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: 10491175
• 负责人: Janet G. Yang
• 金额: $ 10.82万
• 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/10491175
• 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; Crystallization; Crystallography; Cystic Fibrosis; Cytoplasm; Data; Development; Disease; 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; Workplace; Yersinia pestis; base; 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.

项目摘要 所有生物必须选择性地调节环境分子的摄取和扩展。此外 为了识别和进口至关重要的离子，养分和过渡金属，细胞也必须严格选择和 导出广泛的底物，包括肽，多糖和毒素。细胞员工ATP结合 纸盒（ABC）转运蛋白以驱动基材的单向运输抗浓度 梯度。该提案试图了解原核生物ABC进口商如何摄取至关重要的营养 为了生存，这些发现可以为针对细菌病原体的治疗提供新的靶标。向 这一目的，我们将采用生化和生物物理方法来剖析大肠杆菌metni转运蛋白的如何 已建立的模型系统将方法从周期传输到细胞质。虽然最广泛 - 导入的公认模型基于结构研究，新兴的功能研究表明 底物易位的不同模型。这些不同的模型似乎发生了冲突。但是，我们假设 Metni运输蛋白可以采用多种运输模式来响应底物特征和 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建议 可能会合并以前的发现或发现全新作用机理的新模型。在AIM 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 循环。使用不同的底物（L-MET，D-MET和较大的甲糖衍生物）并结合受损 突变体，我们将剖析三个步骤：（a）Metq同源蛋白与metni转运蛋白的结合， （b）ATP结合和水解，以及（c）运输。我们的发现可能揭示了metni-Q机制是 固有的用途，可以优先选择不同的易位途径，具体取决于基板。 对于AIM 2，我们已经生成了异二聚体metni“嵌合体”，以破译两个相同的ATP站点工作 共同实现高效和特定的运输。具体而言，我们将确定一个或两个ATP分子是否 是每个运输周期水解的，如果运输效率受一个ATP部位损害的影响。全面的， 我们的发现将对ABC转运蛋白如何为不同的底物选择以及ATP产生关键见解 用法驱动跨膜的底物易位。