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- PDB-6uun: CryoEM Structure of the active Adrenomedullin 1 receptor G protei... -

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Basic information

Entry
Database: PDB / ID: 6uun
TitleCryoEM Structure of the active Adrenomedullin 1 receptor G protein complex with adrenomedullin peptide
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • ADM
  • Calcitonin gene-related peptide type 1 receptor
  • Nanobody 35
  • Receptor activity-modifying protein 2
KeywordsMEMBRANE PROTEIN / GPCR / adrenomedullin receptor complex
Function / homology
Function and homology information


adrenomedullin receptor binding / positive regulation of progesterone biosynthetic process / basement membrane assembly / cellular response to sucrose stimulus / adrenomedullin binding / CGRP receptor complex / vascular associated smooth muscle cell development / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway ...adrenomedullin receptor binding / positive regulation of progesterone biosynthetic process / basement membrane assembly / cellular response to sucrose stimulus / adrenomedullin binding / CGRP receptor complex / vascular associated smooth muscle cell development / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway / regulation of urine volume / regulation of systemic arterial blood pressure / calcitonin receptor activity / calcitonin gene-related peptide receptor signaling pathway / calcitonin gene-related peptide receptor activity / amylin receptor 2 signaling pathway / bicellular tight junction assembly / positive regulation of vasculogenesis / Calcitonin-like ligand receptors / regulation of G protein-coupled receptor signaling pathway / sprouting angiogenesis / G protein-coupled receptor internalization / negative regulation of vascular permeability / adherens junction assembly / negative regulation of vasoconstriction / vasculogenesis / negative regulation of endothelial cell apoptotic process / adenylate cyclase-activating G protein-coupled bile acid receptor signaling pathway / adenylate cyclase-activating serotonin receptor signaling pathway / cellular response to vascular endothelial growth factor stimulus / regulation of skeletal muscle contraction / developmental growth / hair follicle placode formation / PKA activation in glucagon signalling / renal water homeostasis / intracellular transport / D1 dopamine receptor binding / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / positive regulation of heart rate / vascular endothelial cell response to laminar fluid shear stress / Hedgehog 'off' state / coreceptor activity / cellular response to hormone stimulus / activation of adenylate cyclase activity / adenylate cyclase-activating adrenergic receptor signaling pathway / cellular response to acidic pH / clathrin-coated pit / cellular response to glucagon stimulus / intracellular glucose homeostasis / protein localization to plasma membrane / adenylate cyclase activator activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / trans-Golgi network membrane / positive regulation of insulin secretion / negative regulation of inflammatory response to antigenic stimulus / receptor internalization / response to prostaglandin E / bone development / hormone activity / intracellular protein transport / platelet aggregation / regulation of blood pressure / cognition / G protein-coupled receptor activity / positive regulation of angiogenesis / heart development / G-protein beta/gamma-subunit complex binding / calcium ion transport / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G protein-coupled acetylcholine receptor signaling pathway / sensory perception of smell / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / Prostacyclin signalling through prostacyclin receptor / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / 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 / ADP signalling through P2Y purinoceptor 1 / cellular response to catecholamine stimulus / G beta:gamma signalling through PI3Kgamma / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / positive regulation of cold-induced thermogenesis / protein transport
Similarity search - Function
Pro-adrenomedullin / : / GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / Receptor activity modifying protein / RAMP domain superfamily / Receptor activity modifying family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / GPCR, family 2, calcitonin receptor family / Calcitonin/adrenomedullin ...Pro-adrenomedullin / : / GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / Receptor activity modifying protein / RAMP domain superfamily / Receptor activity modifying family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / GPCR, family 2, calcitonin receptor family / Calcitonin/adrenomedullin / Calcitonin / CGRP / IAPP family / G-protein coupled receptors family 2 signature 1. / : / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / Hormone receptor domain / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like / 7 transmembrane receptor (Secretin family) / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / G-protein alpha subunit, group S / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / Few Secondary Structures / Irregular / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / 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 WD-40 repeat protein / G-protein, beta subunit / WD domain, G-beta repeat / 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. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Receptor activity-modifying protein 2 / Pro-adrenomedullin / 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 / Calcitonin gene-related peptide type 1 receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsBelousoff, M.J. / Liang, Y.L. / Sexton, P. / Danev, R.
Funding support Australia, Japan, 5items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1120919 Australia
National Health and Medical Research Council (NHMRC, Australia)1159006 Australia
National Health and Medical Research Council (NHMRC, Australia)1150083 Australia
Japan Society for the Promotion of Science (JSPS)KAKENHI #18H06043 Japan
Japan Science and Technology#18069571 Japan
CitationJournal: ACS Pharmacol Transl Sci / Year: 2020
Title: Structure and Dynamics of Adrenomedullin Receptors AM and AM Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins.
Authors: Yi-Lynn Liang / Matthew J Belousoff / Madeleine M Fletcher / Xin Zhang / Maryam Khoshouei / Giuseppe Deganutti / Cassandra Koole / Sebastian G B Furness / Laurence J Miller / Debbie L Hay / ...Authors: Yi-Lynn Liang / Matthew J Belousoff / Madeleine M Fletcher / Xin Zhang / Maryam Khoshouei / Giuseppe Deganutti / Cassandra Koole / Sebastian G B Furness / Laurence J Miller / Debbie L Hay / Arthur Christopoulos / Christopher A Reynolds / Radostin Danev / Denise Wootten / Patrick M Sexton /
Abstract: Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal ...Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AMR and AMR, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AMR in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AMR in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
History
DepositionOct 30, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release
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Revision 1.2Oct 16, 2024Group: Data collection / Database references / Structure summary
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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
E: Receptor activity-modifying protein 2
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody 35
P: ADM
R: Calcitonin gene-related peptide type 1 receptor


Theoretical massNumber of molelcules
Total (without water)187,3727
Polymers187,3727
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18470 Å2
ΔGint-121 kcal/mol
Surface area52720 Å2

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

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


Mass: 45683.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38534.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2


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: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein , 3 types, 3 molecules EPR

#3: Protein Receptor activity-modifying protein 2 / Calcitonin-receptor-like receptor activity-modifying protein 2 / CRLR activity-modifying protein 2


Mass: 17839.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAMP2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O60895
#6: Protein ADM


Mass: 6038.813 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P35318
#7: Protein Calcitonin gene-related peptide type 1 receptor / CGRP type 1 receptor / Calcitonin receptor-like receptor


Mass: 56274.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALCRL, CGRPR / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16602

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Antibody , 1 types, 1 molecules N

#5: Antibody Nanobody 35


Mass: 15140.742 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)

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Details

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
IDNameTypeEntity IDParent-IDSource
1AM:CLR:RAMP2:GasDN:Gb1:Gg2:Nb35 GPCR ComplexCOMPLEXall0RECOMBINANT
2Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Receptor activity-modifying protein 2, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2, Calcitonin gene-related peptide type 1 receptorCOMPLEX#1-#4, #71RECOMBINANT
3Nanobody 35COMPLEX#51RECOMBINANT
4ADMCOMPLEX#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Lama glama (llama)9844
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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: 68 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039166
ELECTRON MICROSCOPYf_angle_d0.47412453
ELECTRON MICROSCOPYf_dihedral_angle_d7.2635412
ELECTRON MICROSCOPYf_chiral_restr0.0411439
ELECTRON MICROSCOPYf_plane_restr0.0031598

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