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- EMDB-18442: Structure of active state MC4R in complex with a potent ligand mi... -

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Entry
Database: EMDB / ID: EMD-18442
TitleStructure of active state MC4R in complex with a potent ligand mimicking nanobody
Map data
Sample
  • Complex: MC4R receptor in complex with Gs protein heterotrimer, nanobody-35/ConfoBody 35 and ligand-mimicking nanobody
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: Nanobody-35
    • Protein or peptide: Soluble cytochrome b562,Melanocortin receptor 4,Red fluorescent protein drFP583
    • Protein or peptide: pN162
KeywordsGPCR / ConfoBody / agonistic nanobody / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of eating behavior / response to bisphenol A / response to melanocyte-stimulating hormone / melanocyte-stimulating hormone receptor activity / melanocortin receptor activity / regulation of grooming behavior / energy reserve metabolic process / neuropeptide binding / feeding behavior / insulin secretion ...regulation of eating behavior / response to bisphenol A / response to melanocyte-stimulating hormone / melanocyte-stimulating hormone receptor activity / melanocortin receptor activity / regulation of grooming behavior / energy reserve metabolic process / neuropeptide binding / feeding behavior / insulin secretion / regulation of metabolic process / response to food / negative regulation of feeding behavior / diet induced thermogenesis / peptide hormone binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / positive regulation of bone resorption / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / Transcriptional and post-translational regulation of MITF-M expression and activity / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / bioluminescence / Peptide ligand-binding receptors / trans-Golgi network membrane / generation of precursor metabolites and energy / response to insulin / negative regulation of inflammatory response to antigenic stimulus / electron transport chain / bone development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled 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 / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / 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 / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / positive regulation of cold-induced thermogenesis / G protein activity / GTPase binding / Ca2+ pathway / retina development in camera-type eye / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / periplasmic space / electron transfer activity / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway
Similarity search - Function
Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / G-protein alpha subunit, group S / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein ...Melanocortin 4 receptor / Melanocortin receptor 3-5 / Melanocortin/ACTH receptor / G-protein alpha subunit, group S / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / 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. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Soluble cytochrome b562 / 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 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Red fluorescent protein drFP583
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama) / Discosoma sp. (sea anemone)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBusch A / Jaakola V-P / Masiulis S
Funding support Belgium, 2 items
OrganizationGrant numberCountry
Other government2016-R-49 Belgium
Other governmentHBC_2020.2042 Belgium
CitationJournal: Nat Commun / Year: 2024
Title: Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist.
Authors: Thomas Fontaine / Andreas Busch / Toon Laeremans / Stéphane De Cesco / Yi-Lynn Liang / Veli-Pekka Jaakola / Zara Sands / Sarah Triest / Simonas Masiulis / Lies Dekeyzer / Noor Samyn / ...Authors: Thomas Fontaine / Andreas Busch / Toon Laeremans / Stéphane De Cesco / Yi-Lynn Liang / Veli-Pekka Jaakola / Zara Sands / Sarah Triest / Simonas Masiulis / Lies Dekeyzer / Noor Samyn / Nicolas Loeys / Lisa Perneel / Melanie Debaere / Murielle Martini / Charlotte Vantieghem / Richa Virmani / Kamila Skieterska / Stephanie Staelens / Rosa Barroco / Maarten Van Roy / Christel Menet /
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 ...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.
History
DepositionSep 12, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_18442.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 180 pix.
= 256.5 Å
1.43 Å/pix.
x 180 pix.
= 256.5 Å
1.43 Å/pix.
x 180 pix.
= 256.5 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.425 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.0016747486 - 1.8374246
Average (Standard dev.)0.0011681758 (±0.024647027)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 256.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18442_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_18442_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_18442_half_map_2.map
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Sample components

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Entire : MC4R receptor in complex with Gs protein heterotrimer, nanobody-3...

EntireName: MC4R receptor in complex with Gs protein heterotrimer, nanobody-35/ConfoBody 35 and ligand-mimicking nanobody
Components
  • Complex: MC4R receptor in complex with Gs protein heterotrimer, nanobody-35/ConfoBody 35 and ligand-mimicking nanobody
    • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
    • Protein or peptide: Nanobody-35
    • Protein or peptide: Soluble cytochrome b562,Melanocortin receptor 4,Red fluorescent protein drFP583
    • Protein or peptide: pN162

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Supramolecule #1: MC4R receptor in complex with Gs protein heterotrimer, nanobody-3...

SupramoleculeName: MC4R receptor in complex with Gs protein heterotrimer, nanobody-35/ConfoBody 35 and ligand-mimicking nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 1 / Details: Dominant-negative / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.699434 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV R ACYERSNE ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV R ACYERSNE YQLIDCAQYF LDKIDVIKQA DYVPSDQDLL RCRVLTSGIF ETKFQVDKVN FHMFDVGAQR DERRKWIQCF ND VTAIIFV VASSSYNMVI REDNQTNRLQ AALKLFDSIW NNKWLRDTSV ILFLNKQDLL AEKVLAGKSK IEDYFPEFAR YTT PEDATP EPGEDPRVTR AKYFIRDEFL RISTASGDGR HYCYPHFTCS VDTENIRRVF NDCRDIIQRM HLRQYELL

UniProtKB: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 38.534062 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHGSS GSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKL IIWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC R FLDDNQIV ...String:
MHHHHHHGSS GSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKL IIWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC R FLDDNQIV TSSGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQTFTGHESD IN AICFFPN GNAFATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DALKADRAGV LAG HDNRVS CLGVTDDGMA VATGSWDSFL KIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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Macromolecule #4: Nanobody-35

MacromoleculeName: Nanobody-35 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 15.140742 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLEWVSD ISQSGASISY TGSVKGRFTI SRDNAKNTLY LQMNSLKPE DTAVYYCARC PAPFTRDCFD VTSTTYAYRG QGTQVTVSSH HHHHHEPEA

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Macromolecule #5: Soluble cytochrome b562,Melanocortin receptor 4,Red fluorescent p...

MacromoleculeName: Soluble cytochrome b562,Melanocortin receptor 4,Red fluorescent protein drFP583
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Discosoma sp. (sea anemone)
Molecular weightTheoretical: 82.025141 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: FADYKDDDDK AADLEDNWET LNDNLKVIEK ADNAAQVKDA LTKMRAAALD AQKATPPKLE DKSPDSPEMK DFRHGFDILV GQIDDALKL ANEGKVKEAQ AAAEQLKTTR NAYIQKYLLE VLFQGPMVNS THRGMHTSLH LWNRSSYRLH SNASESLGKG Y SDGGCYEQ ...String:
FADYKDDDDK AADLEDNWET LNDNLKVIEK ADNAAQVKDA LTKMRAAALD AQKATPPKLE DKSPDSPEMK DFRHGFDILV GQIDDALKL ANEGKVKEAQ AAAEQLKTTR NAYIQKYLLE VLFQGPMVNS THRGMHTSLH LWNRSSYRLH SNASESLGKG Y SDGGCYEQ LFVSPEVFVT LGVISLLENI LVIVAIAKNK NLHSPMYFFI CSLAVADMLV SVSNGSETIV ITLLNSTDTD AQ SFTVNID NVIDSVICSS LLASICSLLS IAVDRYFTIF YALQYHNIMT VKRVGIIISC IWAACTVSGI LFIIYSDSSA VII CLITMF FTMLALMASL YVHMFLMARL HIKRIAVLPG TGAIRQGANM KGAITLTILI GVFVVCWAPF FLHLIFYISC PQNP YCVCF MSHFNLYLIL IMCNSIIDPL IYALRSQELR KTFKEIICCY PLGGLCDLSS RYLEVLFQGP VSKGEEDNMA IIKEF MRFK VHMEGSVNGH EFEIEGEGEG RPYEGTQTAK LKVTKGGPLP FAWDILSPQF MYGSKAYVKH PADIPDYLKL SFPEGF KWE RVMNFEDGGV VTVTQDSSLQ DGEFIYKVKL RGTNFPSDGP VMQKKTMGWE ASSERMYPED GALKGEIKQR LKLKDGG HY DAEVKTTYKA KKPVQLPGAY NVNIKLDITS HNEDYTIVEQ YERAEGRHST GGMDELYKHH HHHHHHHHGL NDIFEAQK I EWHEEPEA

UniProtKB: Soluble cytochrome b562, Melanocortin receptor 4, Red fluorescent protein drFP583

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Macromolecule #6: pN162

MacromoleculeName: pN162 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 12.655162 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQPGGSLRL SCAASGSAFS LNVMGWYRQT PGNQRALVAE ITKDGSTNYA DSAKGRFTIS RDNAKNTVHL QMDSLKPED TAVYYCKART GRIVRPLDYW GQGTQVTV

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10.4 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 30.3 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 219891
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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