Monitoring the antibiotic darobactin modulating the β-barrel assembly factor BamA

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• The beta-barrel assembly machinery (BAM) complex is crucial for inserting and folding outer membrane proteins (OMPs) in Escherichia coli.
• The antibiotic darobactin targets BamA, the central component of the BAM complex.

Purpose of the Study:

• To investigate the structure-function relationship of BamA using dynamic single-molecule force spectroscopy (SMFS).
• To understand how darobactin inhibits BamA function.

Main Methods:

• Dynamic single-molecule force spectroscopy (SMFS) was employed to probe BamA's mechanical properties.
• Mechanical, kinetic, and energetic stabilities of BamA domains were analyzed.

Main Results:

• The N-terminal polypeptide transport (POTRA) domains exhibit low mechanical stability.
• The linker region connecting POTRA domains to the beta-barrel shows high stiffness and low kinetic stability, suggesting a mechano-functional role.
• The N-terminal beta-hairpins (H1-H4) within the barrel are mechanically stable, while C-terminal hairpins (H5-H6) are more flexible.
• This structural asymmetry suggests a mechanism for substrate-induced conformational changes in the C-terminal beta-hairpins.

Conclusions:

• BamA possesses distinct mechanical properties across its domains, highlighting a structure-function relationship.
• The linker region and the asymmetric beta-barrel structure are critical for BamA's function and potential conformational changes during OMP insertion.
• These findings provide insights into BamA's mechanism and its inhibition by darobactin.