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

项目摘要 所有生物体都必须选择性地调节从环境中吸收和排出分子。此外 为了识别和输入关键的离子、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，包括肽、多糖和毒素。细胞利用ATP结合 盒（ABC）转运蛋白，以驱动底物逆其浓度单向转运 梯度。这项提议旨在了解原核生物ABC进口商如何摄取至关重要的营养物质 这些发现可以为治疗细菌病原体提供新的靶点。朝向 为此，我们将采用生物化学和生物物理的方法来剖析如何E。大肠杆菌MetNI转运蛋白， 建立的模型系统，将蛋氨酸从周质转运到细胞质。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， 不同的底物转运模式。这些不同的模型似乎相互冲突;然而，我们假设 MetNI转运蛋白可以响应于底物特征而采用多种转运模式， 空房的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 新的模型可能会合并以前的发现或揭示全新的作用机制。在目标1中，我们 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 周期使用不同的底物（L-Met、D-Met和较大的甲硫氨酸衍生物）和结合受损 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，和（c）运输。我们的研究结果可以揭示MetNI-Q机制是 固有地通用，并且可以根据底物优先选择不同的易位途径。 对于目标2，我们已经产生了异二聚体MetNI“嵌合体”，以破译两个相同的ATP位点是否起作用。 以实现高效和特定的运输。具体来说，我们将确定是否有一两个ATP分子 每个运输周期水解，如果运输效率受到一个ATP位点受损的影响。总体而言， 我们的发现将对ABC转运蛋白如何选择不同的底物以及ATP如何选择不同的底物产生重要的见解。 使用驱动底物跨膜移位。

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.