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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066775-03
Application #
6891675
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$357,277
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Lookadoo, Daniel B; Beyersdorf, Matthew S; Halkides, Christopher J (2018) Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY. Methods Mol Biol 1729:337-343
Merz, Gregory E; Borbat, Peter P; Muok, Alise R et al. (2018) Site-Specific Incorporation of a Cu2+ Spin Label into Proteins for Measuring Distances by Pulsed Dipolar Electron Spin Resonance Spectroscopy. J Phys Chem B 122:9443-9451
Sukomon, Nattakan; Widom, Joanne; Borbat, Peter P et al. (2017) Stability and Conformation of a Chemoreceptor HAMP Domain Chimera Correlates with Signaling Properties. Biophys J 112:1383-1395
Samanta, Dipanjan; Widom, Joanne; Borbat, Peter P et al. (2016) Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor. J Biol Chem 291:25809-25814
Samanta, Dipanjan; Borbat, Peter P; Dzikovski, Boris et al. (2015) Bacterial chemoreceptor dynamics correlate with activity state and are coupled over long distances. Proc Natl Acad Sci U S A 112:2455-60
Greenswag, Anna R; Muok, Alise; Li, Xiaoxiao et al. (2015) Conformational Transitions that Enable Histidine Kinase Autophosphorylation and Receptor Array Integration. J Mol Biol 427:3890-907
Greenswag, Anna R; Li, Xiaoxiao; Borbat, Peter P et al. (2015) Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation. Biochemistry 54:3454-68
Merz, Gregory E; Borbat, Peter P; Pratt, Ashley J et al. (2014) Copper-based pulsed dipolar ESR spectroscopy as a probe of protein conformation linked to disease states. Biophys J 107:1669-74
Pratt, Ashley J; Shin, David S; Merz, Gregory E et al. (2014) Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes. Proc Natl Acad Sci U S A 111:E4568-76
Li, Xiaoxiao; Fleetwood, Aaron D; Bayas, Camille et al. (2013) The 3.2 Å resolution structure of a receptor: CheA:CheW signaling complex defines overlapping binding sites and key residue interactions within bacterial chemosensory arrays. Biochemistry 52:3852-65

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