Translocation through TonB-dependent transporters

通过依赖于 TonB 的转运蛋白进行易位

• 批准号: 8580549
• 负责人: Karen Jakes
• 金额: $ 15.42万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8580549
• 关键词: Antibiotics; Bacteria; Binding; Biological Warfare; Boxing; C-terminal; Calorimetry; Cell membrane; Cell surface; Cells; Colicin Ia; Complex; Cytosol; Epitopes; Escherichia coli; Face; Family; Freezing; Goals; Gram-Negative Bacteria; Iron; Lead; Ligands; Measurement; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; N-terminal; Nature; Nutrient; Pathway interactions; Process; Protein C; Proteins; Research; Side; Signal Transduction; Structure; Tertiary Protein Structure; Testing; Toxin; Vitamin B 12; Vitamins; Work; Yersinia pestis; colicin; crosslink; deletion analysis; design; killings; mutant; novel; pathogen; periplasm; protein protein interaction; public health relevance; receptor; receptor binding; research study; response; small molecule; uptake

DESCRIPTION (provided by applicant): Translocation through TonB-dependent transporters E. coli bacteria, as well as pathogens such as Yersinia pestis, acquire essential nutrients such as iron and vitamin B12 through a group of ligand-gated outer membrane receptor proteins called TonB-dependent transporters. These receptors share a common structure; they are all 22-stranded b-barrels with a plug that faces the inside of the outer membrane. Nutrient uptake through these receptors depends on energy transduction from the inner membrane via the TonB protein, which makes contact with a sequence called the TonB box on the plug domain of the receptor, to move it out of the way and allow substrate entry. Protein toxins called colicins that are made by some E. coli to kill other E. coli have co-opted a number of TonB-dependent transporters as their outer membrane receptors, to attach to target bacteria. They must then cross the outer membrane, in a process called translocation, to deliver their toxic domain inside the target cell. It was recently discovered that colicin Ia uses its TonB-dependent outer membrane receptor, Cir, not just as its primary receptor, to attach to the cell surface, but also as its translocator across the membrane. The interaction of the colicin with Cir that initiates translocation is via its N-terminal translocation, or T, domain, rather than via the better-characterized interaction with the colicin's receptor-binding domain. This proposal is aimed at using the translocation domain of the colicin as a probe to identify the minimal signals that lead to the opening or unplugging of the Cir receptor in response to T domain binding, a process believed to mimic the opening of the receptors by their natural substrates. The minimal translocation peptide domain(s) that bind to the receptor will be identified by deletion analysis and isothermal calorimetry binding measurements. Mutant translocation-domain peptides will be used to freeze the translocation process at various steps, with cross-linking of binding partners, in order to better understand the entire process by which the colicin binds to its translocator and triggers its opening.

描述（由申请人提供）：通过 TonB 依赖性转运蛋白进行易位 大肠杆菌以及鼠疫耶尔森氏菌等病原体通过一组称为 TonB- 的配体门控外膜受体蛋白获取铁和维生素 B12 等必需营养物质。依赖性转运蛋白。这些受体具有共同的结构；它们都是 22 股 b 桶，带有面向外膜内侧的插头。通过这些受体的营养吸收取决于通过 TonB 蛋白从内膜进行的能量转导，TonB 蛋白与受体插头结构域上称为 TonB 盒的序列接触，将其移开并允许底物进入。一些大肠杆菌产生的称为大肠菌素的蛋白质毒素可以杀死其他大肠杆菌，它选择了许多依赖于 TonB 的转运蛋白作为其外膜受体，以附着在目标细菌上。然后，它们必须穿过外膜，在一个称为易位的过程中，将其毒性结构域传递到靶细胞内。最近发现，大肠杆菌素 Ia 使用其 TonB 依赖性外膜受体 Cir 不仅作为其主要受体附着在细胞表面，而且还作为其跨膜转运蛋白。引发易位的大肠菌素与 Cir 的相互作用是通过其 N 端易位（或 T）结构域，而不是通过与大肠菌素受体结合结构域的更好表征的相互作用。该提案旨在使用大肠菌素的易位结构域作为探针来识别导致 Cir 受体响应 T 结构域结合而打开或拔出的最小信号，这一过程被认为是通过其天然基质。与受体结合的最小易位肽结构域将通过缺失分析和等温量热法结合测量来鉴定。突变的易位结构域肽将用于冻结易位过程的各个步骤，并与结合配偶体交联，以便更好地了解大肠菌素与其易位蛋白结合并触发其打开的整个过程。