Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

Abstract

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor

“…GHSR was expressed in inclusion bodies in E. coli using batch-fed fermentation (Schrottke et al, 2017). GHSR was reconstituted into lipid bicelles formed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC).…”

“…We used bicelles as they provide the best lipid systems for the reconstitution of high amounts of functional GHSR. Receptor functionality was confirmed by saturation ligand binding using atto520-labeled ghrelin peptide as in Figure S1 (Schrottke et al, 2017).…”

“…A unique challenge of peptide ligands is the large degree of freedom associated with each amino acid. In addition, GPCRs possess a vast degree of internal flexibility, as seen in spectroscopic studies (Isogai et al, 2016;Kofuku et al, 2012;Manglik et al, 2015;Schafer and Farrens, 2015;Schrottke et al, 2017;Sounier et al, 2015;Ye et al, 2016). Flexibility within the receptor loops is likely to select for different ligand types, as seen in crystal structures of the C5a receptor, in which ECL2 adopted different conformations for small-molecule-bound versus peptide-bound states (Liu et al, 2018;Robertson et al, 2018).…”

“…13C/15N-amino acids were coupled manually with 5 equivalents of Fmoc-protected amino acid, 5 equic HOBt and 5 equivalents of DIC in DMF overnight. Fluorescent tracer [Dpr 3 -Oct; Dpr 16 -atto520]-Ghrelin (Dpr, diaminopropionic acid; Oct, octanoic acid) for in vitro functionality assay was prepared as described (Schrottke et al, 2017). Briefly, 15 mmol of Boc-[Dpr(Mtt) 3 , Dpr(ivDde) 16 ]-ghrelin was synthesized automatically on R-Wang resin.…”

“…Ghrelin is best characterized as an orexigenic peptide, as increased levels stimulate hunger via GHSR activation (Cummings et al, 2001). GHSR has high constitutive activity (Holst et al, 2004) and dynamism in the apo state (Schrottke et al, 2017). Mutational work has examined the structural basis for this basal activity (Goze et al, 2010;Holst et al, 2010;Valentin-Hansen et al, 2012) and identified ligand binding sites (Els et al, 2012;Holst et al, 2004Holst et al, , 2007Holst et al, , 2009Liu et al, 2007;Mokrosinski et al, 2012).…”