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- PDB-7yj4: Cryo-EM structure of the INSL5-bound human relaxin family peptide... -

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Entry
Database: PDB / ID: 7yj4
TitleCryo-EM structure of the INSL5-bound human relaxin family peptidereceptor 4 (RXFP4)-Gi complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • (Insulin-like peptide INSL5 ...) x 2
  • Relaxin-3 receptor 2
  • scFv16
KeywordsSTRUCTURAL PROTEIN / human relaxin family peptide receptor 4 / G protein-coupled receptor / ligand recognition
Function / homology
Function and homology information


Relaxin receptors / negative regulation of adenylate cyclase-activating adrenergic receptor signaling pathway involved in heart process / positive regulation of feeding behavior / Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G protein-coupled adenosine receptor signaling pathway / negative regulation of calcium ion-dependent exocytosis / positive regulation of urine volume / Activation of the phototransduction cascade ...Relaxin receptors / negative regulation of adenylate cyclase-activating adrenergic receptor signaling pathway involved in heart process / positive regulation of feeding behavior / Olfactory Signaling Pathway / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G protein-coupled adenosine receptor signaling pathway / negative regulation of calcium ion-dependent exocytosis / positive regulation of urine volume / Activation of the phototransduction cascade / negative regulation of adenylate cyclase activity / C-C chemokine receptor activity / C-C chemokine binding / positive regulation of neural precursor cell proliferation / G protein-coupled peptide receptor activity / gamma-aminobutyric acid signaling pathway / negative regulation of synaptic transmission / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / G alpha (q) signalling events / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / neuronal dense core vesicle / regulation of calcium ion transport / negative regulation of apoptotic signaling pathway / Adenylate cyclase inhibitory pathway / neuropeptide signaling pathway / positive regulation of insulin receptor signaling pathway / positive regulation of vascular associated smooth muscle cell proliferation / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / response to nutrient / cell chemotaxis / positive regulation of superoxide anion generation / Regulation of insulin secretion / G protein-coupled receptor binding / calcium-mediated signaling / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G protein-coupled acetylcholine receptor signaling pathway / hormone activity / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / extracellular vesicle / signaling receptor complex adaptor activity / midbody / G alpha (i) signalling events / cell body / positive regulation of cytosolic calcium ion concentration / G alpha (s) signalling events / cell population proliferation / Extra-nuclear estrogen signaling / positive regulation of ERK1 and ERK2 cascade / positive regulation of cell migration / immune response / cell cycle / G protein-coupled receptor signaling pathway / cell division / external side of plasma membrane / GTPase activity / centrosome / synapse / dendrite / positive regulation of cell population proliferation / GTP binding / signal transduction / extracellular exosome / extracellular region / nucleoplasm / membrane / metal ion binding / plasma membrane
Similarity search - Function
Angiotensin II receptor family / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily / G-protein alpha subunit, group I / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit ...Angiotensin II receptor family / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily / G-protein alpha subunit, group I / 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 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(i) subunit alpha-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Relaxin-3 receptor 2 / Insulin-like peptide INSL5
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsChen, Y. / Zhou, Q.T. / Wang, J. / Xu, Y.W. / Wang, Y. / Yan, J.H. / Wang, Y.B. / Zhu, Q. / Zhao, F.H. / Li, C.H. ...Chen, Y. / Zhou, Q.T. / Wang, J. / Xu, Y.W. / Wang, Y. / Yan, J.H. / Wang, Y.B. / Zhu, Q. / Zhao, F.H. / Li, C.H. / Chen, C.W. / Cai, X.Q. / Bathgate, R.A.D. / Shen, C. / Liu, H. / Xu, H.E. / Yang, D.H. / Wang, M.W.
Funding support China, 11items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81872915 China
National Natural Science Foundation of China (NSFC)82073904 China
National Natural Science Foundation of China (NSFC)82121005 China
National Natural Science Foundation of China (NSFC)81973373 China
National Natural Science Foundation of China (NSFC)82130105 China
National Natural Science Foundation of China (NSFC)21704064 China
Other government2021ZD0203400
Other government2018YFA0507000
Other governmentZDKJ2021028
Other government21JC1401600
Other government1135837
CitationJournal: Nat Commun / Year: 2023
Title: Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4).
Authors: Yan Chen / Qingtong Zhou / Jiang Wang / Youwei Xu / Yun Wang / Jiahui Yan / Yibing Wang / Qi Zhu / Fenghui Zhao / Chenghao Li / Chuan-Wei Chen / Xiaoqing Cai / Ross A D Bathgate / Chun Shen ...Authors: Yan Chen / Qingtong Zhou / Jiang Wang / Youwei Xu / Yun Wang / Jiahui Yan / Yibing Wang / Qi Zhu / Fenghui Zhao / Chenghao Li / Chuan-Wei Chen / Xiaoqing Cai / Ross A D Bathgate / Chun Shen / H Eric Xu / Dehua Yang / Hong Liu / Ming-Wei Wang /
Abstract: Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and ...Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4-G protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.
History
DepositionJul 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Insulin-like peptide INSL5 A chain
B: Insulin-like peptide INSL5 B chain
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
I: Guanine nucleotide-binding protein G(i) subunit alpha-2
R: Relaxin-3 receptor 2
S: scFv16
T: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1


Theoretical massNumber of molelcules
Total (without water)158,9377
Polymers158,9377
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Insulin-like peptide INSL5 ... , 2 types, 2 molecules AB

#1: Protein/peptide Insulin-like peptide INSL5 A chain


Mass: 2221.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INSL5, UNQ156/PRO182 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y5Q6
#2: Protein/peptide Insulin-like peptide INSL5 B chain


Mass: 2850.365 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INSL5, UNQ156/PRO182 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y5Q6

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

#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.) Bos taurus (cattle) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63212
#4: Protein Guanine nucleotide-binding protein G(i) subunit alpha-2 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40502.863 Da / Num. of mol.: 1 / Mutation: S47N,G204A,E246A,A327S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI2, GNAI2B / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P04899
#7: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37915.496 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: P62871

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Protein / Antibody , 2 types, 2 molecules RS

#5: Protein Relaxin-3 receptor 2 / RLN3 receptor 2 / G-protein coupled receptor 100 / G-protein coupled receptor GPCR142 / Insulin- ...RLN3 receptor 2 / G-protein coupled receptor 100 / G-protein coupled receptor GPCR142 / Insulin-like peptide INSL5 receptor / Relaxin family peptide receptor 4


Mass: 41177.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RXFP4, GPCR142, GPR100, RLN3R2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TDU9
#6: Antibody scFv16


Mass: 26408.492 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Spodoptera frugiperda (fall armyworm)

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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
1Cryo-EM structure of the human relaxin family peptide receptor 4 in complex with INSL5 and G proteinCOMPLEXall0RECOMBINANT
2INSL5COMPLEX#1-#21RECOMBINANT
3human relaxin family peptide receptor 4COMPLEX#51RECOMBINANT
4G proteinCOMPLEX#3-#4, #71MULTIPLE SOURCES
5scFv16COMPLEX#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
44Bos taurus (cattle)9913
55synthetic construct (others)32630
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding 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: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.19 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Num. of particles: 52435 / Symmetry type: POINT

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