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

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<mark>Journal publication date</mark>7/03/2015
<mark>Journal</mark>Organic and Biomolecular Chemistry
Issue number9
Volume13
Number of pages5
Pages (from-to)2664-2668
Publication StatusPublished
Early online date23/12/14
<mark>Original language</mark>English

Abstract

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 Mn2+ 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 13C labelled acetonide groups in the rhamnose sugar and steroid moieties ([13C2]ODA), is 1000-fold less potent than the parent compound. It is shown that the 13C CP-MAS solid-state NMR spectra of the NKA-[13C2]ODA complex exhibit distinct signals for the two 13C 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 Mn2+ in a transmembrane site close to the high-affinity ouabain site. Here, complexation of NKA with Mn2+ broadens the resonance line from the rhamnose group substantially more than the steroid peak, indicating that the rhamnose group is closer to the Mn2+ 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.