Simulating Ion Modulated Stability of Retroviral Kissing-Loop Complexes

模拟逆转录病毒接吻环复合物的离子调制稳定性

• 批准号: 8387712
• 负责人: Alan Austin Chen
• 金额: $ 5.39万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-11-15 至 2013-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8387712
• 关键词: Affinity; Agreement; Anti-Retroviral Agents; Base Pairing; Binding; Cereals; Chemicals; Complex; Computer Simulation; DNA; Data; Dependence; Dimerization; Dinucleoside Phosphates; Dissociation; Ensure; Environment; Equilibrium; Error Sources; Event; Future; Genome; Goals; HIV; Hydrogen Bonding; Individual; Ions; Kinetics; Lead; Maps; Measurement; Measures; Mechanics; Mediating; Methods; Modeling; Molecular; Moloney Leukemia Virus; Mutation; Nucleic Acids; Nucleosides; Nucleotides; Pathway interactions; Pharmaceutical Preparations; Physiological; Process; Purines; RNA; RNA Folding; Research; Resolution; Retroviridae; Route; Sampling; Scheme; Simulate; Site; Sodium Chloride; Solutions; Solvents; Structure; System; Testing; Therapeutic; Torsion; Viral; Virus Replication; Water; Work; adjudicate; base; design; driving force; in vivo; inhibitor/antagonist; insight; interstitial; laser tweezer; molecular dynamics; optical traps; predictive modeling; public health relevance; purine; research study; simulation; single molecule; small molecule

DESCRIPTION (provided by applicant): The goal of this project is to uncover the physical basis for the ion-mediated interactions of retroviral RNA kissing-loop complexes and to explain the unusual sequence requirements for maximum mechanical stability; specifically that of the Dimerization Initiation Site (DIS) of HIV and Moloney Leukemia Virus (MMLV). This high stability is known to be crucial for retroviral genome dimerization, as mutations to the DIS loop always result in greatly reduced virus replication and infectivity rates in vivo. Therefore, interfering with kissing-loop mediated genome dimerization may prove to be a successful route to designing new anti-retroviral therapeutics. However, current attempts to target this interface have actually resulted in increased kissing-loop stability with no detectible inhibition of viral replication. It would therefore be useful to determine the physical basis of the enhanced kissing loop stability in order to inform future attempts at designing targeted inhibitors. Mutational analysis has shown that several bases flanking the loop residues are crucial for high complex stability, but both structural and chemical mapping experiments confirm that these bases are not base paired, do not participate in intra or inter-molecular hydrogen bonds, and actually appear to be flipped out into solution. Lastly, the observed separation distances at the transition state are too large to be explained by partial base-pairing or the presence of interstitial water molecules. We hypothesize that the flanking residues effect neigboring base pair dissociation kinetics through modulation of the local ionic environment. We also predict that the release of partially dehydrated ions at the transition state constitutes the rate-limiting step for kissing-loop dissociation. Using explicit ion, implicit solvent Monte Carlo simulations, the kinetic pathways of kissing loops dissociation will be determined. A Markov state model of the dominant dissociation pathway will be created, and then examined in detail using large numbers of short, all-atom molecular dynamics simulations of transitions along dissociation intermediates. These simulations will utilize an applied external force to enhance dissociation, analagous to single-molecule pulling experiments. The accuracy of the simulations will be ascertained by comparison of the predicted force-extension curves, separation at the transition state, critical force, and dissociation rates with the actual experimental measurements. In this way, the extent to which the unpaired flanking residues indirectly contribute to the overall mechanical stability of the complex through ion-mediated modulation of base pairing kinetics at the loop-loop interface will be ascertained. Understanding ion-mediated driving forces for complex formation should allow better prediction of stabilizing and destabilizing mutations, as well as identify specific ion-mediated interactions which may be exploitable in the design of small molecule inhibitors.

描述（由申请人提供）：该项目的目的是揭示逆转录病毒RNA接吻环复合物的离子介导的相互作用的物理基础，并解释最大机械稳定性的异常序列要求；特别是HIV和Moloney白血病病毒（MMLV）的二聚化启动位点（DIS）。已知这种高稳定性对于逆转录病毒基因组二聚化至关重要，因为对循环的突变总是导致病毒复制大大降低，并在体内降低了感染率。因此，干扰亲吻环介导的基因组二聚化可能被证明是设计新的抗返回病毒疗法的成功途径。但是，当前针对该界面的尝试实际上导致了接吻环的稳定性增加，而没有可检测到的病毒复制抑制。因此，确定增强的接吻环稳定性的物理基础是有用的，以告知未来设计目标抑制剂的尝试。突变分析表明，循环残基两侧的几个碱基对于高复杂的稳定性至关重要，但是结构和化学映射实验都证实了这些碱基不是基本配对，不要参与分子内或分子间氢键，并且实际上似乎将其倒入溶液中。最后，在过渡态下观察到的分离距离太大，无法通过部分碱基对或存在间质水分子来解释。我们假设侧翼残基会通过调节局部离子环境来影响奈尼孔基对分解动力学。我们还预测，在过渡状态下部分脱水离子的释放构成了接吻环解离的速率限制步骤。使用显式离子，隐式溶剂蒙特卡洛模拟，将确定接吻环解离的动力学途径。将创建主要的分离途径的马尔可夫状态模型，然后使用大量的短而全原子的分子动力学模拟沿解离中间体进行详细检查。这些模拟将利用施加的外力来增强解离，与单分​​子拉力实验相似。通过比较预测的力扩展曲线，在过渡态下的分离，关键力和解离速率与实际的实验测量值，可以确定模拟的准确性。通过这种方式，将确定不成对的侧翼残基间接地通过离子介导的基础配对动力学在Loop-Loop界面上的离子介导的调节来促进复合物的整体机械稳定性。了解复合形成的离子介导的驱动力应可以更好地预测稳定和破坏稳定的突变，并识别特定的离子介导的相互作用，这些相互作用可能在小分子抑制剂的设计中可以利用。