Molecular conformation affects the interaction of the Pseudomonas quinolone signal with the bacterial outer membrane.

Gram-negative bacteria produce outer-membrane vesicles (OMVs) that package genetic elements, virulence factors, and cell-to-cell communication signaling compounds. Despite their importance in many disease-related processes, how these versatile structures are formed is incompletely understood. A self-produced secreted small molecule, the Pseudomonas quinolone signal (PQS), has been shown to initiate OMV formation in Pseudomonas aeruginosa by interacting with the outer membrane (OM) and inducing its curvature. Other bacterial species have also been shown to respond to PQS, supporting a common biophysical mechanism. Here, we conducted molecular dynamics simulations to elucidate the specific interactions between PQS and a model P. aeruginosa OM at the atomistic scale. We discovered two characteristic states of PQS interacting with the biologically relevant membrane, namely attachment to the membrane surface and insertion into the lipid A leaflet. The hydrogen bonds between PQS and the lipid A phosphates drove the PQS-membrane association. An analysis of PQS trajectory and molecular conformation revealed sequential events critical for spontaneous insertion, including probing, docking, folding, and insertion. Remarkably, PQS bent its hydrophobic side chain into a closed conformation to lower the energy barrier for penetration through the hydrophilic headgroup zone of the lipid A leaflet, which was confirmed by the potential of mean force (PMF) measurements. Attachment and insertion were simultaneously observed in the simulation with multiple PQS molecules. Our findings uncover a sequence of molecular interactions that drive PQS insertion into the bacterial OM and provide important insight into the biophysical mechanism of small molecule-induced OMV biogenesis.