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- EMDB-16252: Structure of Adenylyl cyclase 8 bound to stimulatory G-protein, C... -

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Entry
Database: EMDB / ID: EMD-16252
TitleStructure of Adenylyl cyclase 8 bound to stimulatory G-protein, Ca2+/Calmodulin, Forskolin and MANT-GTP
Map data
Sample
  • Complex: Structure of Adenylyl cyclase 8 bound to stimulatory G protein, Ca2+/Calmodulin, Forskolin, and MANT-GTP
    • Complex: Adenylyl cyclase 8
      • Protein or peptide: Adenylate cyclase type 8
    • Complex: G protein alpha S
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Ligand: FORSKOLIN
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsAdenylyl Cyclase / cAMP signaling / G proteins / Calmodulin / MEMBRANE PROTEIN
Function / homology
Function and homology information


cellular response to morphine / Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / positive regulation of long-term synaptic depression / glucose mediated signaling pathway / calcium- and calmodulin-responsive adenylate cyclase activity / regulation of cellular response to stress / PKA activation / neuroinflammatory response / sensory perception of chemical stimulus ...cellular response to morphine / Adenylate cyclase activating pathway / Adenylate cyclase inhibitory pathway / positive regulation of long-term synaptic depression / glucose mediated signaling pathway / calcium- and calmodulin-responsive adenylate cyclase activity / regulation of cellular response to stress / PKA activation / neuroinflammatory response / sensory perception of chemical stimulus / adenylate cyclase / positive regulation of synaptic plasticity / Hedgehog 'off' state / cAMP biosynthetic process / adenylate cyclase activity / G alpha (z) signalling events / neuronal cell body membrane / cellular response to glucagon stimulus / presynaptic active zone / positive regulation of insulin secretion involved in cellular response to glucose stimulus / excitatory synapse / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / D1 dopamine receptor binding / long-term memory / beta-2 adrenergic receptor binding / adenylate cyclase-activating adrenergic receptor signaling pathway / regulation of cytosolic calcium ion concentration / adenylate cyclase activator activity / hippocampal mossy fiber to CA3 synapse / positive regulation of long-term synaptic potentiation / locomotory behavior / ionotropic glutamate receptor binding / insulin-like growth factor receptor binding / Schaffer collateral - CA1 synapse / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / adenylate cyclase-activating dopamine receptor signaling pathway / heterotrimeric G-protein complex / presynaptic membrane / basolateral plasma membrane / postsynaptic density / intracellular signal transduction / apical plasma membrane / axon / GTPase activity / glutamatergic synapse / dendrite / GTP binding / ATP binding / metal ion binding / 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 / Adenylyl cyclase class-3/4/guanylyl cyclase / Adenylate and Guanylate cyclase catalytic domain ...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 / Adenylyl cyclase class-3/4/guanylyl cyclase / Adenylate and Guanylate cyclase catalytic domain / Guanylate cyclase domain profile. / Nucleotide cyclase / G-protein alpha subunit, group S / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Adenylate cyclase type 8 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKhanppnavar B / Korkhov VM / Mehta V
Funding support Switzerland, 2 items
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, 2022-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16252.png

Downloads & links

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Map

FileDownload / File: emd_16252.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 420 pix.
= 277.2 Å		0.66 Å/pix.
x 420 pix.
= 277.2 Å		0.66 Å/pix.
x 420 pix.
= 277.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.03261464 - 0.05055138
Average (Standard dev.)0.00006151891 (±0.0009610565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 277.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half2

Fileemd_16252_half_map_1.map
Annotationhalf2
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half1

Fileemd_16252_half_map_2.map
Annotationhalf1
Projections & Slices
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Sample components

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Entire : Structure of Adenylyl cyclase 8 bound to stimulatory G protein, C...

EntireName: Structure of Adenylyl cyclase 8 bound to stimulatory G protein, Ca2+/Calmodulin, Forskolin, and MANT-GTP
Components
  • Complex: Structure of Adenylyl cyclase 8 bound to stimulatory G protein, Ca2+/Calmodulin, Forskolin, and MANT-GTP
    • Complex: Adenylyl cyclase 8
      • Protein or peptide: Adenylate cyclase type 8
    • Complex: G protein alpha S
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Ligand: FORSKOLIN
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Structure of Adenylyl cyclase 8 bound to stimulatory G protein, C...

SupramoleculeName: Structure of Adenylyl cyclase 8 bound to stimulatory G protein, Ca2+/Calmodulin, Forskolin, and MANT-GTP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Adenylyl cyclase 8

SupramoleculeName: Adenylyl cyclase 8 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)

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Supramolecule #3: G protein alpha S

SupramoleculeName: G protein alpha S / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Adenylate cyclase type 8

MacromoleculeName: Adenylate cyclase type 8 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: adenylate cyclase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 140.192328 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MELSDVRCLS GSEELYTIHP TPPAGDSGSG SRPQRLLWQT AVRHITEQRF IHGHRGGGGG GGNGSSSKAS DPGGGGPNHH HASQLSGDS ALPLYALGPG ERAHGTGGPK VFPERSGSGS ASGSGGGGDL GFLHLDCAPS NSDFFLNGGY SYRGVIFPTL R NSFKSRDL ...String:
MELSDVRCLS GSEELYTIHP TPPAGDSGSG SRPQRLLWQT AVRHITEQRF IHGHRGGGGG GGNGSSSKAS DPGGGGPNHH HASQLSGDS ALPLYALGPG ERAHGTGGPK VFPERSGSGS ASGSGGGGDL GFLHLDCAPS NSDFFLNGGY SYRGVIFPTL R NSFKSRDL ERLYQRYFLG QRRKSEVVMN VLDVLTKLTL LVLHLSLASA PMDPLKGILL GFFTGIEVVI CALVVVRKDT TS HTYLQYS GVVTWVAMTT QILAAGLGYG LLGDGIGYVL FTLFATYSML PLPLTWAILA GLGTSLMQVV LQAVIPRLAV ISI NQVVAQ AVLFMCMNTA GIFISYLSDR AQRQAFLETR RCVEARLRLE TENQRQERLV LSVLPRFVVL EMINDMTNVE DEHL QHQFH RIYIHRYENV SILFADVKGF TNLSTTLSAQ ELVRMLNELF ARFDRLAHEH HCLRIKILGD CYYCVSGLPE PRQDH AHCC VEMGLSMIKT IRYVRSRTKH DVDMRIGIHS GSVLCGVLGL RKWQFDVWSW DVDIANKLES GGIPGRIHIS KATLDC LNG DYNVEEGHGK ERNEFLRKHN IETYLIKQPE ESLLSLPEDI VKESVSSSDR RNSGATFTEG SWSPELPFDN IVGKQNT LA ALTRNSINLL PNHLAQALHV QSGPEEINKR IEHTIDLRSG DKLRREHIKP FSLMFKDSSL EHKYSQMRDE VFKSNLVC A FIVLLFITAI QSLLPSSRVM PMAIQFSILI MLHSALVLIT TAEDYKCLPL VLRKTCCWIN ETYLARNVII FASILINFL GAILNILWCD FDKSIPLKNL TFNSSAVFTD ICSYPEYFVF TGVLAMVTCA VFLRLNSVLK LAVLLIMIAI YALLTETIYA GLFLRYDNL NHSGEDFLGT KEASLLLMAM FLLAVFYHGQ QLEYTARLDF LWRVQAKEEI NEMKELREHN ENMLRNILPS H VARHFLEK DRDNEELYSQ SYDAVGVMFA SIPGFADFYS QTEMNNQGVE CLRLLNEIIA DFDELLGEDR FQDIEKIKTI GS TYMAVSG LSPEKQQCED KWGHLCALAD FSLALTESIQ EINKHSFNNF ELRIGISHGS VVAGVIGAKK PQYDIWGKTV NLA SRMDST GVSGRIQVPE ETYLILKDQG FAFDYRGEIY VKGISEQEGK IKTYFLLGRV QPNPFILPPR RLPGQYSLAA VVLG LVQSL NRQRQKQLLN ENNNTGIIKG HYNRRTLLTP SGPEPGAPAE GADKPELP

UniProtKB: Adenylate cyclase type 8

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Macromolecule #2: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 47.003801 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG GEGGEEDPNA AKSNSDGEK ATKVQDIKNN LKEAIETIVA AMSNLVPPVE LANPENQFRV DYILSVMNVP DFDFPPEFYE HAKALWEDEG V RACYERSN ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG GEGGEEDPNA AKSNSDGEK ATKVQDIKNN LKEAIETIVA AMSNLVPPVE LANPENQFRV DYILSVMNVP DFDFPPEFYE HAKALWEDEG V RACYERSN EYQLIDCAQY FLDKIDVIKQ DDYVPSDQDL LRCRVLTSGI FETKFQVDKV NFHMFDVGGQ RDERRKWIQC FN DVTAIIF VVASSSYNMV IREDNQTNRL QEALNLFKSI WNNRWLRTIS VILFLNKQDL LAEKVLAGKS KIEDYFPEFA RYT TPEDAT PEPGEDPRVT RAKYFIRDEF LRISTASGDG RHYCYPHFTC AVDTENIRRV FNDCRDIIQR MHLRQYELLG GHHH HHHHH

UniProtKB: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Macromolecule #3: FORSKOLIN

MacromoleculeName: FORSKOLIN / type: ligand / ID: 3 / Number of copies: 1 / Formula: FOK
Molecular weightTheoretical: 410.501 Da
Chemical component information

ChemComp-FOK:
FORSKOLIN

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Macromolecule #4: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GSP
Molecular weightTheoretical: 539.246 Da
Chemical component information

ChemComp-GSP:
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab-initial 3D model reconstruction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77575
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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