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- PDB-7pd8: Structure of Adenylyl cyclase 9 in complex with DARPin C4 and MANT-GTP -

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Basic information

Entry
Database: PDB / ID: 7pd8
TitleStructure of Adenylyl cyclase 9 in complex with DARPin C4 and MANT-GTP
Components
  • Adenylate cyclase 9
  • DARPin C4
KeywordsSIGNALING PROTEIN / membrane protein / adenylyl cyclase / signalling transduction.
Function / homology
Function and homology information


Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / PKA activation / adenylate cyclase / Hedgehog 'off' state / cAMP biosynthetic process / adenylate cyclase activity / G alpha (z) signalling events / adenylate cyclase-activating adrenergic receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway ...Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / PKA activation / adenylate cyclase / Hedgehog 'off' state / cAMP biosynthetic process / adenylate cyclase activity / G alpha (z) signalling events / adenylate cyclase-activating adrenergic receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / in utero embryonic development / intracellular signal transduction / ATP binding / metal ion binding / plasma membrane / cytosol
Similarity search - Function
Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylate and Guanylate cyclase catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Guanylate cyclase domain profile. / Nucleotide cyclase
Similarity search - Domain/homology
Biological speciesBos taurus (cattle)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsQi, C. / Korkhov, V.M.
Funding support2items
OrganizationGrant numberCountry
Swiss National Science Foundation150665
Swiss National Science Foundation176992
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of adenylyl cyclase 9 activation.
Authors: Chao Qi / Pia Lavriha / Ved Mehta / Basavraj Khanppnavar / Inayathulla Mohammed / Yong Li / Michalis Lazaratos / Jonas V Schaefer / Birgit Dreier / Andreas Plückthun / Ana-Nicoleta Bondar / ...Authors: Chao Qi / Pia Lavriha / Ved Mehta / Basavraj Khanppnavar / Inayathulla Mohammed / Yong Li / Michalis Lazaratos / Jonas V Schaefer / Birgit Dreier / Andreas Plückthun / Ana-Nicoleta Bondar / Carmen W Dessauer / Volodymyr M Korkhov /
Abstract: Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the ...Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.
History
DepositionAug 4, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Source and taxonomy / Structure summary
Category: citation / em_entity_assembly ...citation / em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Mar 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Assembly

Deposited unit
A: Adenylate cyclase 9
B: DARPin C4


Theoretical massNumber of molelcules
Total (without water)167,1862
Polymers167,1862
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1490 Å2
ΔGint-7 kcal/mol
Surface area46540 Å2

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Components

#1: Protein Adenylate cyclase 9


Mass: 151142.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Production host: Homo sapiens (human) / References: UniProt: E1BM79
#2: Protein DARPin C4


Mass: 16042.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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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
1Adenylyl cyclase 9 bound to MANT-GTPCOMPLEXall0RECOMBINANT
2Adenylate cyclase 9COMPLEX#11RECOMBINANT
3DARPin C4COMPLEX#21RECOMBINANT
Molecular weightValue: 146 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bos taurus (cattle)9913
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606Human Embryonic Kidney Cells 293
33Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding 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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
7RELIONmodel fitting
9RELIONmodel refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 210729 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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