Translocation through TonB-dependent transporters

通过依赖于 TonB 的转运蛋白进行易位

• 批准号: 8023328
• 负责人: Karen Jakes
• 金额: $ 16.6万
• 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8023328
• 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; 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. PUBLIC HEALTH RELEVANCE: The TonB-dependent receptors used for colicin uptake are used by virtually all Gram-negative bacteria, including pathogens like Yersinia pestis, to acquire iron, essential to their viability. Better understanding the mechanisms that mobilize that uptake will allow design of means to inhibit that essential process.

描述（由申请人提供）：通过TONB依赖性转运蛋白大肠杆菌细菌的易位，以及诸如Yersinia Pestis等病原体，通过一组称为配体的外膜受体受体蛋白（TONB依赖性转运蛋白）获得了必需的营养素，例如铁和维生素B12。这些受体共有一个共同的结构；它们都是22链的B桶，带有插头，面向外膜的内部。通过这些受体的营养吸收取决于通过TONB蛋白从内膜转导的能量转导，这使得与受体插头域上称为TONB盒的序列接触，以将其移开并允许底物进入。蛋白质毒素称为大肠杆菌杀死其他大肠杆菌的蛋白质毒素已将许多依赖性tonb依赖性转运蛋白作为其外膜受体，以附着于靶细菌上。然后，他们必须在一个称为易位的过程中越过外膜，以在目标细胞内传递其毒性结构域。最近发现，Colicin IA使用其TONB依赖性外膜受体CIR，不仅是其主要受体，也附着在细胞表面上，而且还作为其在整个膜上的转运剂。结肠素与引发易位的CIR的相互作用是通过其N末端易位或T，域的域，而不是通过与Colicin的受体结合域的特征性相互作用。该建议旨在使用结肠蛋白的易位结构域作为探针，以识别导致CIR受体开放或拔下t域的最小信号，以响应T结构域的结合，这一过程被认为可以模仿其自然底物。结合与受体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识别。突变体易位域肽将用于在各个步骤（通过结合伴侣的交联）中冻结易位过程，以便更好地理解结肠素与其转运剂结合并触发其开口的整个过程。 公共卫生相关性：几乎所有革兰氏阴性细菌（包括诸如Yersinia Pestis）的病原体，用于获得铁的所有革兰氏阴性细菌，用于获取铁，对它们的生存能力所必需。更好地理解动员吸收的机制将允许设计手段以抑制这一基本过程。