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

项目摘要 耶鲁大学的共同PI Loria和Batista将研究温度对变构途径的影响。 嗜热细菌T.maritima的咪唑甘油磷酸合成酶(IGPS)，在 分子水平，重点是小分子调节剂结合到IGPS和 影响酶催化活性与效应子N‘-[(5’-)]同步的分子机制 核糖核糖基)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide(PrFAR)结合 变构部位。IGPS是一种蛋白质异源二聚体，由 HISH和HISF蛋白，具有经典变构酶的大部分性质，包括低聚物 结构，多个配体结合部位，在没有配体的情况下的多重构象平衡，以及 配体对特定蛋白质构象的稳定作用。它是一个潜在的治疗靶点，因为它没有被发现 存在于哺乳动物中，存在于细菌以及一些植物和真菌中。尤其是许多植物病原体 以及感染的条件人类病原体，如隐球菌、念珠菌和味觉霉菌 免疫缺陷个体的免疫球蛋白与酿酒酵母和毛滴虫高度同源。 酵素。此外，最近研究表明，HISF基因敲除来自不动杆菌和 假鼻疽伯克霍尔德菌增加前者对β-内酰胺类抗生素的敏感性并减少 后者的传染性。然而，由变构的非恒定温度效应所反映的熵的作用 可能代表药物发现目标的机制尚未建立。这项研究 假设是：(I)更高的温度和PRFAR结合增加了IGPS启用的灵活性 一种活性酶形式的构象采样；(Ii)PRFAR诱导的运动通过井- 定义和保守的氨基酸残基；(Iii)这些运动的调节和随后的功能 改变可以通过小分子变构配体来实现。提出的方法结合了巴蒂斯塔的 计算建模，包括微秒级分子动力学模拟、网络分析、模拟 核磁共振波谱和计算机药物筛选，使用洛里亚最先进的核磁共振松弛技术， 定量药物或配体结合引起的微秒到毫秒的构象运动 原子分辨率、诱变研究和等温滴定量热法。该研究计划涉及 迭代方法的多个循环，其中在每个循环中，通过 液体核磁共振弛豫法探测的差动分析和计算(MD和网络 分析)，获得关于关键氨基酸残基和负责的特定相互作用的宝贵信息 传递跨越变构和活性中心的结构或动力学变化。由此产生的洞察力 为下一轮突变体和调节子的联合实验和研究提供指导 从理论上阐明了熵在IGPS变构机制中的作用。

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.