The protein histidine kinase CheA is central to signal transduction pathways that allow prokaryotes to sense and respond to their environments. During chemotaxis, CheA couples changes in ligand occupancy of transmembrane chemoreceptors to phosphorylation of the response regulator CheY; CheY controls the flagellar motor. In understanding the well-characterized phosphorelay of chemotaxis, central unanswered questions concern how receptor occupancy regulates kinase activity and how CheA coordinates phosphate flow from ATP to response regulators via an internal phosphorylation site. To address these issues we propose synergistic biophysical experiments centered on the crystallographic structure determinations of dimeric CheA in complex with receptor fragments and the receptor coupling protein CheW. To complement structures, solution studies will probe CheA dynamics and partner interactions. Relative CheA domain motions and subunit associations likely control interactions with its signaling partners. Crystals of CheA in complex with CheW and nucleotides will reveal new aspects of CheA catalysis and regulation. Soluble fragments of transmembrane receptors that activate the CheA kinase have been identified and overexpressed for crystallization with CheA. Resonance energy transfer and electron-spin interactions between CheA domains tagged with probes will correlate solution conformations to crystallographic structures and define limits of movement in the presence of receptor and CheW. Kinetic studies will interrogate CheA subunit exchange to determine its potential role in transmembrane signaling and response regulator activation. Our experiments will employ chemotaxis proteins from Thermatoga maritima, which due to their thermostability offer distinct advantages in crystallography, dynamical studies and kinetics. This work will lead to novel strategies for design of small molecules capable of modulating CheA activity. Given the absence of histidine kinases in eukaryotes and their essential role in bacterial virulence, they are ideal targets for a new class of antibiotics. Thus, we are also pursuing structure determinations of CheA from Helicobacter pylori, a known pathogen linked to ulcers and stomach cancer.
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