Conformational Dynamics of the Escherichia coli TonB Protein and Implications for Energy Transduction
Open Access
- Author:
- Gresock, Michael George
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 06, 2014
- Committee Members:
- Kathleen Postle, Dissertation Advisor/Co-Advisor
Kathleen Postle, Committee Chair/Co-Chair
Donald Ashley Bryant, Committee Member
B Tracy Nixon, Committee Member
Lorraine C Santy, Committee Member
Timothy W Mcnellis, Committee Member - Keywords:
- TonB
FepA
signal transduction
iron transport - Abstract:
- To cross the outer membrane of Gram-negative bacteria such as Escherichia coli, large, scarce, and important nutrients such as iron or vitamin B12 first bind to specific outer membrane transporters. A complex of the cytoplasmic membrane proteins ExbB, ExbD, and TonB transduces energy derived from the cytoplasmic membrane protonmotive force (PMF) to permit ligand release into the periplasm, with TonB physically contacting the outer membrane transporter. Although no individual sidechain in the E. coli TonB is irreplaceable, TonB consists of two essential domains: a transmembrane domain (TMD) that interacts with ExbB and ExbD and a periplasmic carboxy-terminal domain that interacts with outer membrane transporters. The mechanisms by which TonB facilitates ligand transport across the outer membrane have been a matter of considerable debate. In one model, the TonB TMD delivers energy to transporters by “shuttling” between cytoplasmic and outer membranes, a process that would require the TMD to be removed from the cytoplasmic membrane. Alternatively, several mechanical models suggest that TonB always remains anchored in the cytoplasmic membrane during energy transduction. The ideal test of the shuttle model is to fuse a stable protein domain to the amino terminus of TonB to prevent it from shuttling and determine if the fusion retains activity. In this study, a ToxR-TonB fusion was proteolytically stable, retained the ability to form PMF-independent and PMF-dependent interactions with ExbD and exhibited wild-type ferrichrome transport rates. Therefore, TonB does not shuttle in vivo. The observation that the TonB TMD did not shuttle suggested that its primary role was to mediate assembly of TonB with ExbB, ExbD, or another TonB. TonB was previously known to dimerize in vivo, yet the contributions of TMD sidechains (e.g. H20) to dimerization were relatively unexplored. Here, an inactive TonB H20A substitution categorically reduced dimerization through the TonB carboxy terminus. TonB H20A also prevents association between TonB and ExbD. Since ExbD forms homodimers before associating with TonB, the results are consistent with the idea that TonB forms homodimers before forming TonB-ExbD heterodimers. Disulfide-linked TonB dimers formed through TonB F125C, located in a disordered region of the periplasmic domain, could fractionate with the outer membrane whereas dimers formed within TonB carboxy-terminal residues 186-230 were unable to associate with the outer membrane. This suggests that dimers formed through TonB residues 186-230 need to be resolved for this region to associate with the outer membrane, which is consistent with the suggestion that the dimers form early in an energy transduction cycle. Finally, this work identified sites of functionally significant interaction between TonB and the FepA cork domain using in vivo photocrosslinking with the photoreactive amino acid p-benzoyl-l-phenylalanine (pBpa). Each of the pBpa substitutions that could crosslink to TonB are located in regions of FepA that are exposed to the periplasm in the FepA crystal structure, suggesting that TonB does not contact other areas of the cork in vivo. The presence of enterochelin strengthened crosslinking through some positions but diminished crosslinking through FepA I14pBpa, V28pBpa and T32pBpa. This suggests that TonB binds more efficiently to these three sidechains in the absence of ligand, and once ligand binds, the transporter undergoes a conformational rearrangement to bind different sites. Deletion of the TonB box did not reduce crosslinking through FepA T32pBpa or I145pBpa, suggesting that formation of these complexes does not require a functional transporter. Overall, this work extended knowledge of how the TonB TMD and carboxy-terminal domain participate in early and late stages of energy transduction and provided more insight into TonB-transporter interactions beyond the characterized TonB-TonB box interaction.