Effect of Ligands and Transducers on the Neurotensin Receptor 1 Conformational Ensemble

Using a discrete, intracellular F-19 nuclear magnetic resonance (NMR) probe on transmembrane helix 6 of the neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptor's structural ensemble in a solution. For apo NTS1, F-19 NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the millisecond to second timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other G protein-coupled receptors (GPCRs), the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to beta-arrestin-1 (beta Arr1) or the C-terminal helix of G alpha(q), which comprises greater than or similar to 60% of the GPCR/G protein interface surface area. Whereas beta Arr1 exclusively selects for pre-existing active-like substates, the G alpha(q) peptide induces a new substate. Both transducer molecules promote substantial line broadening of active-like states, suggesting contributions from additional microsecond to millisecond exchange processes. Together, our study suggests that (i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and (ii) the available static structures do not represent the entire conformational ensemble observed in a solution.