A protein design- and structure-guided interrogation of signal transduction mechanisms

项目摘要/摘要 活细胞已经进化出复杂的机制来检测它们的环境并在 生物膜，在生物行为中诱导反应。尽管这些受体普遍存在， 我们对信号跨膜传播的离散机制的理解仍然是 不断进化。在这一领域，组氨酸激酶(HKS)是细菌、真菌、 以及调节生长、存活或致病性的植物。HKS感测不同的细胞外刺激和转导 跨膜和通过多个亚区的信号，激活磷酸化级联反应和 诱导转录反应。早期的模型认为，HKS是通过大型刚体移动来实现这一点的 感知细胞外刺激。后续工作表明，信号在香港子域之间以 循序渐进的方式，通常通过蛋白质动力学的变化，告知信号转导的假设 是HK复合体亚区之间热力学耦合的结果。这进一步意味着许多 在HK信号传递过程中可能采用构象。研究其分子和生物物理基础 由于HK的特异性和信号转导，我们提出了一种结构和蛋白质设计方法来询问 跨膜信号传递中的能量阈值、传感器特异性和构象偏差。第一，理性和 从头开始的设计将用来产生非本地的、热力学可调的传感器域，以确定 什么样的配体诱导的能量反应足以启动信号传递。这一点将得到补充 通过序列识别的合成的、正交感应域的实验表征--和 结构制导神经网络算法。同时，我们将继续研究X射线结晶学和低温电子。 显微镜以阐明处于不同信号状态的HK复合体或分离的亚域的结构，以 通知组件结构-构象-功能关系。这项研究将大大推进我们的 理解跨膜信号转导中的能量学和动力学，同时提高我们的能力 使用蛋白质设计和计算生物学来询问和设计复杂的生物物理机制。这个 拟议的努力也将直接实现我博士后任期的培训目标，提供必要的技能 让我为独立的研究生涯做好准备，学习和设计信号转导机制。

Project Summary/Abstract Living cells have evolved intricate mechanisms to detect their environment and transduce signals across biological membranes, inducing responses in organismal behavior. Despite the prevalence of these receptors, our understanding of the discrete mechanisms by which signals are propagated across membranes is still evolving. In this area, histidine kinases (HKs) are a predominant class of membrane receptors in bacteria, fungi, and plants that regulate growth, survival, or pathogenicity. HKs sense diverse extracellular stimuli and transduce a signal across the membrane and through multiple subdomains, activating a phosphorylation cascade and inducing a transcriptional response. Early models proposed that HKs do so through large, rigid body shifts after sensing extracellular stimuli. Subsequent work indicates that signals are passed between HK subdomains in a step-wise manner, often through changes in protein dynamics, informing the hypothesis that signal transduction is the result of thermodynamic coupling between subdomains of the HK complex. This further implies that many conformations may be adopted in the course of HK signaling. To investigate the molecular and biophysical basis of HK specificity and signal transduction, we propose a structure and protein design approach to interrogate energetic thresholds, sensor specificity, and conformational bias in transmembrane signaling. First, rational and de novo design will be used to generate non-native, thermodynamically tunable sensor domains to determine what ligand-induced energetic response is sufficient to initiate signaling. This will be complemented with experimental characterization of synthetic, orthogonal sensor domains identified through a sequence- and structure-guided neural network algorithm. In parallel, we will pursue X-ray crystallography and cryo-electron microscopy to elucidate the structure of HK complexes or isolated subdomains in various signaling states, to inform assembly of structure-conformation-function relationships. This research will significantly advance our understanding of energetics and dynamics in transmembrane signal transduction while advancing our ability to use protein design and computational biology to interrogate and engineer complex biophysical mechanisms. The proposed efforts will also directly fulfill the training goals of my postdoctoral tenure, affording the necessary skills to prepare me for an independent research career studying and engineering signal transduction mechanisms.