Studies of Allostery between Multi-domain Proteins and Nucleic Acid Complexes

多结构域蛋白与核酸复合物的变构研究

• 批准号: 10331326
• 负责人: Victor S Batista
• 金额: $ 34.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331326
• 关键词: Active Sites; Allosteric Regulation; Allosteric Site; Amino Acids; Binding; Biological; Biological Assay; Biomedical Engineering; CRISPR/Cas technology; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Complex; Computer Simulation; Computing Methodologies; Coupling; DNA; Deoxyribonucleases; Distant; Drug Targeting; Engineering; Ensure; Enzymes; Gaussian model; Guidelines; Joints; Liquid substance; Macromolecular Complexes; Mediating; Methodology; Methods; Modernization; Molecular; Motion; Mutagenesis; Mutation; Nucleic Acids; Pathway Analysis; Pathway interactions; Process; Property; Protein Engineering; Proteins; RNA; Relaxation; Research; Research Personnel; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Spliceosomes; Structure; System; Techniques; Tertiary Protein Structure; Therapeutic; Universities; base; biophysical analysis; biophysical techniques; computer studies; drug discovery; endonuclease; experimental study; flexibility; genome editing; improved; innovation; insight; interest; macromolecule; millisecond; molecular dynamics; mutant; network models; novel; nuclease; precision medicine; programs; protein complex; rational design; response; simulation; small molecule inhibitor; tool

Project Summary The PI Batista from Yale and co-investigators (Lisi, Brown University, and Palermo, UC Riverside) will investigate allosteric pathways in the CRISPR-Cas9 system – composed of the multi-domain endonuclease Cas9 in complex with RNA and DNA. The system allows for studies of long-range signaling critical for allosteric mechanisms that achieve enhanced selectively and tunability of the protein/nucleic acid complex response. CRISPR-Cas9 is an innovative therapeutic tool with widely demonstrated capabilities for genome editing. An outstanding challenge of great research interest is to develop a detailed understanding of allosteric signals in CRISPR-Cas9 responsible for the DNA editing capability. Such understanding would have profound implications for bioengineering and precision medicine, as well as for establishing modern paradigms of allosteric regulation in protein/nucleic acid machines. A substantial hurdle in investigating the mechanisms of large protein/nucleic acid complexes is the inherent difficulty of adapting experimental and computational methodologies to capture the intrinsic flexibility of these structures essential for functionality. We propose to implement a synergistic approach of solution NMR and molecular dynamics (MD) in combination with established and novel methods for analysis of allosteric networks to elucidate the structural and dynamic determinants of allosteric signaling in CRISPR-Cas9. We have recently identified a pathway of dynamic communication connecting multiple domains of Cas9 through millisecond timescale motion that spans its critical nucleases, consistent with a regulatory signal proposed through experimental characterization. Thus, the following hypotheses guide our specific aims: (i) A well-defined allosteric pathway controls the CRISPR-Cas9 functionality; (ii) The allosteric interplay between spatially distant protein domains activates the DNA nuclease function; (iii) Modulation of the allosteric motions through the mutation of critical residues achieves altered specificity; and (iv) Dynamically-driven signaling is an intrinsic property of protein-nucleic acid macromolecular complexes. Our specific aims are: Aim 1: Characterize the allosteric control of the HNH nuclease; Aim 2: Determine the allosteric pathway from HNH to RuvC and the allosteric role of the PAM recognition sequence; and Aim 3: Characterize the effect of mutations on the allosteric pathway. The research program involves multiple cycles of an iterative approach where, in each cycle, allosteric pathways are explored through the analysis of differential motions probed by liquid-NMR relaxation methods and computation (MD and network analysis), obtaining valuable information on key amino acid residues and specific interactions responsible for transmitting structural or dynamical changes spanning the allosteric and active sites. The resulting insight provides guidelines for the next round of studies of mutants and modulators in a joint experimental and theoretical effort to elucidate the CRISPR-Cas9 allosteric mechanisms.

项目摘要 来自耶鲁大学的PI巴蒂斯塔和合作研究者（布朗大学的Lisi和加州大学滨江分校的巴勒莫）将 研究CRISPR-Cas9系统中的变构途径-由多结构域核酸内切酶组成 Cas9与RNA和DNA复合。该系统允许研究对变构至关重要的长距离信号传导。 实现蛋白质/核酸复合物应答的增强的选择性和可调谐性的机制。 CRISPR-Cas9是一种创新的治疗工具，具有广泛的基因组编辑能力。一个 巨大的研究兴趣的一个突出的挑战是发展一个详细的了解变构信号， CRISPR-Cas9负责DNA编辑能力。这样的理解将产生深远的影响 生物工程和精准医学，以及建立现代变构调节范式 在蛋白质/核酸机器中。研究大蛋白质/核酸的机制的一个实质性障碍 酸络合物是适应实验和计算方法来捕获的固有困难 这些结构的内在灵活性对于功能性是必不可少的。我们建议实施一项协同 溶液NMR和分子动力学（MD）的方法与已建立的和新的方法相结合， 分析变构网络，以阐明变构信号传导的结构和动力学决定因素， CRISPR-Cas9.我们最近发现了一条连接多个域的动态通信路径 Cas9通过毫秒级时间尺度运动跨越其关键核酸酶，与调节信号一致 通过实验表征提出。因此，以下假设指导我们的具体目标：（i）A 明确定义的变构途径控制CRISPR-Cas9功能性;（ii）CRISPR-Cas9与CRISPR-Cas9之间的变构相互作用 空间上远离的蛋白质结构域激活DNA核酸酶功能;（iii）变构运动的调节 通过关键残基的突变实现改变的特异性;和（iv）动态驱动的信号传导是一种 蛋白质-核酸大分子复合物内在性质。我们的具体目标是：目标1： 目的2：确定从HNH到RuvC的变构途径和HNH核酸酶的变构控制; 目的3：表征突变对PAM识别序列的变构作用的影响， 通路该研究计划涉及迭代方法的多个循环，其中在每个循环中，变构 通过液体NMR弛豫方法探测的微分运动分析探索途径， 计算（MD和网络分析），获得关键氨基酸残基和特定氨基酸残基的有价值的信息。 负责传递跨越变构和活性位点的结构或动力学变化的相互作用。 由此产生的洞察力为下一轮的突变体和调节剂的研究提供了指导方针， 实验和理论努力来阐明CRISPR-Cas9变构机制。