases), have been identified in a majority of sequenced bacterial genomes, and to date no orthologues have been found in Eukarya. The aim of this review was to present the most recent knowledge about BY-kinases by focusing primarily on their evolutionary origin, structural and functional aspects, and emerging regulatory potential based on recent bacterial phosphoproteomic studies.

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

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.

A cell–cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group

In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signalling peptides and regulators of the RNPP family. We investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. We show that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. The transcription start site of the nprA gene has been identified and the minimal region necessary for full activation has been characterized by promoter mutagenesis. We demonstrate that the NprX peptide is secreted, processed and then reimported within the bacterial cell. Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. Alignment of available NprR sequences from different species of the B. cereus group defines seven NprR clusters associated with seven NprX heptapeptide classes. This cell–cell communication system was found to be strain‐specific with a possible cross‐talk between some pherotypes. The phylogenic relationship between NprR and NprX suggests a coevolution of the regulatory protein and its signalling peptide.

Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR

The quorum-sensing regulator PlcR is the master regulator of most known virulence factors in Bacillus cereus. It is a helix-turn-helix (HTH)-type transcription factor activated upon binding of its cognate signaling peptide PapR on a tetratricopeptide repeat-type regulatory domain. The structural and functional properties of PlcR have defined a new family of sensor regulators, called the RNPP family (for Rap, NprR, PrgX, and PlcR), in Gram-positive bacteria. To fully understand the activation mechanism of PlcR, we took a closer look at the conformation changes induced upon binding of PapR and of its target DNA, known as PlcR-box. For that purpose we have determined the structures of the apoform of PlcR (Apo PlcR) and of the ternary complex of PlcR with PapR and the PlcR-box from the plcA promoter. Comparison of the apoform of PlcR with the previously published structure of the PlcR–PapR binary complex shows how a small conformational change induced in the C-terminal region of the tetratricopeptide repeat (TPR) domain upon peptide binding propagates via the linker helix to the N-terminal HTH DNA-binding domain. Further comparison with the PlcR–PapR–DNA ternary complex shows how the activation of the PlcR dimer allows the linker helix to undergo a drastic conformational change and subsequent proper positioning of the HTH domains in the major groove of the two half sites of the pseudopalindromic PlcR-box. Together with random mutagenesis experiments and interaction measurements using peptides from distinct pherogroups, this structural analysis allows us to propose a molecular mechanism for this functional switch.

HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate

HPr kinase/phosphorylase (HprK/P), the sensor enzyme for catabolite repression in gram-positive bacteria, phosphorylates HPr, a phosphocarrier protein of a sugar transport and phosphorylation system ([41][1]), at a serine residue ([10][2]). To carry out its catalytic function in sugar transport and