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）：将分子和蛋白质等必需化合物主动膜转运至细胞内是细胞生理学中的基本过程，因此受到许多不同转运途径的调节。革兰氏阴性细菌、线粒体和叶绿体的外膜 (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发挥重要作用。一个重要的 OMP 家族是 TonB 依赖性转运蛋白 (TBDT)。 TBDT 主要参与铁的吸收，铁是几乎所有生物体生长和发育所必需的金属。此外，TBDT 还转运天然存在的抗生素、大肠菌素和噬菌体。由于铁转运系统对于体内大量病原菌的生存至关重要，因此 TBDT 是治疗干预的有吸引力的候选者。此外，目前正在生产针对 TBDT 的抗生素，并依赖它们在细胞内运输。因此，该系统因医学和生物技术原因引起了极大的兴趣。许多研究都集中在剖析底物通过 TBDT 孔传输的机制。 TBDT 具有共同的结构，由跨膜 β 桶和球状结构域（即所谓的塞子）组成，该球状结构闭塞桶的内腔。 TBDT 的周质 N 末端包含一个序列，即所谓的 TonB 盒，它招募 TonB 的周质结构域。这种结合事件对于运输来说是绝对必要的。有超过 50 种不同配位态的 TBDT 晶体结构可供选择，但所有这些晶体结构的孔总是被封闭的。 TonB 与 TBDT 结合如何实现底物易位仍然是个谜。我们建议使用核磁共振波谱来剖析变构相互作用并揭示 FhuA（一种原型 TBDT）中的传输机制。我们将获得生理底物与 FhuA 转运蛋白之间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 如何实现底物易位。我们提供了非常有力的支持数据，表明关键过程可以通过使用先进的 NMR 和同位素标记方法在原子水平上通过 NMR 进行表征。我们的目标是：（i）通过 NMR 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) 表征大肠菌素的转运机制。成功完成本提案中概述的具体目标将为大家族 TBDT 实现基质传输的基本机制提供前所未有的、令人着迷的见解。对这些蛋白质运作的结构和机制基础的全面描述将进一步加深我们对变构膜转运蛋白如何发挥作用及其如何调节的理解。