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

Abstract

Using 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 receptors 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 b-arrestin-1 or the C-terminal helix of Gaq, which comprises >60% of the GPCR/G protein interface surface area, abolishes the inactive substate. But whereas b-arrestin-1 selects for preexisting active-like substates, the Gaq peptide induces two new substates. Both transducer molecules promote substantial line-broadening of active states suggesting contributions from additional us-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.

A Method for Selective 19F-Labeling Absent of Probe Sequestration (SLAPS)

“…We sonicated cell pellets containing enNTS1(Q301 6.28 /C172S . 16 Intact and protease-digested LC-MS confirmed that 19 F-enNTS1(Q301C 6.28 /C172S 3.55 ) was exclusively-labeled at Q301C 6.28 with no observable unreacted 19 F-BTFMA [Fig. 3(C) and Table S1].…”

“…no offsite labeling). 9,13,16 As a negative control, we engineered 19 F-enNTS1(Q301 6.28 /C172S 3.55 ) with residue 301 reverted to glutamine and repeated the experiment. The spectrum contained two resonances that were present in the 19 F-enNTS1(Q301C 6.28 /C172S 3.55 ) spectrum indicative of offsite labeling [Fig.…”

“…In particular, the ability of NMR to access motional regimes covering more than 15 orders of magnitude (ps-s) makes it especially attractive for this task, although the challenges associated with uniform incorporation of NMR-active isotopes has somewhat limited its application. 10 Exogenous cysteine-reactive fluorine ( 19 F) probes have proven an effective alternative to uniform labeling [11][12][13][14][15][16] owing to their high gyromagnetic ratio (i.e. sensitivity), 100% natural abundance, large chemical shift range, and absence of background signals in biomolecular samples.…”

“…Offsite 19 F-probe incorporation is an additional source of signal overlap that is specifically problematic when the target protein contains critical cysteine residues that cannot be mutated. 9,18,19 In our previous work labeling the neurotensin receptor 1 (NTS1) Class A GPCR with cysteine-reactive 19 F-probes, 16 we uncovered a second source of offsite labeling: non-covalent sequestration by detergent micelles. Conventional labeling methods solubilize the receptor in detergent micelles without the prior removal of excess 19 F-probe molecules.…”