The role of NMR spectroscopy in mapping the conformational landscape of GPCRs.

Over recent years, nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful mechanistic tool for the investigation of G protein-coupled receptors (GPCRs). NMR provides insights which underpin the dynamic nature of these important receptors and reveals experimental evidence for a complex conformational energy landscape that is explored during receptor activation resulting in signalling. NMR studies have highlighted both the dynamic properties of different receptor states as well as the exchange pathways and intermediates formed during activation, extending the static view of GPCRs obtained from other techniques. NMR studies can be undertaken in realistic membrane-like phospholipid environments and an ever-increasing choice of labelling strategies provides comprehensive, receptor-wide information. Combined with other structural methods, NMR is contributing to our understanding of allosteric signal propagation and the interaction of GPCRs with intracellular binding partners (IBP), crucial to explaining cellular signalling.

Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound ß1-adrenergic receptor.

A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and ß-arrestin. Using 13C methyl methionine NMR for the ß1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive µs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.