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

蛋白质设计和结构引导的信号转导机制询问

• 批准号: 10537123
• 负责人: Anna Katherine Hatstat
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537123
• 关键词: Address; Adopted; Algorithms; Area; Bacteria; Behavior; Binding; Biochemical; Biological; Biological Assay; Biophysical Process; Biophysics; Cells; Chimera organism; Collaborations; Complement; Complex; Computational Biology; Coupling; Cryoelectron Microscopy; Data; Data Collection; Engineering; Environment; Exhibits; Fingerprint; Genetic Transcription; Goals; Growth; In Vitro; Knowledge; Libraries; Ligand Binding; Ligands; Machine Learning; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Mutate; Output; Pathogenicity; Pathway interactions; Phosphorylation; Plants; Preparation; Prevalence; Process; Protein Dynamics; Protein Engineering; Proteins; Receptor Signaling; Research; Signal Transduction; Specificity; Stimulus; Structure; Structure-Activity Relationship; System; Testing; Thermodynamics; Training; Translating; Universities; Work; X-Ray Crystallography; base; biological systems; career; conformer; data modeling; design; extracellular; fitness; fungus; generative adversarial network; insight; interest; mimetics; neural network algorithm; non-Native; particle; post-doctoral training; protein structure; protein-histidine kinase; receptor; reconstitution; response; scaffold; sensor; sensor histidine kinase; skills; structural biology

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.

项目概要/摘要 活细胞已经进化出复杂的机制来检测其环境并在不同的细胞之间传递信号 生物膜，诱导有机体行为反应。尽管这些受体很普遍， 我们对信号跨膜传播的离散机制的理解仍然是 不断发展。在这一领域，组氨酸激酶 (HK) 是细菌、真菌、 以及调节生长、存活或致病性的植物。 HKs 感知多种细胞外刺激并进行转导 信号穿过膜并通过多个子域，激活磷酸化级联， 诱导转录反应。早期的模型提出，HK 在运动后通过大的、刚体的变化来做到这一点。 感知细胞外刺激。后续工作表明信号在 HK 子域之间传递 逐步的方式，通常通过蛋白质动力学的变化，告知信号转导的假设 是 HK 复合体子域之间热力学耦合的结果。这进一步意味着许多 HK信令过程中可以采用构造。研究分子和生物物理基础 为了研究 HK 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探究 跨膜信号传导中的能量阈值、传感器特异性和构象偏差。第一、理性与 从头设计将用于生成非本地、热力学可调的传感器域，以确定 配体诱导的能量反应足以启动信号传导。这将得到补充 通过序列和识别的合成正交传感器域的实验表征 结构引导神经网络算法。与此同时，我们将研究 X 射线晶体学和冷冻电子学 显微镜阐明不同信号状态下 HK 复合物或分离子结构域的结构， 告知结构-构象-功能关系的组装。这项研究将极大地推进我们的 了解跨膜信号转导的能量学和动力学，同时提高我们的能力 使用蛋白质设计和计算生物学来探究和设计复杂的生物物理机制。这 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 为我的独立研究生涯做好准备，研究和工程信号转导机制。