Membrane transport

Biology

Receptor

Biochemistry

Protein structure

Computational biology

Stereochemistry

Membrane

Molecular dynamics

Computational chemistry

Crystallography

Chemistry

Transmembrane domain

Biophysics

Transmembrane protein

Efflux

Journal of Structural Biology

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.

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