| Abstract: | P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP bindingcassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancercells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy,we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state andfound that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree ofseparation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs inthe equilibrium simulations covers a range of at least 20 Å, including, both, more open andmore closed NBD configurations than the crystal structure. The double electron-electron resonancemeasurements on spin-labeled Pgp mutants also show wide distributions covering both longer andshorter distances than those observed in the crystal structure. Based on structural and sequenceanalyses, we propose that the transmembrane domains of Pgp might be more flexible than otherstructurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only inclose agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABCexporters and possibly represents a fundamental difference in the transport mechanism between ABCexporters and ABC importers. In addition, during the simulations we have captured partial entranceof a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site.The location of the protruding lipid suggests a putative pathway for direct drug recruitment fromthe membrane. |
