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

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 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.

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…There are several examples where solid-state NMR methods have been used to characterize the structures of ligands bound to GPCRs (41)(42)(43)(44) as well as GPCRs themselves. Most prior studies of membrane proteins or their ligands have involved the direct detection of signals from labeled 13 C or 15 N sites; this continues to be a fruitful approach and we applied it in our earlier studies of CXCR1 (23,33,45).…”

Solid-state NMR structures of integral membrane proteins

“…Impressively though, structures determined exclusively by MAS methods include that of the seven transmembrane spanning Anabaena sensory rhodopsin in a trimeric form (Wang et al, 2013) (see below). MAS solid-state NMR is also very useful for elucidating the atomicresolution structures, molecular conformations, positions and orientations of ligands and drug compounds in the binding sites of membrane proteins from measurement of accurate interatomic distances and torsion angles (Cady et al, 2010;Edwards et al, 2010;Lakatos et al, 2012;Lopez et al, 2008;Middleton et al, 2011;Patching et al, 2008Patching et al, , 2013Whittaker et al, 2015;Williamson et al, 2007). Such measurements can contribute important information for the design and discovery of drugs involving membrane-embedded targets (Ding et al, 2013a;Tapaneeyakorn et al, 2011;Watts, 2005;Williamson, 2009).…”

On‐Cell NMR Contributions to Membrane Receptor Binding Characterization

“…Additionally, measurement of relaxation parameters and dipolar couplings allows the probing of structural dynamics in the bound state. It is important to note, that with the same technique (provided sensitivity allows) observation of the membrane receptor is feasible, as has been reported recently [49,56,57] …”

“…It is important to note, that with the same technique (provided sensitivity allows) observation of the membrane receptor is feasible, as has been reported recently. [49,56,57] Upon binding to a membrane receptor, the ligand behaves as a part of a huge macromolecule (i. e., slow molecular tumbling), where LS-NMR is not capable to directly detect any signal from the bound state due to broad signals and decreased signal-to-noise ratios, and one has to resort to indirect observables. Ligands that display weaker binding affinities and fast exchange rates are easily accessible to LS-NMR methods such as trNOEs, water LOGSY and STD NMR spectroscopy, which, although observing the free ligand, provide valuable information about the receptor-bound state.…”