Detecting Substrates Bound to the Secondary Multidrug Efflux Pump EmrE by DNP-Enhanced Solid-State NMR

Escherichia coli EmrE, a homodimeric multidrug antiporter, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It contains four transmembrane helices and forms a functional dimer. We have probed the specific binding of substrates TPP+ and MTP+ to EmrE reconstituted into 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes by 31P MAS NMR. Our NMR data show that both substrates occupy the same binding pocket but also indicate some degree of heterogeneity of the bound ligand population, reflecting the promiscuous nature of ligand binding by multidrug efflux pumps. Direct interaction between 13C-labeled TPP+ and key residues within the EmrE dimer has been probed by through-space 13C–13C correlation spectroscopy. This was made possible by the use of solid-state NMR enhanced by dynamic nuclear polarization (DNP) through which a 19-fold signal enhancement was achieved. Our data provide clear evidence for the long assumed direct interaction between substrates such ...