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 来自耶鲁大学和共同研究员的Pi Batista（Lisi，Brown University和Palermo，UC Riverside）将 研究CRISPR-CAS9系统中的变构途径 - 由多域内切酶组成 Cas9与RNA和DNA复杂。该系统允许研究对变构至关重要的远程信号传导 实现蛋白质/核酸复合物反应的可鼠能力的机制。 CRISPR-CAS9是一种创新的治疗工具，具有广泛的基因组编辑功能。一个 重大研究兴趣的杰出挑战是对变构信号的详细理解 CRISPR-CAS9负责DNA编辑功能。这种理解将具有深远的影响 用于生物工程和精确医学，以及建立变构法规的现代范式 在蛋白质/核酸机器中。研究大蛋白/核的机理的重大障碍 酸络合物是对适应实验和计算方法捕获的继承困难 这些结构的固有灵活性对于功能必不可少。我们建议实施协同作用 溶液NMR和分子动力学（MD）的方法与已建立的新方法 分析变构网络，以阐明变构信号的结构和动态确定器 CRISPR-CAS9。我们最近确定了连接多个域的动态通信的途径 cas9通过毫秒的时间尺度运动跨越其关键核，与调节信号一致 通过实验表征提出。那就是以下假设指导我们的具体目的：（i） 定义明确的变构途径控制CRISPR-CAS9功能； （ii）之间的变构相互作用 空间远处的蛋白质结构域激活DNA核酸酶功能。 （iii）调节变构运动 通过关键残差的突变实现了特异性的改变； （iv）动态驱动的信号是 蛋白质核酸大分子复合物的内在特性。我们的具体目的是：目标1：特点 HNH核酸酶的变构控制； AIM 2：确定从HNH到RUVC的变构途径和 PAM识别序列的变构作用；和目标3：表征突变对变构的影响 路径。该研究计划涉及迭代方法的多个循环，在每个周期中，变构中 通过分析液体-NMR弛豫方法探测的差分运动，探索了途径 计算（MD和网络分析），获得有关关键氨基酸残差和特定的价值信息 负责传输结构或动态变化的相互作用跨越了变构和活性位点。 由此产生的见解为关节中突变体和调节剂的下一轮研究提供了指南 实验和理论上的努力阐明了CRISPR-CAS9变构机制。