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

项目摘要 来自耶鲁大学的合作PI Loria和Batista将研究温度对变构途径的影响， 酶咪唑甘油磷酸合酶（IGPS）从超嗜热菌T。马里蒂马 分子水平，重点是与IGPS结合的小分子调节剂的影响， 影响使酶催化活性与效应物N ′-[（5 ′- 磷酸核酮糖基）甲亚胺基]-5-氨基咪唑-4-甲酰胺-核糖核苷酸（PRFAR）结合 变构位点IGPS理想地适合于变构的研究，因为它是一种蛋白质异源二聚体，由 HisH和HisF蛋白质，具有经典变构酶的大部分特性，包括寡聚体 结构，多个配体结合位点，在配体不存在的情况下的多个构象平衡，以及 通过配体稳定特定蛋白质构象。它是一个潜在的治疗靶点，因为它没有被发现 在哺乳动物中，在细菌以及一些植物和真菌中发现。特别是许多植物病原体 和机会性人类病原体，如隐球菌、念珠菌和放线菌， 免疫受损个体具有与S. cerevisiae和T. maritima 内切酶此外，最近已经表明，来自不动杆菌属的HisF的基因敲除和 类鼻疽伯克霍尔德菌增加了前者对β-内酰胺类抗生素的敏感性， 后者的感染力。然而，熵的作用，反映了非寻常的温度效应对变构 可能代表药物发现目标的机制尚未建立。研究 假设是：（i）较高的温度和PRFAR结合增加IGPS的灵活性， 活性酶形式的构象采样;（ii）PRFAR诱导的运动通过良好的 确定的和保守的氨基酸残基;（iii）这些运动和随后的功能性调节 可以通过小分子变构配体实现改变。所提出的方法结合了联合收割机巴蒂斯塔的 计算建模，包括微秒分子动力学模拟，网络分析，模拟 核磁共振光谱和计算药物筛选，与洛里亚的国家的最先进的核磁共振弛豫技术， 定量由药物或配体结合诱导的微秒至毫秒的构象运动， 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 用液体核磁共振弛豫方法和计算（分子动力学和网络）探测的微分运动分析 分析），获得关键氨基酸残基和负责特定相互作用的有价值的信息， 传递跨越变构和活性位点的结构或动力学变化。由此产生的洞察力 为联合实验中突变体和调节剂的下一轮研究提供了指导方针， 理论上的努力，以阐明熵的作用，对IGPS变构机制。

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.