Modeling substrate efflux in human P-glycoprotein at the atomic level

Human P-glycoprotein (hP-gp) is an ATP-binding cassette (ABC) exporter that actively extrudes a wide range of xenobiotics from the cell, thus limiting drug delivery and contributing to multidrug resistance (MDR) in cancers. Recent structural studies have provided insights into how hP-gp binds diverse compounds, but how they are translocated through the membrane remains poorly understood at the atomic level. In this work, we used steered molecular dynamics (SMD) simulations to investigate the molecular mechanism of how hP-gp expels structurally different compounds and which molecular features favor this efflux step. The potential of mean force (PMF) and structural dynamics analysis showed that the bending of TM1 favored the translocation of vincristine, whereas the high flexibility of tariquidar made it easier to pass through the narrow exit tunnel, suggesting a wide opening of the extracellular gate is not required for the efflux of both compounds. Moreover, an alternating-site hydrolysis mechanism may be shared in which ATP bound in the second nucleotide-binding site was preferentially hydrolyzed to provide chemical energy for the flexible-to-rigid transition of TM10. A conserved salt bridge between the fourth intracellular loop and the flexible X-loop was formed in response to ATP binding, which may participate in the interdomain communication. Furthermore, the SMD trajectories revealed two translocation pathways in the hP-gp cavity, one of which is preferentially but non-exclusively taken by a set of compounds. These findings provide deep insights into the efflux mechanism of hP-gp and will help rational design and development of more selective and effective inhibitors.