Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist

Abstract

The melanocortin receptor 4 (MC4R) belongs to the melanocortin receptor family of G-protein coupled receptors and is a key switch in the leptin-melanocortin molecular axis that controls hunger and satiety. Brain-produced hormones such as α-melanocyte-stimulating hormone (agonist) and agouti-related peptide (inverse agonist) regulate the molecular communication of the MC4R axis but are promiscuous for melanocortin receptor subtypes and induce a wide array of biological effects. Here, we use a chimeric construct of conformation-selective, nanobody-based binding domain (a ConfoBody Cb80) and active state-stabilized MC4R-β2AR hybrid for efficient de novo discovery of a sequence diverse panel of MC4R-specific, potent and full agonistic nanobodies. We solve the active state MC4R structure in complex with the full agonistic nanobody pN162 at 3.4 Å resolution. The structure shows a distinct interaction with pN162 binding deeply in the orthosteric pocket. MC4R peptide agonists, such as the marketed setmelanotide, lack receptor selectivity and show off-target effects. In contrast, the agonistic nanobody is highly specific and hence can be a more suitable agent for anti-obesity therapeutic intervention via MC4R.

Fig. 1. In vitro signaling potency of agonistic nanobody pN162 to melanocortin receptors.

a Dose-dependent human MC4R -induced cAMP signaling (GloSensor) of pN162 and control ligands (endogenous agonist α-MSH and clinical benchmark peptide agonist Setmelanotide). Each data point represents the mean of two replicates. Experiments were performed minimally twice (the number of experiment repeats is indicated in Table 1). Exemplary graphs are shown. Table 1 provides the average EC₅₀s of pN162, α-MSH, and setmelanotide. b Cell surface expression of human MC4R measured by flow cytometry. Overlays of fluorescence histograms obtained by anti-Flag mAb or isotype IgG control detection on Flag-tagged MC4R or mock GPCR transiently transfected HEK293T cells. Cell surface binding of 100 nM c-Myc-tagged pN162 to human MC4R (c) or melanocortin receptor subtypes (d) measured by flow cytometry. Overlays of fluorescence histogram obtained by pN162 or mock nanobody detection on human MC4R or mock GPCR transfected HEK293T cells (c). pN162 specific melanocortin receptor subtype staining, normalized for MC4R expressing HEK293 cells (d). Only intact cells were gated following Topro staining. MC2R was expressed in absence and presence of its expression chaperone MRAPα. e Dose dependent 125I-SHU9119 radioligand displacement with pN162. Within an experiment, each data point is an average of 2 repeats. Experiments were performed three times, and a representative graph is shown. Dose-dependent human MC1R (f), MC3R (g), and MC5R (h) induced cAMP signaling (GloSensor) of pN162 and control ligands (endogenous agonist α-MSH and clinical benchmark peptide agonist setmelanotide). Each data point represents the mean of two replicates. Experiments were performed minimally twice (the number of experiment repeats is indicated in Table 1). Exemplary graphs are shown. Table 1 provides the average EC₅₀s of pN162, α-MSH, and setmelanotide.

Fig. 2. Induction of human MC4R signaling pathways by pN162.

Dose-dependent Gs ConfoSensor (a) or β-arrestin recruitment (b) for agonists pN162, α-MSH, and setmelanotide in the absence (dashed lines) or presence (full lines) of 1 mM Ca²⁺. c Dose-dependent Gq signaling (production of secondary messenger IP1) of pN162, α-MSH, and setmelanotide. Data are depicted as the mean. of two replicates. Experiments were performed twice, and exemplary graphs are shown. Tables 2 and 3 provide the average EC₅₀s of pN162, α-MSH, and setmelanotide.

Fig. 3. Cryo-EM structure of pN162 bound MC4R-Gs-Cb35 complex.

a Cryo-EM density map (sharpened with DeepEMhancer, 8QJ2, and EMD_18442). b Final model submitted to the Protein Data Bank (PDB ID 8QJ2). The color code for the proteins is light blue for pN162, purple for MC4R, salmon pink for DNGαs (Gαs), light green for Gβ, orange for Gγ, and magenta for Cb35.

Fig. 4. Structural comparison between active and inactive MC4R.

a, b Side views of the overlay (MC4R receptor only) of the SHU9119-bound (dark blue), inactive state MC4R structure (gray, PDB ID 6W25) with the setmelanotide- (orange, 7PIU) or pN162-bound (cyan, 8QJ2) active state MC4R structures (receptor in pink and purple, respectively). c Bottom view of the same overlay showing the hallmark TM6 outward and the TM5 inward movement upon receptor activation (indicated with arrows). d Top view of the same 6W25-7PIU-8JQ2 overlay. e MIF motif rearrangement and impact on W258^6.48 of the CWxP toggle switch motif and L133^3.36. f D/NPxxY and ionic lock DRY motif rearrangements upon activation and its impact on residues Y212^5.58 and Y302^7.53. Side chain rearrangements between inactive and active states are indicated with arrows.

Fig. 5. Structural comparison of pN162, α-MSH, and setmelanotide active state MC4R structures in the Gαs interaction vestibule.

Two different views of the interacting residues between G-protein (green) and MC4R in complex with either α-MSH (a, d, light blue, 7F53), setmelanotide (b, e, pink, 7PIU) or pN162 (c, f, purple, 8QJ2) are shown. Red dots represent water molecules. Black dashed lines indicate H-bond interactions. Where highlighted, individual atom colors follow the standard CPK rules (red = oxygen, blue = nitrogen). All sidechains of residues ≤4 Å distance in the MC4R-Gαs interface are depicted.

Fig. 6. Binding modes of agonist and antagonist ligands to MC4R.

a–d Sphere representations of setmelanotide (green, 7PIU), α-MSH (orange, 7F53), pN162 (cyan, 8QJ2), and SHU9119 (light blue, 6W25) in their respective MC4R binding sites (pink, blue, purple and gray, respectively). The horizontal dashed blue line across panels a-d shows the maximum depth reached by all ligands. e–h Side view of the main interactions of each ligand with their respective receptor. Only residue sidechains within 4 Å distance in the respective MC4R-ligand interfaces are shown; dashed lines represent Ca²⁺ coordination and hydrogen bonds. The blue dashed line represents the same height as in (a–d). The orange shaded circle in (d, h) highlights the deep interaction of D-Nal4 from the antagonist SHU9119 that forces L133^3.36 into the inactive state conformation (see active-inactive state overlay Supplementary Fig. 6a). Top view of the key interactions involved in Ca²⁺ binding (i, j, l) and the position of pN162 CDR3 R101 residue sidechain (k) occupying the Ca²⁺ space in the binding pocket. For pN162 only CDR3 interactions are shown. For CDR1-TM7 and framework interaction with ECL3, see Supplementary Fig. 7b, d, respectively. Where highlighted, individual atom colors follow the standard CPK rules (red = oxygen, blue = nitrogen, yellow = sulfur).