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

T. maritima IGPS 变构的计算和生化研究

• 批准号: 8853887
• 负责人: Victor S Batista
• 金额: $ 28.74万
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8853887
• 关键词: Acinetobacter; Active Sites; Affect; Agonist; Ajellomyces; Allosteric Site; Amino Acids; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Burkholderia pseudomallei; Calorimetry; Candida; Computer Simulation; Cryptococcus; Cyclization; Development; Docking; Drug Targeting; Enzyme Kinetics; Enzymes; Equilibrium; Glutamine; Glycerol; Greek; Guidelines; Health; Human; Hydrolysis; Imidazole; Immunocompromised Host; Individual; Joints; Kinetics; Lead; Ligand Binding; Ligands; Liquid substance; Mammals; Methods; Molecular; Molecular Conformation; Monobactams; Motion; Mutagenesis; Pathway Analysis; Pathway interactions; Pharmaceutical Preparations; Physiological; Plants; Preclinical Drug Evaluation; Predisposition; Property; Protein Conformation; Proteins; Relative (related person); Relaxation; Relaxation Techniques; Research; Resolution; Ribonucleotides; Role; Saccharomyces cerevisiae; Safety; Site; Site-Directed Mutagenesis; Structure; Supercomputing; System; Techniques; Tissues; Titrations; base; biomedical resource; computer studies; dimer; drug development; drug discovery; imidazole glycerol phosphate synthase; inhibitor/antagonist; inorganic phosphate; insight; interest; knockout gene; millisecond; molecular dynamics; mutant; pathogen; plant fungi; prevent; programs; receptor function; research study; response; simulation; small molecule; supercomputer; therapeutic target

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.

描述(申请人提供)：来自耶鲁大学的合作者Loria和Batista将在分子水平上研究Maritima的咪唑甘油磷酸合成酶(IGPS)的变构途径，重点是结合IGPS变构界面的小分子调节剂的影响，以及影响同步酶催化活性与变构位点上的效应物结合的分子机制。IGPS是一种由HISH和HISF蛋白组成的蛋白质杂二聚体，具有经典变构酶的大多数性质，包括寡聚体结构、多配体结合位点、在没有配体的情况下的多重构象平衡以及通过配体稳定特定的蛋白质构象，因此iGPS非常适合于别构作用的研究。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发现。特别是，许多感染免疫低下个体的植物病原体和条件人类病原体，如隐球菌、念珠菌和味觉霉菌，都有一个与酿酒酵母和毛霉菌酶高度同源的IGPS。此外，最近的研究表明，不动杆菌和假鼻疽伯克霍尔德菌的HISF基因敲除增加了前者对β-内酰胺类抗生素的敏感性，并降低了后者的感染性。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PRFAR结合引起的特定氨基酸残基的运动；(Ii)HISF中的运动传递到HISF并产生HISH活性部位的活性构象；(Iii)HISF和HISH活性部位之间的通讯运动受药物结合或定点突变的影响。建议的方法结合了巴蒂斯塔的计算模型，包括来自David E.Shaw Research的Anton超级计算机系统的微秒分子动力学模拟，匹兹堡超级计算中心国家生物医学超级计算资源的LLC，网络分析，核磁共振谱模拟和计算性药物筛选，以及Loria最先进的核磁共振松弛技术，量化药物或配体与原子分辨率结合引起的微秒到毫秒的构象运动，突变研究，以及等温滴定量热法。该研究计划涉及多个循环的迭代方法，在每个循环中，通过分析液体-核磁共振松弛方法和计算(MD和网络分析)探测的差异运动来探索变构途径，获得关于关键氨基酸残基和负责跨变构和活性中心传递结构或动力学变化的特定相互作用的有价值的信息。所获得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突变影响的IGPS变构机制。