Solid-state NMR chemical shift analysis for determining the conformation of ATP bound to Na,K-ATPase in its native membrane

• Published in: RSC advances Vol. 13; no. 49; pp. 34836 - 34846
• Main Authors: Middleton, David A, Griffin, John, Esmann, Mikael, Fedosova, Natalya U
• Format: Journal Article
• Language: English
• Published: Cambridge Royal Society of Chemistry 29-11-2023; The Royal Society of Chemistry
• Subjects: Adenine; Affinity; Chemical equilibrium; Chemistry; Crystallography; Density functional theory; Dipole interactions; Ligands; Membrane structures; Membranes; NMR; Nuclear magnetic resonance; Physiology; Proteins; Ribose; Solid state
• DOI: 10.1039/d3ra06236h
• EISSN: 2046-2069

Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that
13
C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The
13
C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3′-
endo
(N) conformation when bound with high affinity to NKA in the E
1
Na state, in contrast to the C2′-
endo
(S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H-C-C-H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand-protein interactions.
Solid-state NMR and DFT
13
C chemical shift calculations are used to determine the ribose ring conformation of hydrolysable adenosine 5′-triphosphate when freeze-trapped in the high-affinity binding site of Na,K-ATPase.