Computational and Biochemical Studies of Temperature Effects on Allostery in the Imidazole Glycerol Phosphate Synthase (IGPS) from T. maritima

温度对 T. maritima 咪唑甘油磷酸合酶 (IGPS) 变构影响的计算和生化研究

• 批准号: 9978862
• 负责人: Victor S Batista
• 金额: $ 29.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978862
• 关键词: Acinetobacter; Active Sites; Affect; Affinity; Ajellomyces; Allosteric Site; Amino Acids; Area; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Burkholderia pseudomallei; Calorimetry; Candida; Chemicals; Communication; Computer Models; Computing Methodologies; Cryptococcus; Cyclization; Dependence; Drug Design; Drug Screening; Drug Targeting; Entropy; Environment; Enzymes; Free Energy; Genes; Glutamine; Glycerol; Guidelines; High temperature of physical object; Hydrolysis; Imidazole; Immunocompromised Host; Individual; Joints; Kinetics; Ligand Binding; Ligands; Liquid substance; Mammals; Methods; Molecular; Molecular Conformation; Monobactams; Motion; Mutagenesis; Mutate; NMR Spectroscopy; Organism; PRFAR; Pathogenicity; Pathway Analysis; Pathway interactions; Pharmaceutical Preparations; Physiological; Plants; Play; Predisposition; Process; Production; Property; Protein Conformation; Proteins; Relaxation; Relaxation Techniques; Research; Resolution; Ribonucleotides; Role; Saccharomyces cerevisiae; Sampling; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Techniques; Temperature; Testing; Thermodynamics; Thermotoga maritima; Titrations; Work; base; beta-Lactams; computer studies; drug discovery; experimental study; flexibility; human pathogen; imidazole glycerol phosphate synthase; inorganic phosphate; insight; interest; knockout gene; millisecond; molecular dynamics; mutant; novel; pathogen; plant fungi; prevent; programs; response; screening; simulation; small molecule; therapeutic target

Project Summary The co-PIs Loria and Batista from Yale will investigate the effect of temperature on allosteric pathways in the enzyme imidazole glycerol phosphate synthase (IGPS) from the hyperthermophilic bacterium T. maritima, at the molecular level, with emphasis on the influence of small molecule modulators that bind to the IGPS and affect the molecular mechanisms that synchronize the enzyme catalytic activity with effector N'-[(5'- phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binding at the allosteric site. IGPS is ideally suited for studies of allostery since it is a protein heterodimer, composed of the HisH and HisF proteins, with most of the properties of classical allosteric enzymes, including an oligomeric structure, multiple ligand binding sites, multiple conformational equilibria in the absence of ligand, and the stabilization of specific protein conformations by ligands. It is a potential therapeutic target since it is not found in mammals and is found in bacteria as well as in some plants and fungi. In particular many plant pathogens and opportunistic human pathogens such as Cryptococcus, Candida, and Ajellomyces that infect immunocompromised individuals have an IGPS that is highly homologous to the S. cerevisiae and T. maritima enzymes. Additionally, it has recently been shown that gene knockouts of HisF from Acinetobacter and Burkholderia pseudomallei increase the susceptibility of the former to β-lactam antibiotics and lessen the infectivity of the latter. However, the role of entropy as reflected by unsual temperature effects on allosteric mechanisms that could represent targets for drug discovery has yet to be established. The research hypotheses are: (i) Higher temperatures and PRFAR binding increase flexibility in IGPS enabling conformational sampling of an active enzyme form; (ii) PRFAR-induced motions propagate through well- defined and conserved amino acid residues; (iii) Modulations of these motions and subsequent functional alteration can be achieved by small molecule allosteric ligands. The proposed methods combine Batista's computational modeling, including microsecond molecular dynamics simulations, network analysis, simulations of NMR spectra and computational drug screening, with Loria's state-of-the-art NMR relaxation techniques, quantifying the microsecond-to-millisecond conformational motions induced by drug or ligand binding with atomic resolution, mutagenesis studies, and isothermal titration calorimetry. 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 role of entropy on IGPS allosteric mechanisms.

项目摘要 耶鲁大学的Co-Pis Loria和Batista将研究温度对变形途径的影响 来自高疗细菌T. maritima的酶咪唑甘油磷酸合酶（IGP） 分子水平，重点是与IGP和IGP结合的小分子调节剂的影响 影响将酶催化活性与效应n'同步的分子机制 - （5'-- 磷酸糖基）formimino] -5-氨基咪唑-4-羧酰胺 - 核苷核苷酸（PRFAR）在该结合处的结合 变构网站。 IGP非常适合于对变构的研究，因为它是由蛋白质异二聚体组成的 HISH和HISF蛋白，具有经典变构酶的大多数特性，包括寡聚 结构，多种配体结合位点，在没有配体的情况下多构象平衡，并且 配体特异性蛋白质构象的稳定。这是一个潜在的治疗靶标 在哺乳动物中，在细菌以及某些植物和真菌中发现。特别是许多植物病原体 和机会性的人类病原体，例如隐孢子，念珠菌和ajellomyces感染 免疫功能低下的个体的IGP与酿酒酵母和Maritima高度同源 酶。此外，最近已经证明了来自acinetobacter和 Burkholderia pseudomallei增加了前者对β-内酰胺抗生素的敏感性，并减少了 后者的感染力。但是，熵的作用反映出异常温度对变构的影响 可以代表药物发现目标的机制尚未确定。研究 假设是：（i）较高的温度和PRFAR结合提高了IGP的灵活性 活性酶形式的构象采样； （ii）Prfar诱导的动作通过良好传播 定义并构成氨基酸残基； （iii）调制这些动作和随后的功能 小分子变构配体可以实现改变。提出的方法结合了Batista的 计算建模，包括微秒的分子动力学模拟，网络分析，模拟 NMR光谱和计算药物筛查，以及Loria的最先进的NMR松弛技术， 量化由药物或配体结合引起的微秒至毫秒构象运动 原子分辨率，诱变研究和等温滴定量热法。研究计划涉及 迭代方法的多个循环，在每个循环中，通过探索变构途径通过 通过液体-NMR松弛方法和计算探测的差分运动的分析（MD和网络 分析），获取有关关键氨基酸残差和负责特定相互作用的价值信息 传输跨越变构和活性位点的结构或动态变化。由此产生的见解 在联合实验和 理论上的努力以阐明熵在IGPS寄发力机制上的作用。