MOLECULAR MECHANISMS OF SIGNAL TRANDUCTION BY CHEY

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Cells use signal transduction pathways to convert external stimuli into an internal form that can generate an appropriate response. In both prokaryotic and eukaryotic organisms this vital task is frequently accomplished by a cascade of transient protein phosphorylation and dephosphorylation events. A fundamental understanding of the mechanism of phosphoryl group transfer among proteins, of the regulation of this process, and of the impact phosphorylation has on protein activity is thus of broad interest The long term objective behind this application is to define the molecular mechanisms employed in bacterial signal transduction. The existence in bacteria of a widespread family of "two-component regulatory systems" that utilize an apparently common signal transduction mechanism, together with the superior experimental accessibility offered by bacteria, suggests this objective is feasible. The present application takes advantage of the best understood two- component system, that governing chemotaxis by Escherichia coli. The phosphorylated form of the CheY protein interacts with the flagellar motor to control swimming behavior. CheY obtains phosphoryl groups from either the CheA kinase or small molecules such as acetyl phosphate, and releases phosphoryl groups by either a self-catalyzed route or with the assistance of the CheZ protein. The mechanism of each of these reactions is unknown. The four specific aims of this project are to determine (i) how phosphorylation activates CheY, and the mechanisms of the CheY (ii) phosphorylation, (iii) autodephosphorylation, and (iv) CheZ-mediated dephosphorylation reactions. An integrated genetic, biochemical, and physical approach is proposed. The primary strategy will be to deduce the critical features of CheY signal transduction by isolating and thoroughly characterizing informative mutant CheY proteins. Numerous mutants are already in hand. Schemes are described to construct or identify additional cheY mutations that either test the current model of activation, are analogous to mutations that affect related signal transduction proteins, or alter the ability of CheY to support the various reactions in which it participates. Mutant CheY proteins with interesting in vivo phenotypes will be examined in further detail using appropriate in vitro assays chosen from a large battery of established biochemical and physical tests, up to and including complete structural determination by X-ray crystallography or multidimensional proton NMR. Two aspects of this basic research proposal are directly relevant to health issues. First, regulatory systems highly analogous to chemotaxis but far less well understood control expression of virulence factors by a variety of bacterial pathogens (e.g. Bordetella pertussis, Pseudomonas aeruginosa, Staphylococcus aureus). A detailed understanding of CheY function could facilitate design of therapeutic agents effective against infection by such bacteria. Second, fundamental insights applicable to eukaryotic signal transduction processes are anticipated. There is ample precedent for inappropriate signaling resulting in pathologies such as cancer.