Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling

Abstract

G protein‐coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein‐stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope‐labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist‐induced activity of the receptor. Furthermore, this assay can be used as a readout when re‐introducing agonist‐dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19F‐labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.

Complexes of the neurotensin receptor 1 with small-molecule ligands reveal structural determinants of full, partial, and inverse agonism

“…R149 3.32 is linked to TM2 via a salt bridge with E124 2.61 and to TM7 via a cation- interaction with F358 7.42 . In a recent nuclear magnetic resonance (NMR) study on NTSR1-H4, it was shown that the mutation E124 2.61 D, which weakens the interaction with R149 3.32 , stabilizes the active state of the receptor (50). In agreement, the mutations R149 3.32 M and F358 7.42 A result in increased affinity for NTS [8][9][10][11][12][13] (Fig.…”

“…Data were normalized to the response of 10 M NTS 8-13 and are shown as means ± SEM from 3 to 11 independent experiments performed in duplicate. EC 50 with the ability to mimic the binding mode of the endogenous peptide agonist NTS. Last, the helical and side-chain rearrangements observed in the extracellular receptor half, in combination with functional data, provide detailed insights into the transmission of the activation trigger to the cytosolic side through a polar network and an aromatic cluster beneath the agonist pocket.…”

The role of structural dynamics in GPCR‐mediated signaling

“…[58]), electron paramagnetic resonance (EPR) (reviewed in Refs [59,60]), and fluorescence spectroscopy (reviewed in Refs [61,62]) supported by molecular dynamics (MD) simulations (reviewed in Refs [63,64]) have been used in complementation to crystallographic and cryo-EM studies to analyze different conformational states of GPCRs and to determine their liganddependent energetics and rates of interconversion. Several NMR studies on the b 1 and b 2 adrenergic receptors (b 1 AR and b 2 AR) [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80], the adenosine A 2A receptor (A 2A R) [81][82][83][84][85][86][87], the l-opioid receptor (lOR) [88,89], the leukotriene B 4 receptor 2 (BLT2R) [90], the a 1A adrenergic receptor (a 1A R) [91], the neurotensin receptor type 1 (NTSR1) [92], and the M 2 R [93,94] have shown that GPCRs are highly dynamic proteins that exist in an equilibrium between multiple functionally relevant conformational states. Among these receptors, the b 2 AR and the A 2A R are the most extensively studied GPCRs in terms of their conformational dynamics.…”

NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17

“…MMTS labeling was done according to a published protocols [29] with specific modifications required for a membrane protein [30]. After chromatographic separation by a semipreparative S200 (300/10) column the pooled fractions were concentrated in Amicon 15 centrifugal devices (10,000 MWCO) to approx.…”