The Effect of Ligands and Transducers on the Neurotensin Receptor 1 (NTS1) Conformational Ensemble

Abstract

ABSTRACTUsing a discrete, intracellular 19F-NMR probe on Neurotensin receptor 1 (NTS1) transmembrane helix (TM) 6, we aim to understand how ligands and transducers modulate the receptor’s structural ensemble in solution. For apo NTS1, 19F-NMR spectra reveal an ensemble of at least three states (one inactive and two active-like) in equilibrium that exchange on the ms-s 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 GPCRs, the full agonist is insufficient to completely stabilize the active state. Receptor coupling to β-arrestin-1 or the C-terminal helix of Gαq, which comprises ⪆60% of the GPCR/G protein interface surface area, abolishes the inactive substate. But whereas β-arrestin-1 selects for preexisting active-like substates, the Gαq peptide induces two new substates. Both transducer molecules promote substantial line-broadening of active states suggesting contributions from additional μs-ms exchange processes. Together, our study suggests i) the NTS1 allosteric activation mechanism is 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 solution.