Solution NMR spectroscopy of GPCRs: Residue-specific labeling strategies with a focus on 13C-methyl methionine labeling of the atypical chemokine receptor ACKR3

Abstract

The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via ¹³CH₃-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.

Keywords: ACKR3; Atypical chemokine receptor 3; Chemokine receptor; G protein-coupled receptor; GPCR; Methionine NMR; Methyl labeling; NMR; Solution nuclear magnetic resonance.

Figure 1.. Comparison of different isotopic labeling approaches for solution NMR studies of GPCRs.

(A) Chemical structures of natural and modified residues used in NMR studies of GPCRs with isotopic labels indicated by colored circles and derivatized group indicated with colored bonds. (B) Comparison of ¹³C, ¹⁹F, and ¹⁵N isotope features in the context of NMR studies of GPCRs. * We designate deuteration as “advisable” for ¹⁵N labeling due to drastic improvements seen with deuteration in Eddy, et al., 2018 (Eddy, Lee, et al., 2018), although we note that Isogai, et al. 2015., perform selective ¹⁵N-Val backbone labeling with excellent results in the absence of deuteration (Isogai et al., 2016). Note that a few studies have seen improvement in ¹³C NMR studies of GPCRs with deuteration (Clark et al., 2017; Kofuku et al., 2014; Kofuku et al., 2018), but a majority of ¹³C studies are performed without deuteration with excellent peak resolution (Kofuku et al., 2012; Nygaard et al., 2013; Solt et al., 2017; Sounier et al., 2015).

Figure 2.. Distribution of residues commonly used for isotopic labeling and NMR studies among class A GPCRs.

(A) Distributions of different residue types among class A GPCRs. Sequences of class A GPCRs were obtained from GPCRdb (http://gpcrdb.org; (Pandy-Szekeres et al., 2018)) and analyzed for the distribution of residue numbers for Ala, Cys, Gly, Ile, Lys, Met, and Val. Cys. Means are shown within each “violin” as diamonds. Among residues that have been used for isotopic labeling (Table 1), Met, Cys, and Lys (highlighted in gray) are represented ~10–15 times in most class A GPCR sequences, such that selection of any of these residues for specific labeling should yield a small but manageable number of residues. Other probes (e.g., Ala and Val) are represented ~27 on average in a GPCR, such that labeling these will yield crowded spectra in the absence of extensive mutagenesis, particularly in ¹³CH₃ experiments where chemical shift ranges are relatively narrow. (B) Probe distribution for different labeling strategies used in NMR studies of GPCRs. Probes that were labeled are represented as spheres with the labeled regions shaded. Whereas Cys and Lys residues are excluded to cytosolic-facing sites, Met labeling is the only of these three labeling strategies that has access to the transmembrane region. PDB IDs 3SN6 (B2AR), 5C1M (μOR), 5G53 (A2A).

Figure 3.. Purification scheme for isotopically labeled GPCRs and validation of ¹³CH₃-methionine-labeled ACKR3 purity and thermostability.

(A) Purification scheme of isotopically-labeled GPCRs for insect cell system. Insect cells are infected with high-titer baculovirus and harvested 48h later. Cells are grown with ¹³CH₃-methionine in methionine-deficient media. Receptors are solubilized by incubating cell pellets with detergent (LMNG/CHS micelles) in the presence of ligand. Detergent-solubilized receptor is purified using a cobalt column, followed by tag removal, deglycosylation, and buffer exchange into NMR-suitable buffer. The data presented in (B) and (C) reflect ¹³CH₃-methionine-labeled ACKR3, but alternatives for isotopic labeling are also depicted. For instance, isotopic labels can be incorporated after affinity column purification by chemical derivatization methods to yield cysteine- or lysine-labeled GPCRs. (B) Representative SDS-PAGE gel stained with Coomassie gel showing pre-cut WT-ACKR3-CCX777 complex, C-terminal FLAG-10x-His cut ACKR3-CCX777, and the final deglycosylated ACKR3-CCX777 sample used for NMR studies. (C) Size exclusion chromatography trace demonstrates a pure, monodisperse ACKR3-CCX777 sample for NMR studies. The sample was run on a Superdex S200 10/300GL Increase column at a flow rate of 0.5ml/min. The column was run using NMR Exchange Buffer, described in Section 2.3.3. (D) CPM melting assay of ¹³CH₃-methionine labeled WT-ACKR3-CCX777 gives a Tm consistent with previously published studies (Gustavsson et al., 2016). See Gustavsson, 2016 for experimental details.

Figure 4.. NMR studies of ACKR3 in detergent micelles and analysis of peak intensity and position.

(A) ¹H-¹³C-HSQC of ¹³CH₃-methionine labeled ACKR3. Positive peaks (solid contours) putatively corresponding to labeled methionines are numbered from 1–9. The construct utilized for these experiments contains 8 methionines. Negative peaks (dashed contours) correspond to natural abundance buffer or detergent signals aliased from outside the ¹³C spectral window. (B) Analysis of relative peak volumes. Peaks were normalized relative to peak 7. (C) ¹³C-ε chemical shift values encode information about methionine χ₃ angles (left, middle) (Butterfoss et al., 2010; London et al., 2008). Left: chemical shift values in the shaded ~15.8–16.8 p.p.m. region correlate with gauche conformations (~ ±67°). The appearance of peaks 1 and 2 in this region suggests that they corresponds to a methionine in gauche conformation. Middle: chemical shift values in the shaded 18.4–19.5 p.p.m. region correlate with a trans conformations (~180°). The appearance of peak 9 in this region suggests that it corresponds to a methionine in trans conformation. Right: ¹H chemical shift values encode information about probe proximity to aromatic sidechains. Upfield deviations from ~2.1 p.p.m. in the ¹H dimension indicate close proximity to aromatic side chains (Kofuku et al., 2012; D. Liu & Wuthrich, 2016). The appearance of peaks 1–4 in this region suggests that these peaks correspond to methionines experiencing ring-current shifts. Left and middle panels were inspired by (Solt et al., 2017).

Figure 5.. Peak assignment of residue Met138 using ACKR3 ΔMethionine reintroduction construct.

(A) Overlay of WT-ACKR3 (blue) with ACKR3 ΔMet construct lacking all methionines. Peaks that disappear are labeled in blue and correspond to signals from the 8 methionines in the ACKR3 construct. Residual peaks 3 and 6a likely correspond to buffer and detergent signal. Peak 6 partially disappears (i.e., 6a), however some portion of that peak remains (i.e., 6b), possibly corresponding to a labeled methionine peak that is obscured by peak 6a. (B) Overlay of WT-ACKR3 (blue) with ACKR3 ΔMet construct with Met138 reintroduced (i.e., ACKR3 ΔMet I138M). As denoted by the red asterisks, one major peak reappears with reintroduction of Met138, suggesting that peak 1 corresponds to residue Met138. Note that two additional peaks (peaks 4 and 5) show some intensity with Met138 reintroduction that may correspond to alternate states of Met138. (C) Zoomed in region from (A), top, and (B), bottom. Overlays of WT-ACKR3 spectrum and ACKR3 ΔMet (top) or ACKR3 ΔMet I138M (bottom) shows reappearance of a peak at approximately 1.30 ppm (¹H) x 16 ppm (¹³C), supporting assignment of this peak as residue 138.