The continuing project investigates the broad class of two-component signaling pathways, recently found to be widespread eukaryotes and ubiquitous in prokaryotes. Two-component pathways play especially critical roles in bacteria, where they control diverse cellular functions including cell division, antibiotic resistance, activation of virulence, and wound detection during infection. The receptors and signaling proteins which comprise these ancient pathways are conserved across species and are considered to be attractive targets for broad-spectrum antibiotics. Thus, a basic mechanistic understanding of the pathways components will have significant impacts on signaling biology and pharmaceutical development. The present research program focuses on two central elements of the chemotaxis pathway of Escherichia coli and Salmonella typhimurium: the transmembrane asperate receptor and the cytoplasmic CheA kinase it regulates. The goals address four fundamental questions. First, how does attractant binding to a chemoreceptor generate a transmembrane signal? Second, how does a receptor adapt to a constant background stimulus? Third, how do these distinct attractant and adaptations signals modulate the activity of a receptor-bound kinase? Fourth, how do the components of the pathway assemble into a cooperative, multi-protein signaling complex? Novel approaches utilizing site-directed cysteine chemistry and spectroscopy are being utilized to address these questions. The progress reports describes the chemical determination of a low- resolution structure for the cytoplasmic domain of the aspartate receptor, and the identification of regions of the cytoplasmic domain critical for CheA kinase regulation. In addition, a highly dynamic region of the cytoplasmic domain is described, and working models for the mechanism of receptor-regulated kinase regulation are proposed.
The specific aims utilize site-directed cysteine chemistry, fluorescence and EPR spectroscopy to further investigate the mechanism by which signals are transmitted through the receptor to the kinase, and the effects of these signals on kinase structure, dynamics and activity. The geometry of the assemble receptor-kinase signaling complex is also probed. Overall, the broad goal of these studies is to understand the mechanisms of transmembrane signaling, receptor adaptation, and kinase regulation in a fully assembled, multi-protein signaling complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040731-15
Application #
6519313
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
1988-07-01
Project End
2005-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
15
Fiscal Year
2002
Total Cost
$244,261
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Parkinson, John S; Hazelbauer, Gerald L; Falke, Joseph J (2015) Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update. Trends Microbiol 23:257-66
Briegel, Ariane; Wong, Margaret L; Hodges, Heather L et al. (2014) New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography. Biochemistry 53:1575-85
Briegel, Ariane; Wong, Margaret L; Hodges, Heather L et al. (2014) Correction to new insights into bacterial chemoreceptor array structure and assembly from electron cryotomography. Biochemistry 53:6624
Falke, Joseph J; Piasta, Kene N (2014) Architecture and signal transduction mechanism of the bacterial chemosensory array: progress, controversies, and challenges. Curr Opin Struct Biol 29:85-94
Piasta, Kene N; Falke, Joseph J (2014) Increasing and decreasing the ultrastability of bacterial chemotaxis core signaling complexes by modifying protein-protein contacts. Biochemistry 53:5592-600
Falke, Joseph J (2014) Piston versus scissors: chemotaxis receptors versus sensor His-kinase receptors in two-component signaling pathways. Structure 22:1219-1220
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
Natale, Andrew M; Duplantis, Jane L; Piasta, Kene N et al. (2013) Structure, function, and on-off switching of a core unit contact between CheA kinase and CheW adaptor protein in the bacterial chemosensory array: A disulfide mapping and mutagenesis study. Biochemistry 52:7753-65
Piasta, Kene N; Ulliman, Caleb J; Slivka, Peter F et al. (2013) Defining a key receptor-CheA kinase contact and elucidating its function in the membrane-bound bacterial chemosensory array: a disulfide mapping and TAM-IDS Study. Biochemistry 52:3866-80
Slivka, Peter F; Falke, Joseph J (2012) Isolated bacterial chemosensory array possesses quasi- and ultrastable components: functional links between array stability, cooperativity, and order. Biochemistry 51:10218-28

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