Abstract
The TonB dependent transporters (TBDTs) are sophisticated machineries which enable active transport across the outer membrane (OM) of Gram-negative bacteria by utilizing the inner membrane’s proton motive force through the TonB complex. Significant research has focused on understanding these transporters within isolated systems that do not truly mimic the physiological OM nor possess the required vital protein players for TBDTs to properly function. However, recently it has been possible to study a TBDT, the vitamin B12 transporter, BtuB, directly in bacteria using electron paramagnetic resonance (EPR) spectroscopy.
Here an alternative spin labeling approach was developed in order to explore BtuB expressed in metabolically active Escherichia coli using continuous wave (CW) and pulsed EPR techniques. We have successfully developed the approach to spin labeled single and double cysteine residues that face the extracellular space of BtuB without significant background labeling. Labeling pairs of spin labels in BtuB proved to be particularly challenging because it required the use of mutants that alter the redox homeostasis in the periplasm. We demonstrated the utility of our approach in several ways. First, single spin labels on BtuB were shown to produce intermolecular double electron-electron resonance (DEER) data that cannot be represented by a purely random distribution of the protein, and that the data are most consistent with a crowding or clustering of the protein in the OM. Such clustering has been a topic of considerable current interest as OM protein (OMP) clustering has been implicated as a mechanism to drive protein turnover in the bacterial OM. There is little direct information on the molecular interactions that drive this association and EPR provides an approach to explore these interactions. In a second set of experiments, double spin-labeled BtuB were used to explore novel conformational states associated with the transport process. The DEER data for a spin pair where one label (T188R1) is in an extracellular loop and a second label is within the protein core (V90R1) reveals two distance population where the short distance represents a previously unseen conformational state placing loop two of BtuB in close proximity to the core. Further investigations support that the core moves towards loop two and that this population increases in the presence of B12. Thus, this new conformational state is likely involved in the substrate transport and/or signal transduction processes. Third, we have shown that this system can be used to the study the binding and positioning of colicin E3, a bacteriocidal protein produced by E. coli that uses BtuB as its primary receptor to target and kill other E. coli. Based on our preliminary work using a short fragment of the colicin E3 receptor domain (E3R), the spin labeled R domain is found to bind to BtuB, both in live E. coli cells and in the reconstituted bilayer systems. The second extracellular loop is unstructured in the crystal structure; however, based on the preliminary DEER data, this loop is in close proximity to colicin E3R.
A final topic addressed in this thesis involves the mechanism of signal transduction through BtuB, which involves a substrate-dependent unfolding of the Ton box, which couples BtuB to TonB. During the signal transduction processes, and possibly during transport, ion pairs may act as molecular switches. Based on site directed spin labeling (SDSL) coupled to EPR, the R14-D316 pair acts as a conformational switch, which is involved in the TonB box unfolding process. However, five conserved ion pair (R36-D515, R47-D548, D53-R526, R69-E419, and R111-E465) mutants inhibit the TonB box unfolding event even in the presence of B12. Interestingly, the R69, R111, E419, and E465 residues may form interchangeable salt bridges, where the signal can be propagated sequentially. Some of these alanine mutants altered the substrate binding, which affected the TonB box unfolding. This work demonstrates that charged side chains and ion pairs within the BtuB play a critical role in the function of BtuB.
