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(申请人提供)：本项目的目标是揭示逆转录病毒RNA吻环复合体离子介导相互作用的物理基础，并解释实现最大机械稳定性的不寻常序列要求；特别是艾滋病毒和莫洛尼白血病病毒(MMLV)的二聚化起始点(DIS)。众所周知，这种高度的稳定性对逆转录病毒基因组二聚化至关重要，因为DIS环的突变总是会导致体内病毒复制和传染性大大降低。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逆转录病毒治疗药物的成功途径。然而，目前针对该界面的尝试实际上已经导致了亲吻环稳定性的提高，而没有检测到对病毒复制的抑制。因此，确定增强的接吻环稳定性的物理基础将是有用的，以便为未来设计靶向抑制剂的尝试提供信息。突变分析表明，环残基两侧的几个碱基对高络合物稳定性至关重要，但结构和化学图谱实验都证实，这些碱基不是碱基配对的，不参与分子内或分子间氢键，实际上似乎翻出到溶液中。最后，观察到的过渡态的分离距离太大，不能用部分碱基配对或间隙水分子的存在来解释。我们假设侧翼残基通过调节局部离子环境来影响邻近碱基对的解离动力学。我们还预测，部分脱水离子在过渡态的释放构成了吻环解离的限速步骤。利用显式离子、隐式溶剂蒙特卡罗模拟，确定了接吻环解离的动力学路径。我们将建立一个主要解离路径的马尔可夫状态模型，然后使用大量沿解离中间产物跃迁的短的、全原子的分子动力学模拟来详细研究该模型。这些模拟将利用施加的外力来加强解离，类似于单分子拉动实验。模拟的准确性将通过将预测力-伸长曲线、过渡态分离、临界力和离解率与实际实验测量结果进行比较来确定。这样，就可以确定未配对的侧翼残基通过离子介导的环-环界面碱基配对动力学的调节，间接对复合体的整体机械稳定性做出贡献的程度。了解离子介导的复合体形成的驱动力应该能够更好地预测稳定和不稳定的突变，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利用。