Dynamic characterization of outer-membrane-protein biogenesis by the BAM and TAM complexes

项目概要/摘要 传染病是世界上第二大死亡原因。新型抗生素药物 几乎不存在，病原菌对当前细菌的耐药性迅速增加，发展- 采用新的方法正成为推进人类健康工作的当务之急。分子建模 将在这些新方法中发挥重要作用，由于关键的原子尺度性质， 细菌蛋白质和抗菌剂功能的基础结构、过程和相互作用。 为了阐明这些结构和过程，PI将专注于革兰氏阴性菌的定义特征 细菌的第二层外膜，以及完整的膜蛋白是如何插入其中的。这些 外膜蛋白（OMPs），几乎所有这些都属于一个特定的类别称为桶，利用 两个关键的插入系统：对于生存能力至关重要的BAM系统，以及必要的相关TAM系统 毒力利用这些系统作为抗菌靶标需要全面了解 结构、动力学和功能之间的关系。在第一个目标中，一种新的机制， 每个系统的组成部分，BamA和TamA，分别通过增强自身的 - 桶将被评估。在分子动力学模拟中将产生插入的中间态 并通过二硫化物交联和电生理学测量进行实验测定。在 第二个目标，BamA和TamA如何扰乱膜，膜本身是插入过程的积极参与者，将 下定决心。模拟表明，存在一种膜缺陷，这种缺陷是由于一种异常的 BamA-桶的薄而不稳定的部分;改变这种扰动的突变，可能会减少 将通过计算机模拟预测OMP插入效率，并进行体内试验。最后，在第三个目标中， BamA和TamA以及BamA与其他BAM组分的相互作用将被表征。基于 通过观察到的构象变化，将选择限制构象变化的小分子候选药物。 稳定性和/或抑制BamA或TamA与其他复合物成员的结合。然后这些候选人将接受测试 抗菌活性的实验。

Project Summary/Abstract Infectious diseases are the second leading cause of death in the world. With novel classes of antibiotic drugs virtually nonexistent, and the resistance of pathogenic bacteria to current ones increasing rapidly, the develop- ment of new approaches is becoming an imperative for advancing human health efforts. Molecular modeling will play an essential role in these new approaches, due to the fundamentally atomic-scale nature of the critical structures, processes, and interactions underlying the function of both bacterial proteins and antibacterial agents. In order to illuminate these structures and processes, the PI will focus on a defining feature of Gram-negative bacteria, namely their second, outer membrane, and how integral membrane proteins are inserted there. These outer-membrane proteins (OMPs), practically all of which belong to a specific class known as -barrels, utilize two key systems for insertion: the BAM system, essential for viability, and the related TAM system, necessary for virulence. Exploiting these systems as antibacterial targets requires a comprehensive understanding of the relationship between structure, dynamics, and function. In the first aim, a novel mechanism in which the key component of each system, BamA and TamA, respectively, catalyzes insertion through augmentation of its own -barrel will be evaluated. Intermediate states of insertion will be generated in molecular dynamics simulations and assayed experimentally through both disulfide cross-linking and electrophysiology measurements. In the second aim, how BamA and TamA perturb the membrane, itself an active participant in the insertion process, will be determined. Simulations have indicated the existence of a membrane defect that forms due to an unusually thin and unstable part of the -barrel of BamA; mutations to alter this perturbation, and presumably decrease OMP insertion efficiency, will be predicted in silico and tested in vivo. Finally, in the third aim, the dynamics of BamA and TamA as well as BamA's interactions with other BAM components will be characterized. Based on the conformational changes observed, small-molecule drug candidates will be selected that limit conformational flexibility and/or inhibit binding of BamA or TamA to other complex members. These candidates will then be tested experimentally for antibacterial activity.