Using a discrete, intracellular 19 F-NMR probe on transmembrane helix 6 (TM6) of the Neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptor’s structural ensemble in solution. For apo NTS1, 19 F-NMR spectra reveal an ensemble of at least three conformational substates (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-like state. The inactive substate is abolished upon coupling to β-arrestin-1 or the C-terminal helix of Gα q, which comprises ⍰60% of the GPCR/G protein interface surface area. Whereas β-arrestin-1 exclusively selects for pre-existing active-like substates, the Gα q peptide induces a new substate. Both transducer molecules promote substantial line-broadening of active-like states suggesting contributions from additional μs-ms exchange processes. Together, our study suggests 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 solution.
G protein-coupled receptors (GPCRs) serve as the primary hubs to relay changes in extracellular environments across the eukaryotic cell membrane. The more than 800 members of this protein superfamily share a conserved seven transmembrane helix (TM) bundle architecture that recognizes a large variety of ligands comprising small molecules, hormones, peptides, and photons. As such, it is no surprise they encompass over 30% of the drug market. Although atomic models are still relatively scarce compared to other protein classes, there are currently 121 unique receptor structures, or ~14% of the total GPCR superfamily. The difficulty of GPCR structural studies primarily reflects inherent protein instability and low recombinant expression. Through the use of detergent membrane mimetics and creative receptor engineering, the rate at which new receptor structures are determined has increased in recent years. These atomic models have revealed conserved, long-range allosteric activation networks that link the receptor orthosteric pocket to the intracellular bundle across the cell membrane. Most notably the DRY, PIF, CWxP, and NPxxY motifs serve as internal molecular “switches” of Class A GPCRs, connecting ligand-binding to downstream effector molecule complexation and activation events, spanning a distance of nearly 50 Å. Modeling of allosteric switches across numerous receptors has led to a putatively conserved structural activation profile.
Neurotensin receptor 1 (NTS1) has quickly become one of the most well-characterized GPCRs with structures of the apo state, complexes with various pharmacological ligands, and ternary complexes with both the heterotrimeric G i protein and β-arrestin-1 (βArr1) transducers. NTS1 is a Class A, β group receptor that is expressed throughout the central nervous system and gastrointestinal tract. Activation by its endogenous tridecapeptide ligand neurotensin (NT) mediates a variety of physiological processes including low blood pressure, high blood sugar, low body temperature, mood, and GI motility.19 It is also a long-standing therapeutic target for Parkinson’s disease, Schizophrenia, obesity, hypotension, psychostimulant substance use disorders, and cancer.
Current atomic models derived from either X-ray crystallography or cryo-EM capture NTS1 in different stages of activation, mediated by bound ligands and transducer proteins. A hallmark of GPCR activation is the outward movement of transmembrane helix 6 (TM6) to accommodate G protein and arrestin complexation. In NTS1, ligand binding at the extracellular orthosteric pocket allosterically induces a ~13 Å lateral displacement at the intracellular tip of TM6. Ultimately, these models remain static. This has left a void in the literature detailing the NTS1 conformational ensemble and the pleiotropic effects ligands and transducers have on individual substates. This inspired us to pursue solution nuclear magnetic resonance (NMR) spectroscopy as a complementary approach to better characterize the allosteric activation mechanism in NTS1.
In this study, we 19 F-label TM6 of a thermostabilized NTS1 construct solubilized in 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG) detergent micelles. Trifluoromethyl NMR probes are an optimal choice for site-selective isotopic labeling due to their low background signal and high spin-1/2 natural abundance. Their observed chemical shift value is dominated by solvent polarity and the local electronic environment, which makes them very sensitive to large conformational rearrangements observed in GPCRs. For example, as the intracellular tip of TM6 moves outward to accommodate transducer proteins, we anticipate an upfield chemical shift perturbation reflecting increased solvent exposure. NMR can provide both qualitative and quantitative information regarding the timescale of structural motions. While very fast rotation about the methyl axis averages any local fluctuations into a single peak, slower “biologically-relevant” motions on approximately the microsecond to milliseconds timescale affect both the resonance linewidth and chemical shift. As conformational exchange slows further into the millisecond to second regime, the averaged resonance will split into distinct peaks with characteristic linewidths and chemical shifts. Herein, we employ the G q C-terminal α5-helix peptide and a pre-activated βArr1 to recapitulate responses to the heterotrimeric G q protein and βArr1. Together, this enables us to develop a dynamic model of NTS1 activation in which ligands and transducers are allosterically coupled.
The well-characterized structure of NTS1 in a variety of pharmacologically-relevant states creates an ideal system for exploring the allosteric mechanisms of GPCR activation. Yet, wildtype NTS1 structural characterization remains challenging due to poor receptor stability following isolation from native membranes. All published NTS1 structures to date incorporate some combination of thermostabilizing mutations, lysozyme fusions, DARPin fusions, or conformationally-selective antibodies. Here, we employed a functional, thermostabilized rat (r)NTS1 variant (termed enNTS1) for solution NMR spectroscopy.
To further validate enNTS1’s functional integrity, we performed a cell-based alkaline phosphatase (AP) reporter assay for G protein activation. Stimulation of Gα q and Gα 12/13 leads to ectodomain shedding of an AP-fused transforming growth factor-α (TGFα), which is then quantified using a colorimetric reporter. HEK293A cells were transfected with AP-TGFα and a NTS1 plasmid construct. A hexapeptide corresponding to NT residues 8-13 (NT8-13) is sufficient to generate a full agonist response in wildtype rNTS1; NT8-13 stimulates robust, concentration-dependent G protein-coupling to enNTS1 in the TGFα shedding assay, though with reduced efficacy compared to human (h)NTS1 (Figure 1A and Figure S1). Both enNTS1 and hNTS1 were equally expressed on the cell surface (Figure S1C). Arr1 recruitment was also measured using a NanoBiT enzyme complementation system. The large and small fragments of the split luciferase were fused to the N-terminus of βArr1 and the C-terminus of NTS1, respectively, and these constructs were expressed in HEK293A cells. As a negative control, we used the vasopressin V2 receptor (V2R) C-terminally fused with the small luciferase fragment. enNTS1 exhibited weak basal βArr1 recruitment that did not increase upon agonist addition (Figure 1B and Figure S1B). Nonetheless, addition of the βArr1-biased allosteric modulator (SBI-553) dose-dependently potentiates NT8-13-mediated βArr1 recruitment (Figure S1D). As SBI-553 alone is unable to substantially stimulate βArr1 recruitment to enNTS1 at the same concentration, we conclude that enNTS1 recruits using the same molecular mechanism as wildtype NTS1, although with reduced potency (Figure S1E).
Figure 1. Orthosteric ligands modulate the enNTS1 conformational ensemble. (A) G protein activation was assessed using a TGFα shedding assay on HEK293A cells transiently-transfected with vasopressin receptor 2 (V2R; Mock), human (h)NTS1, or enNTS1.22 Cells were stimulated with vehicle (brown) or 1 μM NT8-13 (grey). Error bars represent SEM from three independent experiments. (B) βArr1 recruitment to V2R (Mock), hNTS1, and enNTS1 was measured using a NanoBiT-based assay.23 Cells were stimulated with vehicle (brown) or 1 μM NT8-13 (grey). Luminescence counts recorded from 5-10 min following stimulation were averaged and normalized to the initial counts. Error bars represent SEM from four independent experiments. (C) Deconvoluted 19F-NMR spectra of enNTS1[Q301CBTFMA] in various liganded states. All ligands added to receptor at 10 Meq. The relative population and LWHH are indicated for each substate. (D) The chemical shift value of each deconvoluted resonance was confirmed by monitoring the residual error while constraining peak height and LWHH. The chemical shift was constrained to a new value and the procedure repeated. The lowest residual error value for each substate represents the chemical shift used in deconvolution.24
It is unclear which enNTS1 thermostabilizing mutations are responsible for attenuating G protein activation and βArr1 recruitment. We reverted stabilizing mutations adjacent to the connector region (V358F 7.42) and within the sodium binding site (S113
