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 ?-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 ?-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 speci?c 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.
TonB-dependent transport is of high medical importance because the survival of pathogenic bacteria in their hosts depends on their ability to compete for micronutrients such as iron. The possibility of developing antibiotics that penetrate bacteria ef?ciently through high af?nity iron transport receptors is very attractive. Understanding the mechanisms of function of these transporters is crucial towards this goal.
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