Mechanisms of Substrate Selectivity and Transport by a Bacterial Methionine ABC Importer

项目摘要 所有生物体都必须有选择地调节分子从环境中的吸收和排出。此外 为了识别和输入关键离子、营养物质和过渡金属，细胞还必须严格选择和 出口多种底物，包括多肽、多糖和毒素。细胞采用三磷酸腺苷结合 盒(ABC)转运体，根据底物的浓度驱动底物的单向传输 渐变。这项建议旨在了解原核生物ABC进口商如何吸收至关重要的营养 这些发现可能为治疗细菌病原体提供新的靶点。朝向 为此，我们将使用生化和生物物理方法来剖析大肠杆菌Metni转运蛋白和 建立模型系统，将蛋氨酸从周质运输到细胞质。而最广泛的- 公认的进口模型是基于结构研究，新兴的功能研究表明 底物转运模式不同。这些不同的模型似乎相互冲突；然而，我们假设 Metni转运体可以采用多种运输方式以响应衬底特征和 可用性。我们将使用机械酶学的工具来解决这些长期存在的问题，并提出 可能合并以前的发现或揭示全新的行动机制的新模型。在目标1中，我们将 通过测量运输过程中各个步骤的动力学和热力学参数来检验我们的假设 周而复始。使用不同的底物(L蛋氨酸、D-蛋氨酸和更大的蛋氨酸衍生物)和结合受损 对于突变体，我们将剖析三个步骤：(A)Metq同源周质蛋白与Metni转运蛋白的结合， (B)ATP结合和水解，以及(C)转运。我们的发现可能揭示出Metni-Q机制是 固有的多功能性，可以根据底物优先选择不同的转位途径。 对于目标2，我们已经产生了异二聚体Metni“嵌合体”来破译两个相同的ATP位点是否起作用 共同实现高效和具体的运输。具体地说，我们将确定一个或两个ATP分子 如果运输效率因一个ATP位点的损伤而受到影响，则在每个运输周期内都会被水解。总的来说， 我们的发现将为ABC转运蛋白如何选择不同底物以及ATP如何选择提供关键的见解 用法促使底物跨膜转运。

Project Summary All organisms must selectively regulate the uptake and extrusion of molecules from the environment. In addition to identifying and importing crucial ions, nutrients, and transition metals, cells must also rigorously select and export a wide range of substrates, including peptides, polysaccharides, and toxins. The cell employs ATP-binding cassette (ABC) transporters to drive the unidirectional transport of substrates against their concentration gradients. This proposal seeks to understand how prokaryotic ABC importers uptake nutrients that are crucial for survival, and these findings could provide new targets for treatment against bacterial pathogens. Towards this end, we will employ biochemical and biophysical methods to dissect how the E. coli MetNI transporter, an established model system, transports methionine from the periplasm to the cytoplasm. While the most widely- accepted model for import is based on structural studies, emerging functional studies suggest a substantially different model for substrate translocation. These disparate models appear to conflict; however, we hypothesize that the MetNI transporter can adopt multiple modes of transport in response to substrate features and availability. We will use the tools of mechanistic enzymology to resolve these longstanding issues and to propose new models that may merge previous findings or uncover entirely new mechanisms of action. In Aim 1, we will test our hypothesis by measuring the kinetic and thermodynamic parameters of individual steps in the transport cycle. Using different substrates (L-Met, D-Met, and larger methionine derivatives) and binding-impaired mutants, we will dissect three steps: (a) binding of the MetQ cognate periplasmic protein to the MetNI transporter, (b) ATP binding and hydrolysis, and (c) transport. Our findings could reveal that the MetNI-Q mechanism is inherently versatile and could preferentially select different translocation pathways depending on the substrate. For Aim 2, we have generated heterodimeric MetNI “chimeras” to decipher if the two identical ATP sites work together to enable efficient and specific transport. Specifically, we will determine if one or two ATP molecules are hydrolyzed per transport cycle, and if transport efficiency is affected by impairment of one ATP site. Overall, our findings will yield critical insights into how ABC transporters select for different substrates, and how ATP usage drives substrate translocation across membranes.