Computational and Biochemical Studies of Allostery in the IGPS of T. maritima

项目摘要 耶鲁大学的合作者Loria和Batista将研究咪唑甘油酶中的变构途径 Maritima的磷酸合成酶(IGPS)，在分子水平上，重点是小分子的影响 与IGPS变构界面结合并影响分子机制的分子调节剂 使酶的催化活性与变构位点上的效应器结合同步。IGPS非常适合于 变构的研究，因为它是一种蛋白质异二聚体，由HISH和HISF蛋白质组成，大多数 经典变构酶的性质，包括寡聚体结构，多个配体结合位点， 缺乏配基的多重构象平衡和特定蛋白质的稳定 配基的构象。它是一个潜在的治疗靶点，因为它在哺乳动物中没有发现，在 细菌以及一些植物和真菌中的细菌。尤其是许多植物病原体和机会主义人类 感染免疫受损个体的病原体，如隐球菌属、念珠菌属和味精霉菌 一种与酿酒酵母和毛滴虫酶高度同源的免疫球蛋白。此外，它最近还 研究表明，从不动杆菌和假鼻疽伯克霍尔德氏菌中敲除HISF基因会增加 前者对内酰胺类抗生素的敏感性较高，而后者的传染性较低。然而， 可能代表药物发现靶点的潜在变构机制尚未建立 并将在拟议的研究计划中进行探索。研究假设是：(I)变构作用涉及 HISF中PRFAR结合~(II)运动诱导的特定氨基酸残基运动传递给HISH 并产生HISH活动部位的活动构象和(Iii)传递HISH活动部位的运动 HISF和HISH受药物结合或定点突变的影响。建议的方法结合了 Batista的计算模型，包括对Anton的微秒分子动力学模拟 国家生物医学资源有限责任公司David E.Shaw Research的超级计算机系统 匹兹堡超级计算中心的超级计算、网络分析、核磁共振谱模拟和 计算机药物筛选，使用洛里亚最先进的核磁共振松弛技术，量化 由具有原子分辨率的药物或配体结合引起的微秒到毫秒的构象运动， 诱变研究和等温滴定量热法。研究计划涉及多个周期的 迭代方法，在每个循环中，通过对微分的分析来探索变构途径 通过液体核磁共振松弛方法和计算(MD和网络分析)探测的运动，获得 关于关键氨基酸残基和负责传递结构的特定相互作用的有价值的信息 或跨越变构和活性中心的动态变化。由此得到的洞察力为 下一轮突变体和调节子的联合实验和理论研究，以阐明 小分子结合和定点突变对iGPS变构机制的影响。

Project Summary The co-PIs Loria and Batista from Yale will investigate allosteric pathways in the enzyme imidazole glycerol phosphate synthase (IGPS) from T. maritima, at the molecular level, with emphasis on the influence of small molecule modulators that bind to the IGPS allosteric interface and affect the molecular mechanisms that synchronize the enzyme catalytic activity with effector 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 ¿-lactam antibiotics and lessen the infectivity of the latter. However, the underlying allosteric mechanisms that could represent targets for drug discovery have yet to be established and will be explored by the proposed research program. The research hypotheses are: (i) allosterism involves motions of specific amino acid residues induced by PRFAR binding~ (ii) motions in HisF are transmitted to HisH and generate an active conformation of the HisH active site~ and (iii) motions communicating the active sites of HisF and HisH are affected by drug binding, or site-directed mutagenesis. The proposed methods combine Batista's computational modeling, including microsecond molecular dynamics simulations on the Anton supercomputer system from David E. Shaw Research, LLC at the National Resource for Biomedical Supercomputing of the Pittsburgh Supercomputing Center, 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 IGPS allosteric mechanisms as influenced by small molecule binding and site-directed mutagenesis.