Characterization of ExbB Transmembrane Domains for Tonb-dependent Energy Transduction in Escherichia coli
Open Access
- Author:
- Baker, Kristin Renee
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 21, 2012
- Committee Members:
- Kathleen Postle, Dissertation Advisor/Co-Advisor
Sarah Ellen Ades, Committee Member
B Tracy Nixon, Committee Member
Lorraine C Santy, Committee Member
Timothy W Mcnellis, Committee Member - Keywords:
- ExbB
TonB
Energy Transduction
Membrane protein
Escherichia coli - Abstract:
- In Gram negative bacteria, the TonB system serves as an energy conduit, connecting cytoplasmic membrane (CM) proton motive force (pmf) to active transport of ligands at the outer membrane (OM) through TonB-gated transporters. In Escherichia coli, CM proteins TonB, ExbB, and ExbD form a complex which links pmf energy to transport of important nutrients vitamin B12 and iron-siderophore complexes into the cell. TonB and ExbD have a similar topology with a short N-terminal cytoplasmic domain, one transmembrane domain (TMD) and a large C-terminal periplasmic domain. In contrast, ExbB has three TMDs and large cytoplasmic loop and tail domains. Because the majority of TonB/ExbD and ExbB occupy different cellular compartments, important interactions are thought to occur through the TMDs. Characterization of functionally important residues in the TMDs and periplasmic domains of TonB and ExbD have provided mechanistic insight for TonB system function. However, the functional role of ExbB is undefined. Currently, ExbB has three speculative functions, as a scaffolding protein, as a signal transducer and as a proton translocator. Mutagenic studies in the large cytoplasmic loop and tail domains have concluded that these regions are essential for ExbB function. In addition, Cys substitutions in the C-terminal tail identified residues important for signal transduction from the cytoplasm to periplasm. The ExbB TMDs have not been well-studied and it is unclear if all TMDs are essential for ExbB function. This study addressed the functional importance of the ExbB TMDs for TonB-dependent energy transduction. New predictions of ExbB TMD boundaries replaced three prior incongruent ExbB TMD predictions. Substitution of each half ExbB TMD with alanine determined that all TMDs were essential for activity. Additional substitution of individual ExbB TMD residues identified ExbB scaffolding and signal transduction functions. Substitution of all protonatable residues in the TMD ruled out the hypothesis that ExbB residues function in proton translocation. This knowledge was then applied to characterize ExbB ∆120-129-mediated proton leakage via addition of TMD substitutions. The proton leakage rate was reduced by substitution of important ExbB TMD residues, suggesting proton conductance was occurring through ExbB. However, substitution of non-essential ExbB residues had the most profound effect on the ExbB ∆120-129-mediated proton leakage. This implied that the proton leakage was occurring through a non-native ExbB mechanism. The mutagenic studies presented here, reinforce the proposed ExbB scaffolding and signal transducer functions and refute a proton pathway formed through the ExbB TMDs. This work clarified our understanding of ExbB function and what role(s) the TMDs play in TonB-dependent energy transduction.