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- PDB-7piv: Active Melanocortin-4 receptor (MC4R)- Gs protein complex bound t... -

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Basic information

Entry
Database: PDB / ID: 7piv
TitleActive Melanocortin-4 receptor (MC4R)- Gs protein complex bound to agonist NDP-alpha-MSH at 2.86 A resolution.
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Camelid antibody VHH fragment - nanobody 35
  • Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Melanocortin receptor 4
  • NDP-alpha-MSH (other names Afamelanotide; Scenesse)
KeywordsSIGNALING PROTEIN / GPCR / MELANOCORTIN-4 RECEPTOR / MELANOCORTIN RECEPTORS / SETMELANOTIDE / NDP-ALPHA-MSH / ALPHA-18 MSH / ANTAGONISM / AGONISM / APPETITE REGULATION / ANTI-OBESITY TREATMENT
Function / homology
Function and homology information


regulation of eating behavior / melanocyte-stimulating hormone receptor activity / response to melanocyte-stimulating hormone / melanocortin receptor activity / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma ...regulation of eating behavior / melanocyte-stimulating hormone receptor activity / response to melanocyte-stimulating hormone / melanocortin receptor activity / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Prostacyclin signalling through prostacyclin receptor / G alpha (z) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / G beta:gamma signalling through BTK / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thromboxane signalling through TP receptor / Thrombin signalling through proteinase activated receptors (PARs) / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Ca2+ pathway / G alpha (s) signalling events / G alpha (q) signalling events / Extra-nuclear estrogen signaling / G alpha (12/13) signalling events / regulation of grooming behavior / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (i) signalling events / GPER1 signaling / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADP signalling through P2Y purinoceptor 1 / energy reserve metabolic process / neuropeptide binding / regulation of metabolic process / G-protein activation / Activation of the phototransduction cascade / : / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Activation of G protein gated Potassium channels / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G alpha (z) signalling events / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / feeding behavior / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / alkylglycerophosphoethanolamine phosphodiesterase activity / insulin secretion / photoreceptor outer segment membrane / spectrin binding / diet induced thermogenesis / negative regulation of feeding behavior / PKA activation in glucagon signalling / peptide hormone binding / hair follicle placode formation / intracellular transport / photoreceptor outer segment / D1 dopamine receptor binding / developmental growth / positive regulation of bone resorption / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / cardiac muscle cell apoptotic process / activation of adenylate cyclase activity / photoreceptor inner segment / adenylate cyclase activator activity / Peptide ligand-binding receptors / trans-Golgi network membrane / G-protein beta/gamma-subunit complex binding / response to insulin / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / bone development / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / platelet aggregation / Glucagon-type ligand receptors / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding
Similarity search - Function
Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit ...Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / 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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Melanocortin receptor 4 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
Mus musculus (house mouse)
Lama glama (llama)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsHeyder, N.A. / Schmidt, A. / Kleinau, G. / Hilal, T. / Scheerer, P.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Research Foundation (DFG)CRC1423, project number 421152132, subprojects A01, A05, Z03, B02, C01, Z04 Germany
German Research Foundation (DFG)CRC1365, project number 394046635, subproject A03 Germany
German Research Foundation (DFG)Excellence Strategies - EXC2008/1 (UniSysCat) - 390540038 Germany
CitationJournal: Cell Res / Year: 2021
Title: Structures of active melanocortin-4 receptor-Gs-protein complexes with NDP-α-MSH and setmelanotide.
Authors: Nicolas A Heyder / Gunnar Kleinau / David Speck / Andrea Schmidt / Sarah Paisdzior / Michal Szczepek / Brian Bauer / Anja Koch / Monique Gallandi / Dennis Kwiatkowski / Jörg Bürger / ...Authors: Nicolas A Heyder / Gunnar Kleinau / David Speck / Andrea Schmidt / Sarah Paisdzior / Michal Szczepek / Brian Bauer / Anja Koch / Monique Gallandi / Dennis Kwiatkowski / Jörg Bürger / Thorsten Mielke / Annette G Beck-Sickinger / Peter W Hildebrand / Christian M T Spahn / Daniel Hilger / Magdalena Schacherl / Heike Biebermann / Tarek Hilal / Peter Kühnen / Brian K Kobilka / Patrick Scheerer /
Abstract: The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of ...The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R-Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor-Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.
History
DepositionAug 23, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
R: Melanocortin receptor 4
P: NDP-alpha-MSH (other names Afamelanotide; Scenesse)
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: Camelid antibody VHH fragment - nanobody 35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,0017
Polymers144,9616
Non-polymers401
Water2,072115
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14830 Å2
ΔGint-95 kcal/mol
Surface area40800 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules RA

#1: Protein Melanocortin receptor 4 / / MC4-R


Mass: 38698.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MC4R / Plasmid: pOET3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P32245
#3: Protein Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 44326.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Variant: 2 / Plasmid: pFastbac / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092

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

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


Mass: 37728.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnb1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P54311
#5: 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.) Mus musculus (house mouse) / Gene: Gng2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63213

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Protein/peptide / Antibody , 2 types, 2 molecules PN

#2: Protein/peptide NDP-alpha-MSH (other names Afamelanotide; Scenesse)


Mass: 1632.863 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: Antibody Camelid antibody VHH fragment - nanobody 35


Mass: 14714.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli) / Strain (production host): WK6

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Non-polymers , 2 types, 116 molecules

#7: Chemical ChemComp-CA / CALCIUM ION


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


Mass: 18.015 Da / Num. of mol.: 115 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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

ComponentName: Ternary complex of the NDP-alpha-MSH activated melanocortin 4 receptor (MC4R) Gs protein complex stabilized by nanobody 35
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 144.6 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Rattus norvegicus (Norway rat)10116
31Mus musculus (house mouse)10090
41Lama glama (llama)9844
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaClSodium chloride1
30.025 mMTris(2-carboxyethyl)phosphinC9H15O6P1
41 mMcalcium chlorideCaCl21
50.001 mMNDP-alpha-MSHC78H111N21O191
60.00075 %2,2-didecylpropane-1,3-bis-beta-D-maltopyranosideC47H88O221
70.00025 %GDN101C56H92O251
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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 FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40.78 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5618

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.15particle selection
2EPU2.8image acquisition
4cryoSPARC2.15CTF correction
7cryoSPARCmodel fitting
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
11cryoSPARC2.15classification
12cryoSPARC2.183D reconstruction
19Cootmodel refinement
20CCP4 packagemodel refinement
21PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2746119
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 221682 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
13SN6

3sn6

1
26W25

6w25

1

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