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- PDB-6uva: CryoEM Structure of the active Adrenomedullin 2 receptor G protei... -

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Entry
Database: PDB / ID: 6uva
TitleCryoEM Structure of the active Adrenomedullin 2 receptor G protein complex with adrenomedullin 2 peptide
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Calcitonin gene-related peptide type 1 receptor
  • Nanobody 35
  • Protein ADM2
  • Receptor activity-modifying protein 3
KeywordsMEMBRANE PROTEIN / GPCR / adrenomedullin receptor complex
Function / homology
Function and homology information


G protein-coupled receptor signaling pathway involved in heart process / CGRP receptor complex / calcitonin gene-related peptide receptor signaling pathway / adrenomedullin binding / cellular response to sucrose stimulus / cross-receptor inhibition within G protein-coupled receptor heterodimer / amylin receptor complex 3 / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway ...G protein-coupled receptor signaling pathway involved in heart process / CGRP receptor complex / calcitonin gene-related peptide receptor signaling pathway / adrenomedullin binding / cellular response to sucrose stimulus / cross-receptor inhibition within G protein-coupled receptor heterodimer / amylin receptor complex 3 / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway / amylin receptor activity / calcitonin receptor activity / vascular associated smooth muscle cell proliferation / calcitonin gene-related peptide receptor activity / amylin receptor signaling pathway / Calcitonin-like ligand receptors / regulation of systemic arterial blood pressure / feeding behavior / positive regulation of heart rate / positive regulation of receptor recycling / positive regulation of protein kinase A signaling / PKA activation in glucagon signalling / response to amyloid-beta / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / hair follicle placode formation / mu-type opioid receptor binding / developmental growth / corticotropin-releasing hormone receptor 1 binding / intracellular transport / D1 dopamine receptor binding / Hedgehog 'off' state / coreceptor activity / positive regulation of calcium-mediated signaling / beta-2 adrenergic receptor binding / adenylate cyclase-activating adrenergic receptor signaling pathway / cellular response to hormone stimulus / positive regulation of vascular associated smooth muscle cell proliferation / digestion / activation of adenylate cyclase activity / adenylate cyclase activator activity / negative regulation of blood pressure / trans-Golgi network membrane / cellular response to estradiol stimulus / protein localization to plasma membrane / G protein-coupled receptor activity / positive regulation of protein localization to plasma membrane / intracellular protein transport / ionotropic glutamate receptor binding / insulin-like growth factor receptor binding / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / bone development / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G-protein activation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / hormone activity / receptor internalization / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / cognition / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / platelet aggregation / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / adenylate cyclase-activating dopamine receptor signaling pathway / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / positive regulation of angiogenesis / GPER1 signaling / calcium ion transport / G-protein beta-subunit binding / Inactivation, recovery and regulation of the phototransduction cascade / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / sensory perception of smell / protein transport / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / retina development in camera-type eye
Similarity search - Function
GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / Receptor activity modifying protein / RAMP domain superfamily / Receptor activity modifying family / GPCR, family 2, calcitonin receptor family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Calcitonin/adrenomedullin / Calcitonin / CGRP / IAPP family / G-protein coupled receptors family 2 signature 1. ...GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / Receptor activity modifying protein / RAMP domain superfamily / Receptor activity modifying family / GPCR, family 2, calcitonin receptor family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Calcitonin/adrenomedullin / Calcitonin / CGRP / IAPP family / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / 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 / 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 / Few Secondary Structures / Irregular / 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 / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Receptor activity-modifying protein 3 / 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 / Protein ADM2
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 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
DepositionNov 1, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 1, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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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
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody 35
E: Receptor activity-modifying protein 3
P: Protein ADM2
R: Calcitonin gene-related peptide type 1 receptor


Theoretical massNumber of molelcules
Total (without water)185,5137
Polymers185,5137
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21910 Å2
ΔGint-123 kcal/mol
Surface area52680 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: 45699.434 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
#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: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein , 2 types, 2 molecules ER

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


Mass: 16896.650 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAMP3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O60896
#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 / Protein/peptide , 2 types, 2 molecules NP

#4: 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)
#6: Protein/peptide Protein ADM2 / Intermedin


Mass: 5106.779 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q7Z4H4

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Details

Has ligand of interestN

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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
1AM2:CLR:RAMP3: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 3, 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
4Protein ADM2COMPLEX#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: 1.18rc1_3777: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 656000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029780
ELECTRON MICROSCOPYf_angle_d0.49513262
ELECTRON MICROSCOPYf_dihedral_angle_d20.3641298
ELECTRON MICROSCOPYf_chiral_restr0.041475
ELECTRON MICROSCOPYf_plane_restr0.0031684

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