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- PDB-8buz: Structure of Adenylyl cyclase 8 bound to stimulatory G-protein, C... -

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Basic information

Entry
Database: PDB / ID: 8buz
TitleStructure of Adenylyl cyclase 8 bound to stimulatory G-protein, Ca2+/Calmodulin, Forskolin and MANT-GTP
Components
  • Adenylate cyclase type 8
  • Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
KeywordsMEMBRANE PROTEIN / Adenylyl Cyclase / cAMP signaling / G proteins / Calmodulin
Function / homology
Function and homology information


cellular response to morphine / Adenylate cyclase activating pathway / positive regulation of long-term synaptic depression / Adenylate cyclase inhibitory pathway / glucose mediated signaling pathway / calcium- and calmodulin-responsive adenylate cyclase activity / regulation of cellular response to stress / sensory perception of chemical stimulus / PKA activation / neuroinflammatory response ...cellular response to morphine / Adenylate cyclase activating pathway / positive regulation of long-term synaptic depression / Adenylate cyclase inhibitory pathway / glucose mediated signaling pathway / calcium- and calmodulin-responsive adenylate cyclase activity / regulation of cellular response to stress / sensory perception of chemical stimulus / PKA activation / neuroinflammatory response / adenylate cyclase / positive regulation of synaptic plasticity / Hedgehog 'off' state / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / cAMP biosynthetic process / adenylate cyclase activity / beta-2 adrenergic receptor binding / G alpha (z) signalling events / neuronal cell body membrane / positive regulation of insulin secretion involved in cellular response to glucose stimulus / presynaptic active zone / excitatory synapse / D1 dopamine receptor binding / long-term memory / adenylate cyclase-activating adrenergic receptor signaling pathway / regulation of cytosolic calcium ion concentration / ionotropic glutamate receptor binding / cellular response to glucagon stimulus / insulin-like growth factor receptor binding / adenylate cyclase activator activity / hippocampal mossy fiber to CA3 synapse / positive regulation of long-term synaptic potentiation / locomotory behavior / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Schaffer collateral - CA1 synapse / adenylate cyclase-activating dopamine receptor signaling pathway / heterotrimeric G-protein complex / presynaptic membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / basolateral plasma membrane / postsynaptic density / intracellular signal transduction / apical plasma membrane / axon / GTPase activity / glutamatergic synapse / dendrite / GTP binding / ATP binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Adenylate cyclase, conserved domain / Adenylate cyclase / Adenylate cyclase, N-terminal / Adenylate cyclase, conserved domain / Adenylyl cyclase N-terminal extracellular and transmembrane region / 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 ...Adenylate cyclase, conserved domain / Adenylate cyclase / Adenylate cyclase, N-terminal / Adenylate cyclase, conserved domain / Adenylyl cyclase N-terminal extracellular and transmembrane region / 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 / G-protein alpha subunit, group S / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
FORSKOLIN / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / Adenylate cyclase type 8 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKhanppnavar, B. / Korkhov, V.M. / Mehta, V.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation198545 Switzerland
Swiss National Science Foundation184951 Switzerland
CitationJournal: EMBO Rep / Year: 2024
Title: Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.
Authors: Basavraj Khanppnavar / Dina Schuster / Pia Lavriha / Federico Uliana / Merve Özel / Ved Mehta / Alexander Leitner / Paola Picotti / Volodymyr M Korkhov /
Abstract: Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca signalling. Despite the importance of AC8 in physiology, the ...Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.
History
DepositionDec 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: 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

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Adenylate cyclase type 8
B: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
hetero molecules


Theoretical massNumber of molelcules
Total (without water)188,1705
Polymers187,1962
Non-polymers9743
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Adenylate cyclase type 8


Mass: 140192.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: ADCY8 / Production host: Homo sapiens (human) / References: UniProt: E1BQ12, adenylate cyclase
#2: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 47003.801 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: GNAS, GNAS1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P04896
#3: Chemical ChemComp-FOK / FORSKOLIN


Mass: 410.501 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H34O7 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3S / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
1Structure of Adenylyl cyclase 8 bound to stimulatory G protein, Ca2+/Calmodulin, Forskolin, and MANT-GTPCOMPLEX#1-#20RECOMBINANT
2Adenylyl cyclase 8COMPLEX#11RECOMBINANT
3G protein alpha SCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bos taurus (cattle)9913
33Bos taurus (cattle)9913
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4Gctf1.06CTF correction
10RELION3.1.3initial Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77575 / Symmetry type: POINT

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