Paniagua, J. C. | Search bioRxiv

bioRxiv2022

The dynamic nature of neurotensin receptor 1 (NTS1) allostery and signaling bias

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13C{varepsilon}H3-methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highly-conserved activation motifs. {beta}-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A {beta}-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

biochemistry↗

bioRxiv2022

Ligands tune the local and global motions of neurotensin receptor 1 (NTS1): a DFT-guided solution NMR analysis

Unlike many signaling proteins that function as binary switches between on and off states, G protein-coupled receptors (GPCRs) exhibit basal activity that can be increased or decreased by numerous ligands. A given receptor can recognize multiple ligands, allosteric modulators, and transducers to create a complex free energy landscape. Many of the lowest energy states have been captured by static structural techniques while detailing the wells widths, metastable states, and the transition between them, is still in its infancy. Nuclear magnetic resonance (NMR) spectroscopy can monitor the structure and dynamics of GPCR ensembles across fifteen orders-of-magnitude, but technical challenges have limited its application to super-microsecond timescales. Focusing on a prototypical peptide-binding GPCR, the neurotensin receptor 1 (NTS1), we employed NMR and density functional theory (DFT) to probe global sub-nanosecond motions. The near random coil chemical shifts of the apo receptor produced a poor correlation with theoretical predictions that may indicate a high degree of conformational averaging in solution, a crystallization artifact, or both. Whereas orthosteric agonists and antagonists both rigidified the receptor, but to varying degrees, which suggests conformational entropy differentially contributes to their respective pharmacology. The strong correlations of observed and theoretical chemical shifts lend confidence to interpreting spectra in terms of local structure, methyl dihedral angle geometry, and pico-second timescale transitions. Together, our results suggest a role for sub-nanosecond dynamics and conformational entropy in GPCR ligand discrimination.

biophysics↗