GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

Abstract

In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized β1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with β1 adrenergic receptor (β1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tβ1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR

“…Allosteric communication between bound G protein mimetics and the orthosteric ligand-binding pocket was also observed for thermostabilized β 1 AR using 15 N-valine amide backbone NMR probes distributed throughout the receptor. 17 – 19 …”

E. coli “Stablelabel” S30 lysate for optimized cell-free NMR sample preparation

“…Nevertheless, NMR spectra of protein samples labeled with either LVal (4-13 C, 2, 3, 4, 4, 4,-D5) or LLeu (5-13 C, 2, 3, 3, 4, 5, 5, 5-D7) but lacking stereospeci ty of the 13 C methyl groups will still remain more complex if compared to the use of SAIL amino acids 42 . Solubilization in detergent is current standard for the NMR analysis of membrane proteins and even large and complex G-protein coupled receptors can become addressed as demonstrated with the β 1 adrenergic receptor [43][44][45] . L-Val, L-Leu and L-Ile are major residues in receptor and other membrane protein cores and they are frequently involved in the formation of speci c orthosteric ligand binding pockets 46,47 .…”

“…L-Val, L-Leu and L-Ile are major residues in receptor and other membrane protein cores and they are frequently involved in the formation of speci c orthosteric ligand binding pockets 46,47 . The NMR analysis of selectively labeled samples could therefore reveal important insights in G-protein coupled receptor dynamics and ligand interaction [43][44][45]48 .…”

Nanobody GPS by PCS: An Efficient New NMR Analysis Method for G Protein Coupled Receptors and Other Large Proteins

“…They are transmembrane proteins which regulate many vital functions of the human body by recognizing extracellular ligands and transmitting this stimulus to intracellular signaling cascades . Their function is achieved via highly dynamic equilibria between multiple active and inactive conformations, which are modulated by the ligands. − Atomic details of these dynamical equilibria and their driving forces are still largely unknown.…”