A Motif in the ExbD Disordered Domain Mediates TonB System Signal Transduction
Open Access
- Author:
- Kopp, Dale
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 06, 2019
- Committee Members:
- Kathleen Postle, Dissertation Advisor/Co-Advisor
Kathleen Postle, Committee Chair/Co-Chair
B Tracy Nixon, Committee Member
Timothy Iwao Miyashiro, Committee Member
Scott A Showalter, Outside Member
Lorraine C Santy, Committee Member
Wendy Hanna-Rose, Program Head/Chair - Keywords:
- TonB
Signal transduction
ExbD
ExbB
Intrinsic disordered regions
Conserved motifs
protein-protein interactions - Abstract:
- The TonB system provides Gram-negative bacteria an energy source to actively transport essential nutrients across their unenergized outer membranes. The cytoplasmic membrane integral proteins - ExbB, ExbD, and TonB transduce the energy from proton motive force (PMF) of the inner membrane to the TonB-dependent outer membrane transporters. Only when TonB is “energized” can it properly interact with the TonB dependent transporters for ligands to enter the cell. ExbD is an essential protein within the TonB system because its periplasmic domain (44-141) interacts with TonB to define the initial stages of TonB energization. These ExbD-TonB interactions during the energy transduction cycle have been divided into three different Stages based on different molecular characteristics. A scanning 10-amino acid deletion analysis determined that residues 42-61 just following the ExbD transmembrane domain were important for a different Stage of TonB energization than the C-terminal residues 92-121. Interestingly, ExbD residues 42-61 are located within its disordered domain. Disordered proteins are usually involved in important cellular functions including cellular signaling and transient interactions between proteins. Neither the important residues nor the protein-protein interactions within the ExbD disordered domain had been defined. In this work, we identified important determinants of the ExbD periplasmic disordered domain. Using in vivo photo-cross-linking, pBpa substitutions within the disordered domain captured five complexes – an ExbD homodimer, two ExbB-ExbD heterodimers, PMF-independent- and PMF-dependent TonB-ExbD heterodimers. Single pBpa substitutions trapped multiple complexes including some that trapped all five of the identified complexes. This indicated that these residues were transitioning between protein partners during an energy transduction cycle. Through alanine mutagenesis, a novel ΨXΨXLP (Ψ = hydrophobic-branched residues; X = non-hydrophobic residues) motif was identified. This motif contained the only important residues for TonB system activity within the ExbD disordered domain. Interestingly, this motif is also conserved in the ExbD paralog, TolR, providing a possible explanation for crosstalk between the TonB and Tol systems. The physical composition of the ΨXΨXLP motif was similar to that of eukaryotic short-linear motifs called SliMs, which in some cases had been found to initiate transient protein-protein interactions for cellular signaling. In bacteria however, the ΨXΨXLP motif appeared to be part of a novel signaling mechanism that mediates protein interactions in response to PMF. In a follow-up study, the expression of an ExbD disordered domain peptide fused to a periplasmic-localizing signal sequence inhibited TonB system activity. Furthermore, it decreased levels of all ExbD formaldehyde complexes, including the ExbD-TonB PMF-dependent interaction. The peptide was trapped in multiple interactions with ExbD and TonB, in which they were able to be captured with formaldehyde cross-linking. However, a partial deletion or alanine mutations within the conserved ΨXΨXLP motif of peptide were significantly less inhibitory than the ExbD disordered domain peptide with an intact ΨXΨXLP motif. In addition, these peptide variants formaldehyde cross-linked differently with ExbD and TonB. Taken together, these results suggested that the partial deletion or alanine mutations within the conserved ΨXΨXLP motif prevented the peptide from interacting properly with ExbD and TonB. These results demonstrated that the ΨXΨXLP motif within the ExbD disordered domain peptide was important for inhibiting TonB system activity, possibly by competing with and preventing native ExbD and TonB interactions. The TonB system is a virulence factor for many Gram-negative pathogens and is thus an appealing target for potential novel antibiotics. However, currently no antibiotic exists that inhibits this system. The conservation of the ΨXΨXLP motif, its essentiality in TonB-dependent energy transduction, and its localization in the periplasm suggest that it could serve as a viable antibiotic target.