Integrating Solid-State NMR and Computational Modeling to Investigate the Structure and Dynamics of Membrane-Associated Ghrelin

Abstract

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide’s secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide’s positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8–17 form an α-helix, while residues 21–23 and 26–27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.

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

“…Unfortunately, the use of lower tracer concentrations closer to the reported sub-nanomolar K d was obviated by the sensitivity of the fluorescence reader. Ghrelin also binds to empty phosphatidylcholine bicelles, but at much higher concentration as determined before26. As an additional control, we used fluorescently labeled neuropeptide Y (atto520-NPY), which should neither bind to the GHS receptor nor to empty phosphatidylcholine bicelles.…”

“…Here, we report results on the molecular dynamics of the human growth hormone secretagogue (GHS) receptor, which plays a key role in the regulation of appetite and food intake and, therefore, represents an interesting target for intervening with eating disorders25. The ligand of the GHS receptor is the octanoylated growth hormone ghrelin consisting of 28 amino acids262728. We used solid-state NMR spectroscopy to study the molecular dynamics of the GHS receptor reconstituted into DMPC or POPC membranes.…”

Structure and dynamics of G protein-coupled receptor–bound ghrelin reveal the critical role of the octanoyl chain

“…However, this model proposed a helical conformation for residues 9 to 18 of ghrelin, while our results indicate that this region is disordered. Helical propensity in this region was also proposed in a model of membrane-associated ghrelin obtained through a similar approach (30). Whether this difference arises from the distinct lipid models used (bicelles vs. nanodiscs) or from the experimental conditions (e.g., −30°C for the solid-state NMR experiments vs. buffered solution at 7°C) is an open question.…”

“…Interestingly, we observed the specific ordering of the N-terminal region in CG modeling, despite starting from a ghrelin peptide in solution with a minimal set of NMR-derived restraints. Associated with the absence of any well-defined structure in the absence of GHSR (30), this strongly indicates that rigidification of the Nterminal region of ghrelin and formation of the hydrophobic core are intimately associated with the binding process. This mode of interaction is fully consistent with previous structure-activity relationship studies.…”

Side Chain Orientation of Tryptophan Analogues Determines Agonism and Inverse Agonism in Short Ghrelin Peptides

“…[4] It is assumed that the Ser 3 lipidation with octanoic acid is important for membranei nteraction. [5] In as tructuralm odel of ghrelin boundt oi ts receptor,i tw as proposed to extend this minimal binding motif up to His 9 . [6] Therein, amino acids 1t o5a nd His 9 and Arg 11 directly interactw ith the receptor.T he N-terminal motif binds with the centralc avity in the ghrelinr eceptor,a nd the a-helix with His 9 interacts with ECL3.…”