- Ollis, Anne A.
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Doctor of Philosophy
- Document Type:
- Date of Defense:
- August 09, 2011
- Committee Members:
- Kathleen Postle, Dissertation Advisor/Co-Advisor
Kathleen Postle, Committee Chair/Co-Chair
Sarah Ellen Ades, Committee Member
B Tracy Nixon, Committee Member
Lorraine C Santy, Committee Member
William O Hancock, Committee Member
- In Escherichia coli K12, the TonB system serves to couple the electrochemical gradient of the cytoplasmic membrane, protonmotive force (pmf), to high affinity TonB-gated outer membrane transporters, promoting active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Iron is essential for E. coli but bioavailable iron is scarce in the environment, making this a critical process in the competition for survival of these cells. In other Gram negative bacterial species, TonB energizes transport of heme, maltodextrin, sucrose, and nickel. The precise mechanism of TonB-dependent energy coupling is unknown and studies aim to elucidate the details involved in this process. In the cytoplasmic membrane, TonB is thought to form a hetero-oligomeric complex with two other integral membrane proteins, ExbB and ExbD. ExbB and ExbD appear to harness the pmf to energize TonB. TonB spans the periplasm, retaining cytoplasmic membrane association, directly contacts OM transporters, and transmits energy for release of ligands into the periplasm. ExbD and TonB each have a single transmembrane domain (TMD), with the majority of each protein occupying the periplasm, while ExbB has three transmembrane domains with the majority of soluble domains in the cytoplasm. TonB is a conformationally dynamic protein, and ExbD is proposed to direct the conformational changes of TonB. This study addressed the role of ExbD in TonB-dependent energy transduction and provides evidence to support the proposed function of ExbD in modulating TonB conformation. Formaldehyde crosslinking was used to identify ExbD-specific interactions in vivo. ExbD crosslinked into a homodimer and ExbB-ExbD or TonB-ExbD heterodimers. The TonB-ExbD heterodimer was dependent on the presence of ExbB, supporting the predicted formation of a multimeric energy transduction complex. The first mechanistic role for pmf was identified, where pmf is required for specific periplasmic domain interaction that allows crosslinking of ExbD to TonB. The importance of wild-type TMDs of ExbD and TonB for this interaction suggests a role for signaling from the TMD to the periplasmic domain. Pmf-dependent conformational changes in the TonB system were further examined with limited proteolysis studies. ExbD conformation was demonstrated to be responsive to changes in pmf. Pmf appears to be a toggle switch for TonB and ExbD conformational changes. ExbD was also shown to determine the ability of TonB to change conformation in response to pmf, with distinct roles of the ExbD TMD and periplasmic domain. A model is proposed for three stages leading to energization of TonB, emphasizing the importance of the ExbD periplasmic domain for TonB to progress to subsequent stages. The importance of the ExbD periplasmic domain was also apparent in further studies addressing specific regions of ExbD important in its function, using a 10-residue deletion scanning analysis. Functional division of the ExbD periplasmic domain is proposed, with residues 42-61 important in response to pmf and residues 62-141 important for interaction with TonB. The conformation of the region between residues 92-121 was especially important in supporting multiple ExbD protein-protein interactions. Subsequent studies focused on this region with cysteine scanning and disulfide crosslinking. Multiple sites of homodimeric interaction in vivo were identified. Specific interaction with TonB was observed through many of the same sites. Studies of TonB-ExbD interaction were extended to 45 individual cysteine substitutions in the TonB carboxy terminus. Three of six ExbD cys substitutions examined in combination with these TonB substitutions formed multiple disulfide-linked heterodimeric interactions. A wild-type ExbD TMD and ExbB were important for disulfide-linked heterodimer but not homodimer formation. A model is presented where the default configuration of the ExbD periplasmic domain is dimeric with transition from localized homodimeric interactions to heterodimeric interactions with TonB, mediated by the ExbD TMD. Specific interaction sites on TonB further suggested interaction with ExbD may resolve TonB homodimeric interfaces and configure the TonB periplasmic domain for productive interaction with OM transporters. This work has provided significant insight into the functional role of ExbD in TonB energization and expanded knowledge of both the mechanisms and sequence of events involved in the cycle of TonB-dependent energy transduction.