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: 9019305
• 负责人: CHARALAMPOS KALODIMOS
• 金额: $ 38万
• 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 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9019305
• 关键词: Adopted; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacteriophages; Binding; Boxing; Cell physiology; Cells; Chloroplasts; Complement; Complex; Data; Detection; Event; Family; Ferrichrome; Goals; Gram-Negative Bacteria; Growth and Development function; Hydrogen; Iron; Isotope Labeling; Kinetics; Life; Ligands; Lipid Bilayers; MccJ25; Medical; Membrane; Membrane Proteins; Membrane Transport Proteins; Metals; Methodology; Micronutrients; Mitochondria; Molecular Conformation; Motion; Mutagenesis; 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; base; colicin; design; fascinate; in vivo; insight; interest; nanodisk; operation; pathogenic bacteria; periplasm; public health relevance; receptor; reconstitution; research study; 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（由适用提供）：基本化合物（例如分子和蛋白质）进入细胞的主动膜转运是细胞生理学中的一个基本过程，因此受许多不同转运途径的调节。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-桶蛋白，通常称为外膜蛋白（OMP），在货物运输和信号传导中起着基本功能。大而重要的OMP家族是TONB依赖性转运蛋白（TBDTS）。 TBDT主要参与铁吸收，这是一种金属，对于几乎所有活着组织的增长和发展至关重要。此外，TBDT还会运输自然存在的抗生素，绞痛和噬菌体。由于铁运输系统对于体内大量致病细菌的存活至关重要，因此TBDT是治疗干预的有吸引力的候选者。此外，目前正在生产抗生素，以靶向TBDT，并依靠它们在细胞内的运输。因此，出于医学和生物技术原因，该系统引起了极大的兴趣。许多研究集中在解剖通过TBDT孔通过底物运输的机制。 TBDT共享一个共同的结构，该结构由跨膜β-桶和一个全球域，即所谓的插头，它阻塞了枪管的管腔。 TBDT的周质N末端包含一个序列，即所谓的TONB盒子，该盒子募集了TONB的周质结构域。这个绑定事件对于运输绝对是必不可少的。在各种配体状态下，TBDT的50多个晶体结构都可以使用，但在所有配体状态下，孔总是被遮住。 TONB与TBDT的结合如何使底物易位仍然是一个谜。我们建议使用NMR光谱剖定原型TBDT FHUA中的变构相互作用并揭示运输机制。我们将获得物理底物与FHUA转运蛋白之间相互作用的综合结构，动态，动力学和热力学信息，并研究TONB如何实现底物转移。我们提供了非常强大的支持数据，即通过使用先进的NMR和同位素标记方法，在原子级别可以通过NMR来表征关键过程。我们的目标是：（i）确定NMR的FHUA结构动力学； （ii）表征TONB结合对FHUA结构和动力学的影响； （iii）表征了铁载体和抗菌辣椒的转运机制； （iv）表征核激素的运输机制。该提案中概述的特定目的的成功完成将为实现大型TBDT家族的底物运输的基本机制提供前所未有且引人入胜的见解。对这些蛋白质操作的结构和机械基础的全面描述将进一步提高我们对变构膜转运蛋白的功能及其调节方式的理解。