RUI: Biophysical investigation of SH3 domain binding partners: How the binding motif and surrounding disordered sequence affect the finding pathway

RUI：SH3 结构域结合伴侣的生物物理学研究：结合基序和周围无序序列如何影响发现途径

• 批准号: 2324974
• 负责人: K Aurelia Ball
• 金额: $ 44.96万
• 依托单位: Skidmore College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2324974&HistoricalAwards=false
• 关键词: RUI; Biophysical; investigation; SH3; domain

The communication within cells that allows cellular processes to occur is mediated by interactions between proteins. The goal of this research is to understand how these fundamental interactions are controlled at the local site of interaction, as well as the surrounding regions. Understanding the details of these interactions would allow researchers to predict and modify cell behavior. Results of this project will provide deeper insights into how protein binding interactions function in different contexts, including in the presence of multiple sites that can bind the same protein partner, and help explain how a common interaction can specialize to perform many different cellular functions. Undergraduate students working on this project will have the opportunity to learn both computational and experimental biophysics skills. To allow a larger number of students to engage in undergraduate research, the investigator will offer a research-based lab course that will expose students to techniques in computational biophysics and molecular dynamics simulations. In this course, students will develop and carryout a research project contributing to the larger project goals. The investigator will also offer a two-credit course on Minoritized Identities in Science that will encourage natural science majors to consider and grapple with ideas about identity in science and how to make the sciences more inclusive. Students will create specific interventions to improve the experience of underrepresented students in science. The course will prepare students to be leaders on the topic of identity and inclusivity in science.Cellular signaling interactions often involve binding of intrinsically disordered protein regions to small domains. The affinity and specificity of these interactions depend on the binding motif within the disordered region, but are also affected by the flanking regions and surrounding context. Additionally, the binding pathway, rather than just the structure of the bound state, can be important for understanding the functional adaptation of these interactions. Focusing on SH3 domain binding as a model system, the PI have previously shown that the disordered proline-rich peptide ArkA binds to the AbpSH3 domain in a multi-step process using molecular dynamics (MD) simulations. In this project, the PI will now examine how this binding pathway varies in different biologically relevant contexts by systematically studying this pathway at increasing levels of complexity using MD simulations, NMR spectroscopy, and ITC. The first aim will focus on the effects of altering the flexibility and electrostatic interactions of the binding motif itself by simulating the binding with ArkA prolines in the cis conformation, with salt screening electrostatic interactions, and with mutations to charged AbpSH3 residues involved in binding. In the second aim, different physiologically relevant IDR sequences will be compared to explore the effect of the motif-flanking regions on binding. In the third aim, the binding process in the context of multiple binding motifs will be examined. The completion of this project will add a new level of complexity to the understanding of how disordered protein regions bind to SH3 domains, as how both the binding motif and the surrounding sequences influence the binding pathway, and ultimately biological function will be revealed.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.

细胞内允许细胞过程发生的通讯是由蛋白质之间的相互作用介导的。这项研究的目的是了解这些基本相互作用是如何在相互作用的局部部位以及周围区域进行控制的。了解这些相互作用的细节将使研究人员能够预测和修改细胞行为。该项目的结果将更深入地了解蛋白质结合相互作用如何在不同环境中发挥作用，包括存在可以结合相同蛋白质伴侣的多个位点的情况，并有助于解释常见的相互作用如何专门执行许多不同的细胞功能。从事该项目的本科生将有机会学习计算和实验生物物理学技能。为了让更多的学生参与本科研究，研究人员将提供基于研究的实验课程，让学生接触计算生物物理学和分子动力学模拟技术。在本课程中，学生将开发并开展一个研究项目，为更大的项目目标做出贡献。研究人员还将提供一门关于科学中的少数身份的两学分课程，这将鼓励自然科学专业的学生考虑和解决有关科学身份的想法以及如何使科学更具包容性。学生将制定具体的干预措施，以改善代表性不足的学生在科学领域的体验。该课程将使学生成为科学中身份和包容性主题的领导者。细胞信号相互作用通常涉及本质上无序的蛋白质区域与小结构域的结合。这些相互作用的亲和力和特异性取决于无序区域内的结合基序，但也受到侧翼区域和周围环境的影响。此外，结合途径（而不仅仅是结合态的结构）对于理解这些相互作用的功能适应非常重要。 PI 之前以 SH3 结构域结合为模型系统，利用分子动力学 (MD) 模拟表明，无序的富含脯氨酸的肽 ArkA 在多步骤过程中与 AbpSH3 结构域结合。在该项目中，PI 现在将通过使用 MD 模拟、NMR 光谱和 ITC 以不断增加的复杂性系统地研究该途径，从而检查该结合途径在不同的生物学相关环境中如何变化。第一个目标将重点关注通过模拟与顺式构象中的 ArkA 脯氨酸的结合、盐筛选静电相互作用以及对参与结合的带电 AbpSH3 残基的突变来改变结合基序本身的灵活性和静电相互作用的影响。在第二个目标中，将比较不同的生理相关 IDR 序列，以探索基序侧翼区域对结合的影响。在第三个目标中，将检查多个结合基序背景下的结合过程。该项目的完成将为理解无序蛋白质区域如何与 SH3 结构域结合增加新的复杂性，因为结合基序和周围序列如何影响结合途径，并最终揭示生物学功能。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。