Allosteric and Transport Mechanisms in TonB-dependent Transporters

 描述（由申请人提供）：必需化合物（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、线粒体和叶绿体在其外膜（OM）上含有跨膜β-桶蛋白，通常称为外膜蛋白（OMP），其在货物运输和信号传导中起重要作用。一个重要的外膜蛋白家族是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还运输天然存在的抗生素，大肠杆菌素和抗生素。由于铁转运系统对于体内大量病原菌的存活至关重要，因此TBDT是治疗干预的有吸引力的候选者。此外，目前正在生产针对TBDT的抗生素，并依赖它们在细胞内运输。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。大量的研究集中在解剖的机制，通过TBDT的孔隙运输基板的基础。TBDT共有一个共同的结构，由跨膜β-桶和球状结构域组成，即所谓的塞子，其堵塞桶的内腔。TBDTs的周质N末端含有一个序列，即所谓的TonB盒，它募集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TBDT晶体结构，但所有这些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NMR光谱来解剖的变构相互作用，并解开在FhuA，一个原型TBDT的运输机制。我们将获得完整的结构，动力学，动力学和热力学信息之间的相互作用的生理底物和FhuA转运蛋白和研究如何TonB使底物易位。我们提出了非常强有力的支持数据，关键过程可以通过NMR在原子水平上使用先进的NMR和同位素标记方法的特点。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的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）表征大肠杆菌素的转运机制。成功完成本提案中概述的具体目标将为TBDT大家族实现基板传输的基本机制提供前所未有的迷人见解。这些蛋白质的结构和机制的运作基础的全面描述将进一步推进我们的了解如何变构膜转运蛋白的功能和它们是如何调节。

 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.