Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: 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 indicated in superscript for each peptide residue. 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, either from the cytoplasmic view ( e ) or from inside the membrane plane ( f ). Arrows indicate significant relative spatial differences of transmembrane helices (TM5, TM6, and TM7).

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques: Sequencing, Cryo-EM Sample Prep

Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: a – c Sphere representations of NDP-α-MSH (green carbon atoms), setmelanotide (purple carbon atoms), and SHU9119 (blue carbon atoms) bound into their respective binding sites in MC4R (clipped surface, orange, light-yellow, gray, respectively). d – f TM3 and TM6 receptor residues involved in peptide and/or Ca 2+ interactions are highlighted. The bold red dashed line in d , e indicates no interactions of L133 3.36 with agonist residue d -F 1 ; the red dashed bidirectional arrow in e indicates a hydrophobic interaction between F261 6.51 and setmelanotide d -F5 1 (Supplementary information, Table ), and in f indicates an interaction between d -Nal4 1 of the antagonist SHU9119 and L133 3.36 . g – i Top view of d – f , highlighting all intermolecular hydrophilic interactions, assessed by a minimum distance of 3.5 Å (black dashed lines) (Supplementary information, Figs. – and Tables – ). NDP-α-MSH residues 4–11 are shown ( g ).

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques: Binding Assay

Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: Superpositions of NDP-α-MSH (residues 4–11), setmelanotide, and SHU9119 bound at MC4R (orange, light-yellow, and gray backbone cartoons). a Superposition of both agonists. Residues that are shared by the agonists are labeled in pink. b , c The ligand-binding mode of SHU9119 is compared with NDP-α-MSH ( b ) and setmelanotide ( c ). Bi-directional red arrows indicate differences in the relative spatial positioning of residues, the TM3, or the Ca 2+ ion. Detailed intermolecular interactions are summarized in Supplementary information, Tables and . d The relative alterations of TM3 orientation are also found in Ca 2+ and its interacting residues of the EDD motif.

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques: Labeling, Ligand Binding Assay

Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: a – h Signaling data for MC4R variants harboring mutations in the ligand-binding region ( a , c , e , g ) and in the G-protein interface ( b , d , f , h ) are plotted. Concentration-response curves for cAMP accumulation (Gs-activation) upon NDP-α-MSH ( a , b ) and setmelanotide ( e , f ) stimulation. Concentration-response curves of Gq/11 signaling determined by the activation of PLC of wild-type MC4R (WT) or MC4R mutants upon NDP-α-MSH ( c , d ) and setmelanotide ( g , h ) stimulation. Additional signaling data are shown in Supplementary information, Fig. , including α-MSH-induced MC4R signaling. E MAX and EC 50 values are summarized in Supplementary information, Tables − .

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques: Ligand Binding Assay, Concentration Assay, Activation Assay

Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: a Superposition of the agonist NDP-α-MSH (green/orange) and the antagonist SHU9119 (blue/gray) bound to MC4R. The supposed activation-pathway from the LBP to the Gs-protein interface is highlighted by green arrows and is triggered by ligand interactions at TM6, inducing the TM6 opening around W258 6.48 . Antagonistic action is facilitated by the interplay of d -Nal4 1 with L133 3.36 (indicated by blue arrows) and a subsequently blocked “toggle switch” at W258 6.48 . b Top view on ligand pockets highlights (arrows) the relative positioning of transmembrane helices TM1–6 (antagonized versus active structures). c , d Comparison of active and antagonized MC4R structures. d Superposition of active NDP-α-MSH–MC4R and SHU9119–MC4R structures. Relative movements, at the CWxP 6.50 , P 5.50 (M)IF, N(D)P 7.50 xxY, and DR 3.50 Y motifs, that accompany the receptor activation are highlighted by black arrows. Key residues involved in receptor activation are shown as sticks.

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques: Activation Assay

Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: a , b Intramolecular interactions at the IL2–TM3–TM6 site stabilizing the antagonized MC4R (PDB ID: 6w25 ) ( a ), and the inactive β2AR (PDB ID: 2rh1 ) ( b ). c – e Overall view on MC4R–Gs with enlarged areas shown in d and e . Active receptor–Gs complex structures of MC4R bound to NDP-α-MSH ( d ), setmelanotide ( e ). f The corresponding boxed region ( b ) in the β2AR–Gs complex (PDB ID: 3sn6 ) Black dashed lines indicate hydrophilic interactions.

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques:

Journal: Cell Research

Article Title: Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

doi: 10.1038/s41422-021-00569-8

Figure Lengend Snippet: a – c Display of the binding interface between TM3–TM5 and Gs-protein in the NDP-α-MSH ( a ), setmelanotide ( b ) bound MC4R–Gs complexes, and the β2AR–Gs complex (PDB ID: 3sn6) ( c ). Interactions of IL2 and TM3 are displayed as black dashed lines. d – f The superposition of NDP-α-MSH–MC4R–Gs and setmelanotide–MC4R–Gs complexes with the β2AR–Gs complex display the IL2–β2/β3–α5 lock of L155 IL2 (MC4R) with V203 and F362 (Gαs), as well as F139 IL2 (β2AR) with V217 and F376 (Gαs) adjusting the position of the α5 helix. A relative α5 shift can be noticed, which results in a slight rotation of the entire Gs coupled to MC4R compared to that coupled to β2AR, most prominently visible by an αN helix shift. e Superposition of both agonist-bound MC4R–Gs structures highlight changes of the hydrogen bonds between Gαs and IL2 residues (dashed boxes).

Article Snippet: Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer (ABI 3710 XL; Applied Biosystems). α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.

Techniques: Binding Assay