Mikhail N. Levit

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N‐terminal extracytoplasmic sensing domains connected by transmembrane helices to C‐terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with ∼14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus‐induced changes in lateral packing interactions within an array of receptor‐sensing domains at the cell surface perturb an equilibrium between active and inactive receptor–kinase complexes within the cytoplasm.

Dimerization Is Required for the Activity of the Protein Histidine Kinase CheA That Mediates Signal Transduction in Bacterial Chemotaxis

The histidine protein kinase CheA plays an essential role in stimulus-response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a γ-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross-linking studies. The dissociation constant (CheA2 ↔ 2CheA; KD = 0.2-0.4 μM) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (KD values ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (kcat ≈ 10 min−).

Stimulus response coupling in bacterial chemotaxis: receptor dimers in signalling arrays

In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function.

Signal transduction: Hair brains in bacterial chemotaxis

The conserved cytoplasmic domains of bacterial chemotaxis receptors are a fibrous arrangement of alpha-helical coiled coils that look a lot like hair. Such bundles of alpha-helical filaments mediate sensory-motor responses in all prokaryotic cells. How do they work? Very nearly perfectly is probably as good an answer as any.

Active Site Interference and Asymmetric Activation in the Chemotaxis Protein Histidine Kinase CheA

The histidine protein kinase CheA is a multidomain protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem.271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0.10 mM for phosphoacceptor domain; kobs = 17 min−1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.

Interactions between Escherichia coli Nucleoside-diphosphate Kinase and DNA*

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of γ-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (∼1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show thatEscherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.