The long-term objective of this work is to elucidate the molecular signaling mechanisms of the chemoreceptors that mediate chemotactic behavior in E. coli. These transmembrane receptors form stable ternary complexes with two cytoplasmic proteins - CheA, a histidine autokinase, and CheW, which couples CheA to chemoreceptor control. Most of the prodigious signal amplification that occurs in the chemotaxis pathway takes place in these receptor complexes, which are organized into highly cooperative arrays. The proposed studies address two key mechanistic questions: How do receptor molecules control and amplify CheA output signals? How do receptor molecules couple input stimulus information to output signal control? The principal subject of these studies is the serine chemoreceptor, Tsr. To investigate the mechanism of CheA control in Tsr signaling complexes, we will conduct additional tests of the trimer-of-dimers model of receptor signaling teams;characterize the trimer-forming, cluster-forming and signal amplification properties of receptors with trimer contact lesions;identify the CheW-binding and CheA control determinants in receptors by a mutational survey of all polar and hydrophobic residues in the core Tsr signaling domain;construct and characterize soluble single-chain Tsr signaling fragments for ternary complex studies;use Tsr~Tsr constructs to manipulate the geometry and stoichiometry of receptor signaling complexes;and construct mini-CheA and triplet CheW molecules to simplify and stabilize Tsr signaling complexes for high-resolution structural studies. A HAMP domain translates transmembrane piston motions into conformational changes that regulate output from the Tsr signaling domain. To elucidate the role of the HAMP domain in Tsr signaling, we will exploit a large collection of Tsr-HAMP mutants to investigate HAMP interactions with the cytoplasmic membrane;develop in vivo cysteine-directed crosslinking probes for HAMP structures and signaling states;compare the structural and functional consequences of HAMP alterations;investigate the mechanisms of HAMP input and output control with mutations that alter the length and amino acid composition of the segments that link HAMP helices to adjoining Tsr structural elements. These studies should not only provide important conceptual insights into the signaling mechanisms of bacterial chemoreceptors, but may also lead to new therapeutic approaches for combating bacterial pathogens that employ motility and chemotactic behavior during the infection process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM019559-37
Application #
7807095
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Gindhart, Joseph G
Project Start
1991-06-01
Project End
2011-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
37
Fiscal Year
2010
Total Cost
$468,449
Indirect Cost
Name
University of Utah
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Lai, Run-Zhi; Han, Xue-Sheng; Dahlquist, Frederick W et al. (2017) Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme. Proc Natl Acad Sci U S A 114:E7583-E7591
Lai, Run-Zhi; Gosink, Khoosheh K; Parkinson, John S (2017) Signaling Consequences of Structural Lesions that Alter the Stability of Chemoreceptor Trimers of Dimers. J Mol Biol 429:823-835
Frank, Vered; Piñas, Germán E; Cohen, Harel et al. (2016) Networked Chemoreceptors Benefit Bacterial Chemotaxis Performance. MBio 7:
Ames, Peter; Hunter, Samuel; Parkinson, John S (2016) Evidence for a Helix-Clutch Mechanism of Transmembrane Signaling in a Bacterial Chemoreceptor. J Mol Biol 428:3776-88
Piñas, Germán E; Frank, Vered; Vaknin, Ady et al. (2016) The source of high signal cooperativity in bacterial chemosensory arrays. Proc Natl Acad Sci U S A 113:3335-40
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
Mowery, Patricia; Ames, Peter; Reiser, Rebecca H et al. (2015) Chemotactic Signaling by Single-Chain Chemoreceptors. PLoS One 10:e0145267
Kitanovic, Smiljka; Ames, Peter; Parkinson, John S (2015) A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor. J Bacteriol 197:2568-79
Ames, Peter; Zhou, Qin; Parkinson, John S (2014) HAMP domain structural determinants for signalling and sensory adaptation in Tsr, the Escherichia coli serine chemoreceptor. Mol Microbiol 91:875-86
Pedetta, Andrea; Parkinson, John S; Studdert, Claudia A (2014) Signalling-dependent interactions between the kinase-coupling protein CheW and chemoreceptors in living cells. Mol Microbiol 93:1144-55

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