Connecting structure and fitness landscapes to overcome antibiotic resistance

项目总结-将结构和健身环境连接起来以克服抗生素耐药性 抗生素耐药性是一个紧迫的、多方面的挑战。病原体进化超过了新病原体的供应 化合物和类似物，威胁着全球健康危机。理解适应的新方法是 显然是必要的，但这是一个困难的问题。耐药的机制通常是未知的，以及 产生整体微生物适应性变化的变化的总体组合。中国的技术发展 高通量的生物化学使大量的变异库能够被检测，这为 构建抗药性的预测模型，但在我们最近的工作之前，这些模型忽略了复杂的突变， 特别是插入和删除，它们通过对基础内容产生重大更改而发挥巨大作用 具有微小突变的健身景观。为了研究如何，我们将结合实验进化，深入 突变扫描和多温度结晶学，以产生如何插入的集成模型 而缺失可以使蛋白质功能迅速改变。 我们将使用链球菌素A家族作为我们的模型抗生素。这些都是核糖体靶向化合物。 由链霉菌生产。抗性是通过VAT蛋白发生的，这种蛋白特别是失活 链霉素A(SA)通过乙酰化。与加州大学旧金山分校Seiple实验室的合作导致了一个模块化的 可以简单地产生变体的SA的合成，以及几种具有以下特征的新化合物 在体外显示乙酰化程度降低。我们将确定如何适应这些新化合物 以及关键可变底物结合环内的Indels如何对其进行调制。 在我的第一个目标中，我将用实验进化来揭示VAT蛋白如何适应链球菌，以及如何 在此循环中添加插入和删除会改变自适应潜力。在我的第二个目标中，我将 进行高通量稳定性测量以确定阻力变化的机理基础， 然后使用深度突变扫描来测量突变的可及性和生物物理基础 进化的适应轨迹。在我的第三个目标中，我将使用低温和多温结晶学来确定 INDELL增强底物对新SA的专一性改变的静态和动态基础。 这些目标的完成将产生微生物适应的综合图景和基本的新的 洞察复杂但未被充分研究的突变如何从根本上改变基因的适应潜力。十字架- 该项目的纪律性质将使我接触到一些技术和方法，这些技术和方法将为我 在领域定义专家的指导下，有极好的培训机会。新的框架和 我将开发的技术将有助于建立我的科学成熟度，因为我追求我的目标是独立的 大学研究分子蛋白质进化的机制基础的研究职位。

Project summary - Connecting structure and fitness landscapes to overcome antibiotic resistance Antibiotic resistance is a pressing, multifaceted challenge. Pathogen evolution is outstripping the supply of new compounds and analogues, threatening a global health crisis. New approaches to understand adaptation are clearly necessary, but this is a difficult problem. The mechanisms of resistance are often unknown, as well as the overall combination of changes that produce overall microbial fitness changes. Technical developments in high-throughput biochemistry have allowed massive variant libraries to be assayed, which opens the door to constructing predictive models of resistance, but these have until our recent work ignored complex mutations, specifically insertions and deletions, which play a massive role by producing major changes to underlying fitness landscapes with small mutations. To study how, we will combine experimental evolution, deep mutational scanning, and multitemperature crystallography to produce an integrated model for how insertions and deletions permit rapid changes to protein function. We will use the Streptogramin A family as our model antibiotic. These are ribosome-targeting compounds produced by Streptomyces. Resistance occurs through Vat proteins, which specifically inactivate Streptogramin A (SA) via acetylation. A collaboration with the Seiple lab at UCSF has led to a modular synthesis of SA that allows variants to be simply produced, as well as several novel compounds with demonstrated reduced acetylation in vitro. We will determine how adaptation to these novel compounds proceeds, and how indels within a key variable substrate-binding loop modulate it. In my first aim, I will use experimental evolution to uncover how Vat proteins adapt to streptogramins, and how the addition of insertions and deletions within this loop change the adaptive potential. In my second aim, I will conduct high-throughput stability measurements to determine the mechanistic basis for resistance changes, and then use deep mutational scanning to measure the mutational accessibility and biophysical basis for an evolved adaptive trajectory. In my third aim, I will use cryo- and multitemperature crystallography to determine the static and dynamic basis for indel-potentiated changes to substrate specificity towards new SAs. The completion of these aims will produce an integrated picture of microbial adaptation and essential new insight into how complex but understudied mutations radically shift the adaptive potential of genes. The cross- disciplinary nature of the project will expose me to a number of techniques and approaches that will provide me with excellent training opportunities under the mentorship of field-defining experts. The new frameworks and techniques I will develop will help establish my scientific maturity as I pursue my goal of an independent research position studying the mechanistic basis of molecular protein evolution at a university.