Institut for Biomedicin

Mikael Esmann

Ligand orientation in a membrane-embedded receptor site revealed by solid-state NMR with paramagnetic relaxation enhancement

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

  • Christopher A P Whittaker
  • ,
  • Simon G Patching, Ukendt
  • Mikael Esmann
  • David A Middleton

NMR relaxation enhancement by paramagnetic metals provides powerful restraints on the three-dimensional structures of proteins in solution, and this approach has recently been utilized in several NMR structural investigations of proteins in the solid-state. Here we utilize paramagnetic relaxation enhancement (PRE) by Mn(2+) with cross-polarization magic-angle spinning (CP-MAS) solid-state NMR to investigate the interaction of a membrane-embedded protein the Na,K-ATPase (NKA) with a cardiotonic steroid inhibitor. The inhibitor, a diacetonide derivate of the cardiac glycoside ouabain, with (13)C labelled acetonide groups in the rhamnose sugar and steroid moieties ([(13)C2]ODA), is 1000-fold less potent than the parent compound. It is shown that the (13)C CP-MAS solid-state NMR spectra of the NKA-[(13)C2]ODA complex exhibit distinct signals for the two (13)C labels of the inhibitor when bound to the ouabain site of membrane-embedded NKA. Recent crystal structures of NKA indicate that the catalytic α-subunit binds a single Mn(2+) in a transmembrane site close to the high-affinity ouabain site. Here, complexation of NKA with Mn(2+) broadens the resonance line from the rhamnose group substantially more than the steroid peak, indicating that the rhamnose group is closer to the Mn(2+) site than is the steroid group. These observations agree with computational molecular docking simulations and are consistent with ODA adopting an inverted orientation compared to ouabain in the cardiac glycoside site, with the modified rhamnose group drawn toward the transmembrane centre of the protein. This work demonstrates that PRE can provide unique information on the positions and orientations of ligands within their binding pockets of transmembrane proteins.

OriginalsprogEngelsk
TidsskriftOrganic & Biomolecular Chemistry
ISSN1477-0520
DOI
StatusUdgivet - 13 jan. 2015

Se relationer på Aarhus Universitet Citationsformater

ID: 84415126