Efflux of antibiotics by the homotrimeric TolC transmembrane protein.

TolC is an outer membrane efflux protein in Gram-negative bacteria that has a critical role for protection against antibiotics. We investigate its efflux mechanism at molecular level by simulating how different antibiotics pass through the 140 Å long channel. We have performed a series of steered molecular dynamics simulations with three beta-lactam antibiotics that respond differently to efflux, carbenicillin, piperacillin, and oxacillin. This has led to their distinct potential of mean force profiles along the channel. Our findings indicate oxacillin efflux is the easiest of the three, the molecule not experiencing any barriers during expulsion. Conversely, while expelling carbenicillin to extracellular region, a bundle of hydrogen bonds occurring around the initial 40 Å region is determined as the possible cause of limited efflux of carbenicillin. Finally, piperacillin is intermediate to the two in ease of efflux, but the resistance to movement to this molecule has a different mechanism, one that involves the region near extracellular end of TolC. We have also determined the main modes of motion of open and closed forms of TolC by applying principal component analyses to trajectories obtained from classical MD trajectories. By using our perturbation-response scanning method, we have shown which single residue perturbations are effective in shifting the collective motions adopted by the trimeric protein system. Thus, our combined results with computational tools verify residue conferring resistance data from deep mutational scanning and might help engineer antibiotic molecules that can be protected against the bacterial efflux mechanism.