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

项目摘要 来自耶鲁大学的皮巴蒂斯塔和合作调查员(利西，布朗大学，和巴勒莫，加州大学河滨分校)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形成复合体。该系统允许研究对变构至关重要的远程信号 实现蛋白质/核酸复合体反应的选择性和可调性增强的机制。 CRISPR-Cas9是一种创新的治疗工具，具有广泛展示的基因组编辑能力。一个 极具研究兴趣的突出挑战是发展对变构信号的详细理解 CRISPR-CAS9负责DNA编辑能力。这样的理解将产生深远的影响 用于生物工程和精确医学，以及建立变构调节的现代范例 在蛋白质/核酸机器中。研究大蛋白质/大核机制的一个重大障碍 酸性络合物是采用实验和计算方法捕获的固有困难 这些结构的内在灵活性对于功能来说是必不可少的。我们建议实施一种协同 溶液核磁共振和分子动力学(MD)结合已有的和新的方法研究 分析变构网络以阐明变构信号的结构和动态决定因素 CRISPR-CAS9。我们最近发现了一条连接多个域的动态通信路径 通过跨越其关键核酸酶的毫秒时间尺度运动，与调控信号一致 提出通过实验进行表征。因此，以下假设指导我们的具体目标：(一)A 明确定义的变构途径控制CRISPR-Cas9功能；(Ii)变构相互作用 空间距离较远的蛋白质结构域激活DNA核酸酶功能；(Iii)对变构运动的调节 通过突变关键残基获得改变的特异性；以及(Iv)动态驱动的信号是一种 蛋白质-核酸大分子复合体的本征性质。我们的具体目标是：目标1：塑造 HNH核酸酶的变构控制；目标2：确定从HNH到RuvC的变构途径和 PAM识别序列的变构作用；以及目标3：表征突变对变构的影响 路径。研究方案涉及多个循环的迭代方法，其中在每个循环中，变构 通过对液体核磁共振弛豫方法探测的差异运动的分析，探索了路径。 计算(MD和网络分析)，获得有关关键氨基酸残基和特定氨基酸的有价值信息 负责跨变构和活性中心传递结构或动态变化的相互作用。 由此得到的洞察力为下一轮联合突变体和调节子的研究提供了指导方针 实验和理论工作，以阐明CRISPR-Cas9变构机理。

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.