CAREER: The Nature of Interdomain Coupling in the Farnesoid X Receptor

职业生涯：Farnesoid X 受体域间偶联的本质

• 批准号: 2144679
• 负责人: Chiamaka Okafor
• 金额: $ 86.83万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2144679&HistoricalAwards=false
• 关键词: CAREER; Nature; Interdomain; Coupling; Farnesoid

Many hormones and other signaling molecules exert their potent effects by regulating which genes are turned “on” by a process known as transcription. A large family of so-called transcription factors with a similar architecture that includes separate domains for ligand (the hormone/signaling molecule) and DNA binding are known as nuclear hormone receptors. When these proteins bind their ligands, they interact with a specific DNA sequence and allow the machinery necessary to “read” the gene to assemble and proceed. The transcription factors are flexible and the relative positions of the domains can vary dramatically. The PI proposes to understand how the individual domains communicate with each other, so that the DNA-recognition segment can sense that the ligand binding domain is occupied, or how the DNA binding domain might impact the ligand binding domain. Their highly flexible nature is thought to allow for precise biological control of transcription, given that a single member of this family can have distinct responses to very similar ligands. These studies will reveal the key molecular details that drive crucial physiological processes. Additionally, this work will achieve broader impacts by providing educational materials and research training. Curricular materials will be generated to promote teaching and learning of protein dynamics in K-12 and undergraduate courses, while research training will be provided for high school teachers, undergraduate and graduate students. This proposal aims to generate a molecular understanding of how the ligand binding (LBD) and DNA binding (DBD) domains of the farnesoid X receptor (FXR) interact to regulate transcription. The mechanisms of crosstalk between these two domains are uncharacterized in FXR and other nuclear receptors (NR). Thus novel, creative approaches are required to understand how interdomain communication influences transcription. This work will combine molecular experiments with biophysical approaches and computational modeling. This integrative approach will allow molecular-level perturbations of FXR to be linked to structure and dynamic motions, subsequently revealing how FXR function is uniquely regulated and modulated by ligand and/or DNA binding. Molecular dynamics (MD) simulations to model interdomain interactions in various ligand- and DNA-bound states will be followed by hydrogen-deuterium exchange mass spectrometry to experimentally reveal interaction surfaces between the two domains. Small angle X-ray scattering will be used to generate low resolution models of the quaternary structure of FXR. Luciferase reporters of transactivation and nuclear magnetic resonance spectroscopy (NMR) will be used to determine how perturbations of the LBD modulate function in the DBD. Finally, NMR, MD simulations and binding assays will be performed to characterize bidirectional allostery in FXR. By carefully determining how the functions of the NR domains are coupled, this work will provide new insights into how ligands dictate transcriptional outcomes. Importantly, because of the highly conserved functional mechanisms that govern NRs, these studies with FXR will be informative with respect to allosteric mechanisms common to the NR family.This research is funded by the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

许多激素和其他信号分子通过调节哪些基因通过转录过程“打开”来发挥其有效作用。一大类所谓的转录因子具有相似的结构，包括配体（激素/信号分子）和 DNA 结合的独立结构域，被称为核激素受体。当这些蛋白质与其配体结合时，它们会与特定的 DNA 序列相互作用，并允许“读取”基因所需的机器进行组装和继续。转录因子是灵活的，并且域的相对位置可以显着变化。 PI 建议了解各个结构域如何相互通信，以便 DNA 识别片段能够感知配体结合结构域被占用，或者 DNA 结合结构域如何影响配体结合结构域。鉴于该家族的单个成员可以对非常相似的配体产生不同的反应，它们高度灵活的性质被认为可以对转录进行精确的生物控制。这些研究将揭示驱动关键生理过程的关键分子细节。此外，这项工作将通过提供教育材料和研究培训来实现更广泛的影响。将制作课程材料以促进 K-12 和本科生课程中蛋白质动力学的教学和学习，同时将为高中教师、本科生和研究生提供研究培训。该提案旨在从分子角度理解法尼醇 X 受体 (FXR) 的配体结合 (LBD) 和 DNA 结合 (DBD) 结构域如何相互作用以调节转录。这两个结构域之间的串扰机制在 FXR 和其他核受体 (NR) 中尚未表征。因此，需要新颖、创造性的方法来理解域间通信如何影响转录。这项工作将分子实验与生物物理方法和计算模型结合起来。这种综合方法将允许 FXR 的分子水平扰动与结构和动态运动联系起来，随后揭示 FXR 功能如何通过配体和/或 DNA 结合进行独特的调节和调节。 分子动力学 (MD) 模拟用于模拟各种配体和 DNA 结合状态下的域间相互作用，随后将进行氢-氘交换质谱分析，以通过实验揭示两个域之间的相互作用表面。小角度 X 射线散射将用于生成 FXR 四元结构的低分辨率模型。反式激活荧光素酶报告基因和核磁共振波谱 (NMR) 将用于确定 LBD 的扰动如何调节 DBD 的功能。最后，将进行 NMR、MD 模拟和结合测定来表征 FXR 中的双向变构。通过仔细确定 NR 结构域的功能如何耦合，这项工作将为配体如何决定转录结果提供新的见解。重要的是，由于控制 NR 的功能机制高度保守，这些 FXR 的研究将为 NR 家族常见的变构机制提供丰富的信息。这项研究由生物科学理事会分子和细胞生物科学部的分子生物物理学项目资助。该奖项反映了 NSF 的法定使命，并通过使用基金会的评估进行评估，认为值得支持。 智力价值和更广泛的影响审查标准。