title: Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR.
abstract: 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 (31)P 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 (13)C-labeled TPP(+) and key residues within the EmrE dimer has been probed by through-space (13)C-(13)C 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 as TPP(+) and the essential residue E14 in transmembrane helix 1. Our work also demonstrates the power of DNP-enhanced solid-state NMR at low temperatures for the study for secondary transporters, which are highly challenging for conventional NMR detection.
authors.affiliation: Institute of Biophysical Chemistry & Centre for Biomolecular Magnetic Resonance and ‡Institute of Biochemistry, Goethe University Frankfurt , Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany.
authors.forename: Yean Sin
authors.initials: YS
authors.lastname: Ong
authors.forename: Andrea
authors.initials: A
authors.lastname: Lakatos
authors.forename: Johanna
authors.initials: J
authors.lastname: Becker-Baldus
authors.forename: Klaas M
authors.initials: KM
authors.lastname: Pos
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D017920:Antiporters / Q000737:chemistry*; D024901:Drug Resistance, Multiple, Bacterial; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D009861:Onium Compounds / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D014320:Triphenylmethyl Compounds / Q000737:chemistry*
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D014320:Triphenylmethyl Compounds; C075436:EmrE protein, E coli; C011561:triphenylmethylphosphonium; C013289:tetraphenylphosphonium