Cross-linking, DEER-spectroscopy and molecular dynamics confirm the inward facing state of P-glycoprotein in a lipid membrane

[Display omitted] •How well do structural models describe the structure of P-glycoprotein in membranes.•Membrane helices 6 and 12 in link energy provision, drug binding and transport.•Inter-helical distances were measured using DEER spectroscopy and cross-linking.•Data were compared to predictions from several structural models of P-glycoprotein.•The 4m1m model provides the best representation of P-gp structure in a membrane. The drug efflux pump P-glycoprotein (P-gp) displays a complex transport mechanism involving multiple drug binding sites and two centres for nucleotide hydrolysis. Elucidating the molecular mechanism of transport remains elusive and the availability of P-gp structures in distinct natural and ligand trapped conformations will accelerate our understanding. The present investigation sought to provide biochemical data to validate specific features of these structures; with particular focus on the transmembrane domain that provides the transport conduit. Hence our focus was on transmembrane helices six and twelve (TM6/TM12), which are believed to participate in drug binding, as they line the central transport conduit and provide a direct link to the catalytic centres. A series of P-gp mutants were generated with a single cysteine in both TM6 and TM12 to facilitate measurement of inter-helical distances using cross-linking and DEER strategies. Experimental results were compared to published structures per se and those refined by MD simulations. This analysis revealed that the refined inward-facing murine structure (4M1M) of P-gp provides a good representation of the proximity, topography and relative motions of TM6 and TM12 in reconstituted human P-gp.