Hybrid Methods for Dynamic Structure Analysis of Proteins from Pathogenic Microorganisms

项目总结 这项研究计划将调查了解构象多样性的一般假设 对蛋白质的研究将提供对其生物学的新见解，并使医学研究成为可能。它指向两个人 系统分类：整合膜蛋白(IMP)和病毒-宿主相互作用。IMP扮演着关键角色，如 守门人、受体、转运体、动态平衡调节器和药物靶标。这些功能是由 由膜环境中IMP的构象可塑性决定。小鬼们准备起来很有挑战性，而且 更具挑战性的是在适当的膜模拟环境中重建。性价比高 浓缩体积中的同位素浓缩技术，核磁共振与核磁共振相结合的混合方法 进化协变(ECS)、接触预测的新方法以及来自 蛋白质结构预测社区，将应用于IMPS的结构和功能研究。这些小鬼， 选自重要的人类病原体，包括大肠杆菌、肺炎克雷伯菌和铜绿假单胞菌，是潜在的 抗生素发现的目标。ECS还将与核磁共振数据相结合，以确定多个 蛋白质的“天然状态”。我们计划的第二个组成部分是针对病毒宿主生物分子 复合体，以及抗病毒药物的发现。我们将利用创新的顺磁核磁共振方法，以及 小角X射线散射(SAXS)、电子-电子双共振谱(DER)和Förster 共振能量转移(FRET)，以严格定义所赋予的动态域间结构分布 由小鼠Moloney白血病病毒(MLV)整合酶(IN)的部分有序连接子组成。这些数据将是 在最大占用概率(MAXOCC)的背景下进行解释，并用于探索这一角色(S) G-逆转录病毒基因整合机制的灵活性。域间链接器还用于提供 约束伴侣乱交所需的灵活性。我们还将确定域间连接子如何测序 流感非结构蛋白1(NS1)具有适当的可塑性，以确定其特异性和亲和力 宿主蛋白质和RNA。这种结构性和功能性的混杂是NS1的S机制的基础 抑制流感感染的细胞先天免疫反应，并严格表征其 动态结构基础将为减毒活疫苗的研制提供基础信息。 我们还将利用我们的平台研究通过结合SARS-CoV2病毒的主要蛋白酶来抑制其病毒的药物 (MPRO)。我们已经确定了三种药物，已经被批准用于人类，最初设计的目的是抑制 丙型肝炎病毒NSP3/4A蛋白酶，在病毒复制检测中也抑制SARS-CoV2 微摩尔浓度。我们的计算对接研究还发现了其他几个FDA- 可能抑制MPRO的批准药物。酶动力学、生物物理化学和X射线结晶学研究 将用于表征这些酶抑制剂药物与MPRO之间形成的络合物，并 开发其作为新冠肺炎疗法的潜力，或作为新疗法开发的先导化合物。

PROJECT SUMMARY This research program will investigate the general hypothesis that understanding the conformational diversity of proteins will provide new insights into their biology, and enable medical research. It is directed to two classes of systems: Integral Membrane Proteins (IMPs) and viral-host interactions. IMPs play critical roles as gate keepers, receptors, transporters, homeostasis regulators, and drug targets. These functions are mediated by the conformational plasticity of the IMP in the membrane environment. IMPs are challenging to prepare, and even more challenging to reconstitute in appropriate membrane mimicking environments. Cost-effective technologies for isotope-enrichment in condensed volumes, hybrid approaches combining NMR with evolutionary co-variation (ECs), novel methods of contact prediction, and innovative modeling methods from the protein structure prediction community, will be applied to structure-function studies of IMPs. These IMPs, chosen from important human pathogens, including E. coli, K. pneumoniae, and P. aeruginosa, are potential targets for antibiotic discovery. ECs will also be combined with NMR data to determine structures of multiple “native states” of proteins. The second component of our program is directed to viral – host biomolecular complexes, and antiviral drug discovery. We will utilize innovative paramagnetic NMR methods, together with small angle X-ray scattering (SAXS), electron-electron double resonance spectroscopy (DEER), and Förster resonance energy transfer (FRET), to rigorously define dynamic interdomain structural distributions conferred by the partially-ordered linkers of the murine Moloney Leukemia Virus (MLV) integrase (IN). These data will be interpreted in the context of maximum occupancy probabilities (MaxOcc), and used to probe the role(s) of this flexibility in the gene integration mechanisms of g-retroviruses. Interdomain linkers also function to provide flexibility needed for binding partner promiscuity. We will also determine how the interdomain linker sequences of influenza Non-Structural Protein 1 (NS1) confer appropriate plasticity to define its specificity and affinity for host proteins and RNAs. This structural and functional promiscuity underlies NS1’s mechanisms for suppressing the cellular innate immune response to influenza infection, and rigorous characterization of its dynamic structural basis will provide fundamental information for live-attenuated virus vaccine development. We will also apply our platform to investigate drugs that inhibit SARS-CoV2 virus by binding its main protease (Mpro). We have identified three drugs, already approved for use in humans, originally designed to inhibit the NSP3/4A protease of hepatitis C virus, that also inhibit SARS-CoV2 in viral replication assays at low micromolar concentrations. Our computational docking studies have also identified several other FDA- approved drugs that may inhibit Mpro. Enzyme kinetic, biophysical chemistry, and X-ray crystallography studies will be used to characterize complexes formed between these protease inhibitor drugs and Mpro, and to develop their potential as COVID-19 therapeutics, or as lead compounds for new therapeutic development.