Cells use signal transduction pathways to monitor their environment and implement appropriate responses to change. A central feature of this process is the cycling of signal molecules between active and inactive forms. For example, transient protein phosphorylation is used as a dynamic internal representation of external conditions in many systems. A fundamental understanding of the mechanisms and regulation of phosphoryl group transfer among proteins, as well as the impact of phosphorylation on protein activity, is thus of broad interest. The long term objective behind this application is to define the molecular mechanisms of signal transduction in bacteria. ? This proposal takes advantage of a particularly well understood signaling pathway, the two-component regulatory system that governs chemotaxis by Escherichia coli. The CheY and CheB response regulators are activated by self-catalyzed transfer of phosphoryl groups from either small molecules or the CheA sensor kinase, and inactivated by self-catalyzed dephosphorylation. CheY-P also releases phosphoryl groups with the assistance of the CheZ protein. The purpose of this proposal is to develop a comprehensive understanding of the phosphoryl group transactions that occur during chemotactic signal transduction, with the additional intention of clarifying which features are generally applicable to other two-component regulatory systems. Towards that end, Specific Aims 1 and 2 explore the factors that determine the rates and specificity of the reactions catalyzed by representative response regulators, including CheY and CheB.
Specific Aims 3 and 4 build on information derived from the recently determined structure of a CheY/CheZ complex to clarify many aspects of CheZ function. ? An integrated biochemical, genetic, and physical approach is planned. A variety of both established and new biochemical assays will be used to characterize the reactions of wildtype and mutant signaling proteins in vitro. Numerous informative CheY and CheZ mutants are already in hand, and screening strategies to isolate more are described. In appropriate cases, complete structures will be determined by X-ray crystallography. ? Regulatory systems highly analogous to chemotaxis but far less well understood control expression of virulence factors by many bacterial pathogens. The detailed mechanistic understanding of two-component regulatory systems that will result from the proposed research may be relevant to designing new classes of therapeutic agents that interfere with microbial virulence signaling pathways. Fundamental insights applicable to other biological signaling systems are also anticipated. ? ?
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