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.

项目摘要/摘要 活细胞已经发展了复杂的机制，以检测其环境并跨越跨 生物膜，诱导生物行为的反应。尽管这些受体流行，但 我们对跨膜传播信号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（HKS）是细菌，真菌，真菌中的膜受体的主要类别 以及调节生长，生存或致病性的植物。 HKS感应细胞外刺激和散发 跨膜和多个子域的信号，激活磷酸化级联和 引起转录响应。早期模型提出，HKS通过大的，刚性的身体移动 感应细胞外刺激。随后的工作表明，信号是在HK子域之间传递的 经常通过蛋白质动力学的变化来告知信号转导的假设 是HK络合物子域之间热力学耦合的结果。这进一步意味着许多 在香港信号传导过程中可以采用构象。研究分子和生物物理基础 香港特异性和信号转导，我们提出了一种结构和蛋白质设计方法来询问 跨膜信号传导中的能量阈值，传感器特异性和构象偏置。首先，理性和 从头设计将用于生成非本地，热力学调谐传感器域以确定 配体诱导的能量反应足以启动信号传导。这将与 通过序列和 结构引导的神经网络算法。同时，我们将追求X射线晶体学和冷冻电子 显微镜以阐明各种信号态在各种信号状态下的HK复合物或孤立的亚域的结构， 告知结构构成功能关系的组装。这项研究将大大推动我们的 了解跨膜信号转导的能量和动态，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工程复杂的生物物理机制。这 拟议的努力还将直接实现我博士后任期的培训目标，提供必要的技能 为我准备独立的研究职业研究和工程信号转导机制。