Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Gram-negative bacteria utilize proton-motive force to energize active transport via TonB-dependent outer membrane transporters.
• Cytoplasmic proteins ExbB and ExbD couple proton-motive force to TonB, inducing conformational changes for energy transduction.
• Periplasmic domain interactions between TonB and ExbD are crucial for TonB's conformational response to proton-motive force.

Purpose of the Study:

• To pinpoint specific interaction sites between the ExbD periplasmic domain (residues 92-121) and the TonB carboxy terminus.
• To elucidate the structural basis of energy transduction in TonB-dependent transport.

Main Methods:

• Utilized 45 TonB and 6 ExbD individual cysteine substitutions, creating 270 combinations.
• Assessed disulfide-linked heterodimer formation to map protein-protein interactions.
• Investigated the role of specific ExbD residues (A92C, K97C, T109C, F103C, L115C, T121C, D25) in TonB interaction.

Main Results:

• Identified four distinct regions of interaction between ExbD residues A92C, K97C, and T109C and the TonB carboxy terminus.
• Discovered that ExbD positions TonB for optimal interaction with TonB-gated transporters.
• Found that ExbD substitutions F103C, L115C, and T121C did not form heterodimers with any TonB variants.
• ExbD D25 is vital for efficient TonB-ExbD heterodimerization, suggesting involvement in proton translocation.

Conclusions:

• Detailed mapping of ExbD-TonB interactions provides structural insights into energy transduction mechanisms.
• ExbD plays a critical role in modulating TonB conformation and facilitating substrate transport.
• The findings contribute to understanding the molecular machinery of active transport in gram-negative bacteria.