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

Abstract

The β-barrel assembly machinery (BAM) complex is an essential component of Escherichia coli that inserts and folds outer membrane proteins (OMPs). The natural antibiotic compound darobactin inhibits BamA, the central unit of BAM. Here, we employ dynamic single-molecule force spectroscopy (SMFS) to better understand the structure-function relationship of BamA and its inhibition by darobactin. The five N-terminal polypeptide transport (POTRA) domains show low mechanical, kinetic, and energetic stabilities. In contrast, the structural region linking the POTRA domains to the transmembrane β-barrel exposes the highest mechanical stiffness and lowest kinetic stability within BamA, thus indicating a mechano-functional role. Within the β-barrel, the four N-terminal β-hairpins H1-H4 expose the highest mechanical stabilities and stiffnesses, while the four C-terminal β-hairpins H5-H6 show lower stabilities and higher flexibilities. This asymmetry within the β-barrel suggests that substrates funneling into the lateral gate formed by β-hairpins H1 and H8 can force the flexible C-terminal β-hairpins to change conformations.

Keywords: BAM complex; BamA; POTRA domains; antibiotic; atomic force microscopy; darobactin; dynamic force spectroscopy; flexibility; free-energy landscape; inhibit BamA; membrane protein folding and insertion; outer membrane protein; protein unfolding; single-molecule force spectroscopy; stability; stable structural segments; structure-function relationship; β-barrel domain.