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

描述（由申请人提供）：耶鲁大学的合作项目研究员Loria和Batista将在分子水平上研究T. maritima中咪唑甘油磷酸合成酶（IGPS）的变构途径，重点研究与IGPS变构界面结合的小分子调节剂的影响，并影响使酶催化活性与变构位点效应结合同步的分子机制。IGPS非常适合于变构研究，因为它是一种蛋白质异二聚体，由HisH和HisF蛋白组成，具有经典变构酶的大多数特性，包括低聚结构，多个配体结合位点，无配体时的多个构象平衡，以及配体对特定蛋白质构象的稳定。它是一个潜在的治疗靶点，因为在哺乳动物中没有发现它，而在细菌以及一些植物和真菌中发现了它。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Ajellomyces，它们感染免疫功能低下的个体，其IGPS与酿酒葡萄球菌和海洋葡萄球菌酶高度同源。此外，最近有研究表明，从不动杆菌和假伯克霍尔德菌中敲除HisF基因会增加前者对ß-内酰胺类抗生素的敏感性，并降低后者的感染性。然而，潜在的变构机制可能代表药物发现的目标尚未建立，并将通过拟议的研究计划进行探索。研究假设是：(i)变构作用涉及由PRFAR结合引起的特定氨基酸残基的运动~ (ii) HisF中的运动被传递到HisH并产生HisH活性位点的活性构象~ (iii) HisF和HisH活性位点之间的通信运动受到药物结合或位点定向突变的影响。提出的方法结合了巴蒂斯塔的计算建模，包括在匹兹堡超级计算中心国家生物医学超级计算资源David E. Shaw研究有限责任公司的Anton超级计算机系统上的微秒分子动力学模拟，网络分析，核磁共振光谱模拟和计算药物筛选，以及洛里亚最先进的核磁共振松弛技术。定量由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，在每个循环中，通过分析液体核磁共振弛豫方法和计算（MD和网络分析）探测的微分运动来探索变构途径，获得有关关键氨基酸残基和负责传递结构或动态变化的特定相互作用的有价值的信息。由此产生的见解为下一轮突变体和调节剂的联合实验和理论研究提供了指导，以阐明受小分子结合和定点诱变影响的IGPS变构机制。

DESCRIPTION (provided by applicant): 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 heterdimer, 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 potein 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 ß-lactam antibiotics and lessen the infectvity of the latter. However, the underlying allosteric mechanisms that could represent target 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 motion 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.