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- PDB-7f54: Cryo-EM structure of afamelanotide-MC4R-Gs_Nb35 complex -

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Basic information

Entry
Database: PDB / ID: 7f54
TitleCryo-EM structure of afamelanotide-MC4R-Gs_Nb35 complex
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Melanocortin receptor 4
  • Nanobody35
  • afamelanotide
KeywordsMEMBRANE PROTEIN / single particle / Class A G-protein-coupled receptors
Function / homology
Function and homology information


regulation of eating behavior / melanocyte-stimulating hormone receptor activity / response to melanocyte-stimulating hormone / melanocortin receptor activity / regulation of grooming behavior / energy reserve metabolic process / feeding behavior / regulation of metabolic process / neuropeptide binding / insulin secretion ...regulation of eating behavior / melanocyte-stimulating hormone receptor activity / response to melanocyte-stimulating hormone / melanocortin receptor activity / regulation of grooming behavior / energy reserve metabolic process / feeding behavior / regulation of metabolic process / neuropeptide binding / insulin secretion / negative regulation of feeding behavior / diet induced thermogenesis / peptide hormone binding / positive regulation of bone resorption / Transcriptional and post-translational regulation of MITF-M expression and activity / Peptide ligand-binding receptors / response to insulin / Olfactory Signaling Pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Activation of the phototransduction cascade / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / retina development in camera-type eye / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / ubiquitin protein ligase binding / synapse / protein-containing complex binding / signal transduction / extracellular exosome / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs ...Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Melanocortin receptor 4 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsZhang, H. / Chen, L. / Mao, C. / Shen, Q. / Yang, D. / Shen, D. / Qin, J.
CitationJournal: Cell Res / Year: 2021
Title: Structural insights into ligand recognition and activation of the melanocortin-4 receptor.
Authors: Huibing Zhang / Li-Nan Chen / Dehua Yang / Chunyou Mao / Qingya Shen / Wenbo Feng / Dan-Dan Shen / Antao Dai / Shanshan Xie / Yan Zhou / Jiao Qin / Jin-Peng Sun / Daniel H Scharf / Tingjun ...Authors: Huibing Zhang / Li-Nan Chen / Dehua Yang / Chunyou Mao / Qingya Shen / Wenbo Feng / Dan-Dan Shen / Antao Dai / Shanshan Xie / Yan Zhou / Jiao Qin / Jin-Peng Sun / Daniel H Scharf / Tingjun Hou / Tianhua Zhou / Ming-Wei Wang / Yan Zhang /
Abstract: Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of ...Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning MC4R-specific activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric G protein stimulated by the endogenous peptide ligand α-MSH, FDA-approved drugs afamelanotide (Scenesse™) and bremelanotide (Vyleesi™), and a selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and a distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.
History
DepositionJun 21, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 3, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody35
R: Melanocortin receptor 4
L: afamelanotide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,3147
Polymers165,2746
Non-polymers401
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15150 Å2
ΔGint-100 kcal/mol
Surface area45070 Å2

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Components

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Protein , 3 types, 3 molecules ANR

#1: Protein Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 44284.074 Da / Num. of mol.: 1
Mutation: S54N, G226A, E268A, N271K, K274D, R280K, T284D, I287T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63092-2
#4: Protein Nanobody35


Mass: 13711.284 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Protein Melanocortin receptor 4 / MC4-R


Mass: 56053.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MC4R / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P32245

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 41729.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Protein/peptide , 1 types, 1 molecules L

#6: Protein/peptide afamelanotide


Mass: 1634.855 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 5 molecules

#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of afamelanotide bound MC4R-Gs proteinCOMPLEXafamelanotide MC4R Gs protein Nanobody35#1-#60MULTIPLE SOURCES
2Dominant Negative Guanine nucleotide-binding protein G(s) subunit alpha isoformsCOMPLEX#11RECOMBINANT
3Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1COMPLEX#21RECOMBINANT
4Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#31RECOMBINANT
5Nanobody35COMPLEX#41RECOMBINANT
6melanocrotin-4 receptorCOMPLEX#51RECOMBINANT
7afamelanotideCOMPLEX#61SYNTHETIC
Molecular weightValue: 0.18 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44synthetic construct (others)32630
55Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Spodoptera frugiperda (fall armyworm)7108
22Spodoptera frugiperda (fall armyworm)7108
33Spodoptera frugiperda (fall armyworm)7108
44Escherichia coli (E. coli)562
55Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 62 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 217491 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048286
ELECTRON MICROSCOPYf_angle_d0.88111215
ELECTRON MICROSCOPYf_dihedral_angle_d9.9484957
ELECTRON MICROSCOPYf_chiral_restr0.0531280
ELECTRON MICROSCOPYf_plane_restr0.0061425

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