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

许多激素和其他信号分子通过一个称为转录的过程来调节哪些基因被“打开”，从而发挥其强大的作用。一个大家族的所谓的转录因子具有类似的结构，包括单独的结构域的配体（激素/信号分子）和DNA结合被称为核激素受体。当这些蛋白质与它们的配体结合时，它们与特定的DNA序列相互作用，并允许“读取”基因所需的机器组装和进行。转录因子是灵活的，并且结构域的相对位置可以显著变化。PI建议了解各个结构域如何相互通信，以便DNA识别片段可以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。它们高度灵活的性质被认为允许对转录进行精确的生物控制，因为该家族的单个成员可以对非常相似的配体具有不同的反应。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产生更广泛的影响。将生成课程材料，以促进K-12和本科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。该提案旨在从分子水平上了解法尼醇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。在FXR和其他核受体（NR）中，这两个结构域之间的串扰机制尚不清楚。因此，需要新颖的，创造性的方法来了解域间通信如何影响转录。这项工作将结合联合收割机分子实验与生物物理方法和计算建模。这种整合方法将允许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，以模拟在各种配体和DNA结合状态的域间相互作用，随后将通过氢氘交换质谱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四级结构的低分辨率模型。荧光素酶报告的反式激活和核磁共振光谱（NMR）将被用来确定如何扰动的LBD调制功能的DBD。最后，将进行NMR、MD模拟和结合测定以表征FXR中的双向变构。通过仔细确定NR结构域的功能是如何耦合的，这项工作将提供新的见解配体如何决定转录结果。重要的是，由于管理NR的高度保守的功能机制，FXR的这些研究将为NR家族常见的变构机制提供信息。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计划资助。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的学术价值和更广泛的影响评审标准。

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.