New Results
Gunnar Kleinau, David Speck, Andrea Schmidt, Sarah Paisdzior, Michal Szczepek, Brian Bauer, Anja Koch, Monique Gallandi, Dennis Kwiatkowski, Jörg Bürger, Thorsten Mielke, Annette Beck-Sickinger, Peter W.Hildebrand, Christian M. T.Spahn, Daniel Hilger, Magdalena Schacherl, Heike Biebermann, Tarek Hilal, Peter Kühnen, Brian K.Kobilka, Patrick Scheerer
The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R− Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP-α-MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor− Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.
The melanocortin-4 receptor (MC4R) is one of five melanocortin receptor subtypes (MC1-5R) that share a set of similar peptidic ligands and constitute an evolutionarily related group of class A G-protein-coupled receptors (GPCRs). MCRs regulate energy homeostasis, pigmentation, cardiovascular function, and sexual functions. In particular, the MC4R plays a central role in energy balance and appetite regulation. Naturally-occurring human MC4R variants are the most frequent monogenic cause of obesity, with over 160 identified mutants.
Activation of MC4R by its natural agonists α-melanocyte-stimulating hormone (α-MSH) or β-MSH leads to appetite-reducing effects. In contrast, binding of the endogenous inverse agonist agouti-related peptide (AgRP) causes orexigenic effects by reducing high levels of basal signaling activity.
Besides stimulation of heterotrimeric G s αβγ protein (Gs), MC4R can elicit other signaling pathways associated with the recruitment of Gq, Gi, or arrestin.
To date, pharmacological approaches targeting MC4R have failed mostly due to the severe adverse effects of drug candidates caused for instance by a lack of MCR subtype selectivity or G-protein pathway specificity.
Setmelanotide (also termed RM-493, BIM-22493 or IRC-022493) is the first FDA-approved (2020) medication (brand name Imcivree) for the treatment of rare genetic conditions resulting in obesity, including pro-opiomelanocortin deficiency (POMC), proprotein subtilisin/kexin type 1 deficiency (PCSK1), and leptin receptor deficiency (LEPR). Setmelanotide is a cyclic high-affinity peptide with a G-protein signaling profile biased towards Gq. Further, it has a 20-fold subtype selectivity towards MC4R when compared with α-MSH. In contrast to other MC4R agonists, setmelanotide does not cause common Gs-signaling related adverse effects, such as tachycardia or hypertension. Although rare, moderate adverse effects, such as skin hyperpigmentation, have been reported.
Another FDA approved (2019), synthetic but non-selective MCR agonist, NDP-α-MSH (also termed afamelanotide; brand name Scenesse) is a linear high-affinity analog of α-MSH for treatment of MC1R-driven melanogenesis, thereby preventing skin damage from sun exposure (phototoxicity) in individuals with erythropoietic protoporphyria. Moderate adverse effects of NDP-α-MSH exposure include headache, nasopharyngitis, or back pain. The numerous adverse effects of known MCR ligands call for the discovery of more selective ligands for specific MCR subtypes, in particular the MC4R, as the global increasing prevalence of human obesity is a growing medical and socioeconomic problem.
Accordingly, a comprehensive understanding of MC4R-mediated signaling regulation is of fundamental importance. To explore the structural basis of agonist action and receptor-mediated signaling, we determined two cryo-electron microscopy (cryo-EM) structures of human wild-type MC4R− Gs-protein complexes bound to agonists setmelanotide and NDP-α-MSH, with resolutions of 2.6 Å and 2.9 Å, respectively. Comparison of our active structures with the recently solved MC4R crystal structure bound to an antagonist demonstrate the essential role of calcium ion in forming a link between ligands and TM2 and TM3. In addition, the allosteric connection between the peptide agonist binding pocket (LBP) and the G-protein binding cavity (GBC) is mediated by TM3 and facilitated by TM6. Our structural insights combined with signaling data from site-directed mutagenesis reveal mechanistic details of receptor activation and inhibition.
To determine the cryo-EM structure of MC4R-signaling complexes with different agonists, we used NDP-α-MSH and setmelanotide due to their clinical significance and high in vitro binding potency (K i = 0.7 nM and 2.1 nM, respectively) compared with α-MSH (K i = 51 nM), which was essential for generating stable complex samples. Both ligands differ in their general peptide structure. NDP-α-MSH is a linear 13-amino acid peptide and is only modified at two positions (methionine (M4) to norleucine (Nle) and L-phenylalanine (L-F) to D-phenylalanine (D-F)) compared to α-MSH. In contrast, setmelanotide is a cyclic 8-amino acid peptide, which resembles α-MSH at only three positions, in the central H 0 x 1 R 2 W 3 core motif. An unifying MCR ligand peptide numbering scheme based on the conserved H 0 x 1 R 2 W 3 motif is introduced and is used throughout the text.
Figure 1:Cryo-EM complex structures of MC4R− Gs bound with NDP-α-MSH and setmelanotide.
(A) Sequence alignment of MC4R agonists and antagonist SHU9119. For comparison and simplification, a ligand unifying numbering system is introduced based on the α-MSH core HxRW motif and will be indicated in superscript for each peptide residuesD.
(B− C) Cryo-EM densities of MC4R− Gs complexes stabilized by Nb35 and bound agonists, displayed from mirrored perspectives; Gβ, dark blue; Gγ, red; Nb35, yellow.
(D) Superposition of both agonists.
(E− F) Comparison between SHU9119-antagonized (blue) MC4R and the active state structures, (E) from the cytoplasmic view, (F) from inside the membrane plane. Arrows indicate significant relative spatial transmembrane helix differences (TM5, TM6 and TM7).
We confirmed the binding potency of both ligands using a nano-luciferase-based BRET assay. With our established workflow using Sf 9 insect cells, we produced the human wild-type MC4R without modifications in sufficient protein amounts for assembling a complex with the Gs-protein. There was a strong tendency for the MC4R to oligomerize after detergent-based extraction from membranes. This oligomer population was reduced by direct formation of the complex with detergent-purified Gs-protein and MC4R that remained membrane-embedded. After stabilization of the complex through the addition of nanobody-35 (Nb35) and nucleotide hydrolase apyrase, the resulting nucleotide-free agonist-bound MC4R− Gs− Nb35 complexes were extracted from membranes by solubilization and purified. The stable complexes with either agonists NDP-α-MSH or setmelanotide were used for cryo-EM grid preparation and single-particle analysis, yielding cryo-EM maps with a global resolution of 2.9 Å and 2.6 Å, respectively.
The cryo-EM maps revealed well-defined densities that facilitate unambiguous modeling of the secondary structure and side chain orientations of the MC4R− Gs complex as well as the cofactor ion calcium (Ca 2+), several water molecules, and the agonist peptides NDP-α-MSH and setmelanotide bound to the orthosteric LBP.
Only a few components known for their high flexibility, such as the receptor N-(Ntt) and C-termini (Ctt), the intracellular loop (IL) 1, IL3, and the extracellular loop (EL) 1, as well as the alpha-helical domain of Gαs, were not fully built into the final map (modeled residues are listed in suppl. information Methods).
Both agonists are bound foremost with the H 0 x 1 R 2 W 3 motif between the ELs and within the transmembrane bundle of the receptor. The ligands engage MC4R through extensive van der Waals, hydrophobic, and polar interactions, with residues in the transmembrane helices (TMs) as well as EL2. Specific structural MC4R
