Detecting substrates bound to the secondary multidrug efflux pump emre by dnp-enhanced solid-state NMR

Yean Sin Ong, A. Lakatos, Johanna Becker-Baldus, Klaas M. Pos, Clemens Glaubitz

Research output: Contribution to journalArticle

38 Citations (Scopus)

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 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 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.

Original languageEnglish
Pages (from-to)15754-15762
Number of pages9
JournalJournal of the American Chemical Society
Volume135
Issue number42
DOIs
Publication statusPublished - 2013

Fingerprint

Nuclear magnetic resonance
Pumps
Ligands
Antiporters
Phosphorylcholine
Substrates
Liposomes
Spectrum Analysis
Dimers
Escherichia coli
Temperature
Polarization
Population
Spectroscopy

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry
  • Medicine(all)

Cite this

Detecting substrates bound to the secondary multidrug efflux pump emre by dnp-enhanced solid-state NMR. / Ong, Yean Sin; Lakatos, A.; Becker-Baldus, Johanna; Pos, Klaas M.; Glaubitz, Clemens.

In: Journal of the American Chemical Society, Vol. 135, No. 42, 2013, p. 15754-15762.

Research output: Contribution to journalArticle

Ong, Yean Sin ; Lakatos, A. ; Becker-Baldus, Johanna ; Pos, Klaas M. ; Glaubitz, Clemens. / Detecting substrates bound to the secondary multidrug efflux pump emre by dnp-enhanced solid-state NMR. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 42. pp. 15754-15762.
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