Molecular Mechanisms of Signaling in E. coli Chemotaxis

描述（由申请人提供）：细胞使用信号转导途径来监测其环境并对变化实施适当的响应。这个过程的一个中心特征是信号分子在活性和非活性形式之间的循环。例如，在许多系统中，瞬时蛋白质磷酸化被用作外部条件的动态内部表示。因此，对蛋白质之间磷酰基转移的机制和调节以及磷酸化对蛋白质活性的影响的基本理解具有广泛的兴趣。该应用背后的长期目标是确定细菌中信号转导的分子机制。 这个提议利用了一个特别好理解的信号通路，即大肠杆菌趋化性的双组分调控系统。CheY和切布反应调节剂通过磷酰基从小分子或CheA传感器激酶的自催化转移而活化，并通过自催化去磷酸化而失活。CheY-P还在CheZ蛋白的协助下释放磷酰基。本提案的目的是发展一个全面的了解磷酰基基团的交易过程中发生的趋化信号转导，澄清哪些功能通常适用于其他两个组件的监管系统的附加意图。为此，特定目标1和2探索了决定由代表性反应调节剂（包括CheY和切布）催化的反应速率和特异性的因素。具体目标3和4建立在从最近确定的CheY/CheZ复合物的结构中获得的信息的基础上，以澄清CheZ功能的许多方面。 一个综合的生物化学，遗传学和物理方法正在计划中。各种既建立和新的生化测定将被用来表征野生型和突变体信号蛋白在体外的反应。许多信息的CheY和CheZ突变体已经在手，和筛选策略，以隔离更多的描述。在适当的情况下，将通过X射线晶体学确定完整的结构。 调控系统与趋化性高度相似，但对许多细菌病原体的毒力因子表达的控制还远未完全了解。从拟议的研究中产生的双组分调控系统的详细机制的理解可能是相关的设计与微生物毒力信号通路干扰的治疗剂的新类别。也预期适用于其他生物信号系统的基本见解。

DESCRIPTION (provided by applicant): 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.