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

细菌蛋氨酸 ABC 导入器的底物选择性和运输机制

• 批准号: 10668490
• 负责人: Janet G. Yang
• 金额: $ 10.92万
• 依托单位: UNIVERSITY OF SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668490
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Adopted; Affect; Ataxia; Attenuated; Bacillus anthracis; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Cell membrane; Cell physiology; Cells; Cellular Membrane; Chimera organism; Conflict (Psychology); Coupled; Coupling; Crystallography; Cystic Fibrosis; Cytoplasm; Data; Development; Disease; Disparate; Ensure; Environment; Enzymatic Biochemistry; Enzymes; Equilibrium; Escherichia coli; Fluorescence Anisotropy; Goals; Homeostasis; Human; Hydrolysis; Impairment; Individual; Infection; Integral Membrane Protein; Ions; Kinetics; Link; Measures; Mediating; Membrane; Methionine; Modeling; Multi-Drug Resistance; Nature; Nucleotides; Nutrient; Organism; Pathway interactions; Peptides; Periplasmic Proteins; Polysaccharides; Process; Property; Reaction; Role; Salmonella enterica; Sideroblastic Anemia; Site; Structural Models; Structure; System; Testing; Thermodynamics; Toxin; Transition Elements; Transport Process; Uncertainty; Virulence; Work; Yersinia pestis; biophysical techniques; combat; environmental change; experimental study; extracellular; flexibility; in vivo; insight; mutant; pathogenic bacteria; periplasm; proteoliposomes; response; tool; uptake

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.

项目概要 此外，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。 为了识别和导入关键的离子、营养物质和过渡金属，细胞还必须严格选择和 输出多种底物，包括肽、多糖和毒素。细胞采用 ATP 结合。 盒式 (ABC) 转运蛋白驱动底物相对于其浓度的单向转运 该提案旨在了解原核 ABC 进口商如何吸收至关重要的营养物质。 生存，这些发现可以为治疗细菌病原体提供新的目标。 为此，我们将采用生化和生物物理方法来剖析大肠杆菌 MetNI 转运蛋白（一种 建立的模型系统，将蛋氨酸从周质转运到细胞质，而最广泛的是。 公认的进口模型基于结构研究，新兴的功能研究表明， 然而，这些不同的模型似乎存在冲突； MetNI转运蛋白可以根据底物特征采用多种转运模式 我们将使用机械酶学工具来解决这些长期存在的问题并提出建议。 新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，我们将。 通过测量传输中各个步骤的动力学和热力学参数来检验我们的假设 使用不同的底物（L-Met、D-Met 和较大的蛋氨酸衍生物）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) 运输 我们的研究结果可以揭示 MetNI-Q 机制是。 本质上是通用的，并且可以根据底物优先选择不同的易位途径。 对于目标 2，我们生成了异二聚体 MetNI“嵌合体”，以破译两个相同的 ATP 位点是否起作用 具体来说，我们将确定是一个还是两个 ATP 分子。 每个运输周期都会被水解，并且如果运输效率受到一个 ATP 位点损伤的影响，那么总体而言， 我们的研究结果将为 ABC 转运蛋白如何选择不同底物以及 ATP 如何进行提供重要的见解 使用驱动底物跨膜转运。