Illuminating Notch receptor-ligand selectivity through structure-guided protein engineering

通过结构引导的蛋白质工程阐明Notch受体-配体选择性

• 批准号: 10189660
• 负责人: Vincent Christopher Luca
• 金额: $ 43万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10189660
• 关键词: Affinity; Architecture; Area; Binding; Biological Response Modifier Therapy; Biology; Biophysics; Cell Communication; Cell Surface Receptors; Cells; Chemicals; Complex; Crystallization; Cues; Development; Directed Molecular Evolution; Discrimination; Electron Microscopy; Electron Transport Complex III; Engineering; Environment; Funding; Goals; Kinetics; Laboratories; Language; Length; Ligands; Malignant Neoplasms; Mammals; Mediating; Molecular; Mutation; Outcome; Output; Pathway interactions; Post-Translational Protein Processing; Protein Engineering; Proteins; Research; Roentgen Rays; Series; Signal Transduction; Structure; Tissues; Training; Vision; Work; aortic valve disorder; biophysical properties; cell type; design; experimental study; glycosylation; human disease; insight; interdisciplinary approach; mechanical force; mechanical properties; notch protein; novel; novel therapeutics; programs; receptor; reconstitution; response; structural biology

PROJECT SUMMARY Overall vision of the research program. My research is focused on understanding the “molecular language” of cell-to-cell communication. Cell surface receptors transmit signals in response to external cues, and receptor activity is regulated by extrinsic factors such as posttranslational modifications, mechanical force, and chemical environment. Using a multidisciplinary approach that leverages my training in structural biology, biophysics, and protein engineering, my group is attempting to solve difficult problems in the area of receptor biology. We hypothesize that the distinct structural and biophysical parameters governing receptor-ligand interactions control downstream signaling, and that ligands may be engineered to alter these parameters and reprogram signaling outputs. My long-term goal is to harness the insight obtained through our mechanistic studies to design proteins that can fine-tune receptor activity and function as powerful next-generation therapeutics. Overview of research in the laboratory. Notch signaling is initiated when a mechanosensory Notch receptor is “pulled on” by a Delta-like (DLL) or Jagged (Jag) ligand expressed on an adjacent cell. In mammals, the various Notch receptor (Notch1-4) and ligand (DLL1, DLL4, Jag1, Jag2) subtypes are able to induce distinct or even opposing cellular responses. Ligand selectivity is further regulated by Notch receptor glycosylation, which inhibits the activity of Jag ligands yet potentiates the activity of DLL ligands. We currently have a poor understanding of the molecular basis for ligand discrimination in the Notch pathway, and structural studies of Notch receptor-ligand interactions have been difficult because their nearly undetectable binding affinity precludes reconstitution of stable complexes. We recently overcame this obstacle by using directed evolution to strengthen DLL4 and Jag1 binding to Notch1, which allowed us to capture their interacting domains for x-ray crystal structure determination. My lab is now building upon these transformative results to explore how the structural, biophysical, and mechanical properties of various ligand subtypes contribute to their unique functions. Goals for the next five years. Over the five-year funding period, the goals of this project are to: (i) systematically characterize the structure, binding kinetics, and signaling potency of Notch receptor-ligand complexes; (ii) use electron microscopy to visualize the ultrastructural architecture of full-length Notch1-DLL4 and Notch1-Jag1 complexes; (iii) determine the biophysical basis by which Notch glycosylation regulates DLL and Jag activity, and (iv) design tissue-specific Notch antagonists. The successful completion of this work will establish the molecular mechanisms of Notch receptor-ligand selectivity and will guide the engineering of novel modulators of Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.

项目总结 研究计划的总体构想。我的研究重点是理解“分子语言”。 细胞间的通讯。细胞表面受体根据外界信号传递信号，而受体 活性受外部因素的调节，如翻译后修饰、机械力和化学物质 环境。使用多学科方法，利用我在结构生物学、生物物理学和 蛋白质工程，我的团队正在尝试解决受体生物学领域的难题。我们 假设控制受体-配体相互作用的不同结构和生物物理参数控制 下游信号，并且配体可以被设计来改变这些参数和重新编程信号 产出。我的长期目标是利用我们通过机制研究获得的洞察力来设计蛋白质 它可以微调受体的活性，并作为强大的下一代疗法发挥作用。 实验室研究概述。当机械感觉Notch受体启动时，Notch信号被启动 被表达在相邻细胞上的Delta样(Dll)或锯齿状(JAG)配体“拉上”。在哺乳动物中， 不同的Notch受体(Notch1-4)和配体(DLL1、DLL4、Jag1、Jag2)亚型能够诱导不同的或 甚至是相反的细胞反应。配体选择性由Notch受体糖基化进一步调节， 抑制JAG配体的活性，但增强Dll配体的活性。我们目前有一个贫穷的 了解Notch途径中配体识别的分子基础，并对其结构进行研究 缺口受体-配体的相互作用一直很困难，因为它们几乎无法检测到的结合亲和力排除了 稳定络合物的重组。我们最近克服了这个障碍，通过使用定向进化来加强 DLL4和Jag1与Notch1结合，这使得我们能够捕捉到它们相互作用的结构域来进行X射线晶体结构分析 决心。我的实验室现在正在这些变革性的结果的基础上探索结构、生物物理、 不同配体亚型的力学性质决定了其独特的功能。 未来五年的目标。在五年的资助期内，该项目的目标是：(一)系统地 表征Notch受体-配基复合体的结构、结合动力学和信号效力；(Ii)用途 电子显微镜观察全长Notch1-DLL4和Notch1-Jag1的超微结构 化合物；(Iii)确定Notch糖基化调节Dll和JAG活性的生物物理基础， 以及(Iv)设计组织特异性Notch拮抗剂。这项工作的成功完成将建立 Notch受体-配体选择性的分子机制，将指导新型调节剂的设计 在各种生物和治疗环境中具有扩展能力的Notch信号。