Allosteric and Transport Mechanisms in TonB-dependent Transporters

TonB 依赖性转运蛋白的变构和转运机制

• 批准号: 9188052
• 负责人: CHARALAMPOS KALODIMOS
• 金额: $ 38.1万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9188052
• 关键词: Adopted; Affinity; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacteriophages; Binding; Biotechnology; Cell physiology; Cells; Chloroplasts; Complement; Complex; Crystallization; Data; Detection; Event; Family; Ferrichrome; Goals; Gram-Negative Bacteria; Growth and Development function; Hydrogen; Iron; Isotope Labeling; Kinetics; Ligands; Lipid Bilayers; MccJ25; Medical; Membrane; Membrane Proteins; Membrane Transport Proteins; Metals; Methodology; Micronutrients; Mitochondria; Molecular Conformation; Motion; Mutagenesis; Mutation; NMR Spectroscopy; Organism; Pathway interactions; Peptides; Physiological; Physiology; Population; Process; Proteins; Recruitment Activity; Relaxation; Relaxation Techniques; Reporting; Role; Side; Siderophores; Signal Transduction; Structure; System; Techniques; Testing; Therapeutic Intervention; Thermodynamics; Time; Transmembrane Transport; colicin; design; experimental study; fascinate; in vivo; insight; interest; nanodisk; operation; pathogenic bacteria; periplasm; public health relevance; receptor; reconstitution; therapeutic candidate; uptake

 DESCRIPTION (provided by applicant): Active membrane transport of essential compounds such as molecules and proteins into the cell is a fundamental process in cellular physiology and is thus regulated by a number of different transport pathways. Gram-negative bacteria, mitochondria, and chloroplasts contain transmembrane β-barrel proteins on their outer membrane (OM), commonly referred to as outer membrane proteins (OMPs), that serve essential functions in cargo transport and signaling. A large and important family of OMPs are the TonB-dependent transporters (TBDTs). TBDTs are involved primarily in iron uptake, a metal that is essential for the growth and development of almost all living organisms. In addition, TBDTs also transport naturally occurring antibiotics, colicins and phages. Because iron transport systems are critical for the survival of a large number of pathogenic bacteria in vivo, TBDTs are attractive candidates for therapeutic intervention. Moreover, antibiotics are currently being produced that target TBDTs and rely on them for their transport inside the cell. Therefore, this system has attracted tremendous interest for medical and biotechnological reasons. Numerous studies have focused on dissecting the mechanisms underpinning transport of substrates through the pore of TBDTs. TBDTs share a common structure consisting of a transmembrane β-barrel and a globular domain, the so-called plug, that occludes the lumen of the barrel. The periplasmic N terminus of TBDTs contains a sequence, the so-called TonB box, which recruits the periplasmic domain of TonB. This binding event is absolutely essential for the transport. Over 50 crystal structures of TBDTs in various liganded states are available but in all of them the pore is always occluded. How TonB binding to TBDTs enables substrate translocation remains a mystery. We propose to use NMR spectroscopy to dissect the allosteric interactions and unravel the transport mechanisms in FhuA, a prototypic TBDT. We will obtain integrated structural, dynamic, kinetic and thermodynamic information of the interaction between physiological substrates and the FhuA transporter and study how TonB enables substrate translocation. We present very strong supporting data that the key processes can be characterized by NMR at the atomic level by the use of advanced NMR and isotope labeling methodologies. We aim to: (i) determine the structural dynamics of FhuA by NMR; (ii) characterize the effect of TonB binding on the structure and dynamics of FhuA; (iii) characterize the transport mechanisms of siderophores and antibacterial peptides; (iv) characterize the transport mechanisms of colicins. Successful completion of the specific aims outlined in this proposal will provide unprecedented and fascinating insight into the fundamental mechanisms that enable substrate transport by the large family of TBDTs. A comprehensive description of the structural and mechanistic basis of operation of these proteins will further advance our understanding of how allosteric membrane transporters function and how they are regulated.

 描述(由申请人提供)：分子和蛋白质等基本化合物的主动膜转运进入细胞是细胞生理学中的一个基本过程，因此受到许多不同的运输途径的调节。革兰氏阴性细菌、线粒体和叶绿体在其外膜(OM)上含有跨膜β-Barrel蛋白，通常被称为外膜蛋白(OMP)，在货物运输和信号传递中发挥重要功能。一个重要的OMP家族是TonB依赖的转运蛋白(TonB-Dependent Transporter，TBDTs)。TBDTs主要参与铁的吸收，这是一种对几乎所有生物的生长和发育都至关重要的金属。此外，TBDTs还运输自然产生的抗生素、粘菌素和噬菌体。由于铁转运系统对体内大量病原菌的生存至关重要，因此TBDTs是治疗干预的有吸引力的候选对象。此外，目前正在生产针对TBDTs的抗生素，并依赖于它们在细胞内的运输。因此，由于医学和生物技术的原因，该系统引起了极大的兴趣。许多研究都集中在剖析底物通过TBDTs孔道运输的机制。β-TBT共用一个共同的结构，由一个跨膜的TBDTs管和一个球状结构域组成，即所谓的塞子，它堵塞了管腔。TbDTs的周质N末端含有一个序列，即所谓的TonB盒，它招募TonB的周质结构域。这个绑定事件对于传输是绝对必要的。有50多种不同配位状态的TBDTs的晶体结构可用，但所有的晶体结构中的孔总是被封闭的。TonB如何与TBDTs结合使底物易位仍然是一个谜。我们建议使用核磁共振波谱来分析变构相互作用，并揭示原型TBDT-FhuA的输运机制。我们将获得生理底物与FhuA转运蛋白相互作用的综合结构、动力学、动力学和热力学信息，并研究TonB如何实现底物转运。我们提供了非常有力的支持数据，通过使用先进的核磁共振和同位素标记方法，可以在原子水平上用核磁共振来表征关键过程。我们的目标是：(I)通过核磁共振确定FhuA的结构动力学；(Ii)表征TonB结合对FhuA结构和动力学的影响；(Iii)表征铁载体和抗菌肽的转运机制；(Iv)表征结肠素的转运机制。成功完成本提案中概述的SPECIfic目标将为实现TBDT大家族底物运输的基本机制提供前所未有的、令人着迷的见解。对这些蛋白的结构和机制的全面描述将进一步促进我们对变构转运蛋白如何发挥作用以及它们如何被调控的理解。