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

描述（由申请人提供）：来自耶鲁大学的共同PI Loria和Batista将研究来自T.海洋，在分子水平上，重点是小分子调节剂的影响，结合到IGPS变构界面和影响的分子机制，同步酶的催化活性与效应器结合在变构网站。 IGPS理想地适合于变构的研究，因为它是由HisH和HisF蛋白组成的蛋白质异二聚体，具有经典变构酶的大部分性质，包括寡聚体结构、多个配体结合位点、在不存在配体的情况下的多个构象平衡，以及配体对特定蛋白构象的稳定作用。它是一种潜在的治疗靶点，因为它在哺乳动物中没有发现，而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，例如感染免疫功能低下个体的隐球菌属、念珠菌属和阿杰酵母属，其IGPS与S高度同源。cerevisiae和T.海洋酶此外，最近已经表明，来自不动杆菌和类鼻疽伯克霍尔德氏菌的HisF的基因敲除增加了前者对β-内酰胺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，并将探索拟议的研究计划。研究假设是：（i）变构涉及由PRFAR结合诱导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，和（iii）传递HisF和HisH的活性位点的运动受到药物结合或定点诱变的影响。 所提出的方法结合了联合收割机巴蒂斯塔的计算模型，包括大卫E. 匹兹堡超级计算中心生物医学超级计算国家资源的Shaw Research，LLC，网络分析，NMR光谱模拟和计算药物筛选，以及Loria最先进的NMR弛豫技术，用原子分辨率定量由药物或配体结合诱导的微秒至毫秒的构象运动，诱变研究，和等温滴定量热法。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NMR弛豫方法和计算探测的微分运动来探索变构途径（MD和网络分析），获得关于负责传递跨越变构和活性位点的结构或动力学变化的关键氨基酸残基和特异性相互作用的有价值的信息。由此产生的洞察力提供了指导方针，为下一轮的突变体和调制器的联合实验和理论的努力，以阐明的IGPS变构机制的影响，小分子结合和定点诱变的研究。

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.