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- PDB-7jhg: Cryo-EM structure of ATP-bound fully inactive AMPK in complex wit... -

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Basic information

Entry
Database: PDB / ID: 7jhg
TitleCryo-EM structure of ATP-bound fully inactive AMPK in complex with Dorsomorphin (Compound C) and Fab-nanobody
Components
  • (5'-AMP-activated protein kinase subunit ...) x 2
  • 5'-AMP-activated protein kinase catalytic subunit alpha-1
  • Fab heavy chain
  • Fab light chain
  • Maltodextrin-binding protein
  • Nanobody
KeywordsTRANSFERASE/IMMUNE SYSTEM / AMPK / ATP / fully inactive / KD-displaced / TRANSFERASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


negative regulation of glucosylceramide biosynthetic process / positive regulation of mitochondrial transcription / [hydroxymethylglutaryl-CoA reductase (NADPH)] kinase / [hydroxymethylglutaryl-CoA reductase (NADPH)] kinase activity / regulation of stress granule assembly / AMPK inhibits chREBP transcriptional activation activity / histone H2BS36 kinase activity / cold acclimation / lipid droplet disassembly / AMP-activated protein kinase activity ...negative regulation of glucosylceramide biosynthetic process / positive regulation of mitochondrial transcription / [hydroxymethylglutaryl-CoA reductase (NADPH)] kinase / [hydroxymethylglutaryl-CoA reductase (NADPH)] kinase activity / regulation of stress granule assembly / AMPK inhibits chREBP transcriptional activation activity / histone H2BS36 kinase activity / cold acclimation / lipid droplet disassembly / AMP-activated protein kinase activity / Lipophagy / CAMKK-AMPK signaling cascade / protein localization to lipid droplet / negative regulation of hepatocyte apoptotic process / regulation of vesicle-mediated transport / cAMP-dependent protein kinase regulator activity / Carnitine shuttle / Energy dependent regulation of mTOR by LKB1-AMPK / positive regulation of T cell mediated immune response to tumor cell / tau-protein kinase / nucleotide-activated protein kinase complex / protein kinase regulator activity / regulation of vascular permeability / motor behavior / negative regulation of TOR signaling / Activation of PPARGC1A (PGC-1alpha) by phosphorylation / neuron cellular homeostasis / protein localization to membrane / regulation of glycolytic process / cAMP-dependent protein kinase activity / cellular response to ethanol / positive regulation of protein localization to mitochondrion / response to caffeine / cellular response to stress / tau-protein kinase activity / AMP binding / lipid biosynthetic process / Macroautophagy / energy homeostasis / response to UV / negative regulation of ferroptosis / fatty acid homeostasis / carbohydrate transmembrane transporter activity / maltose binding / negative regulation of lipid catabolic process / maltose transport / maltodextrin transmembrane transport / cellular response to glucose starvation / cellular response to nutrient levels / positive regulation of gluconeogenesis / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / positive regulation of adipose tissue development / negative regulation of insulin receptor signaling pathway / Activation of AMPK downstream of NMDARs / response to gamma radiation / positive regulation of protein localization / cellular response to calcium ion / positive regulation of autophagy / positive regulation of glycolytic process / negative regulation of TORC1 signaling / protein localization to plasma membrane / response to activity / cellular response to starvation / cellular response to xenobiotic stimulus / TP53 Regulates Metabolic Genes / cellular response to glucose stimulus / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of microtubule cytoskeleton organization / positive regulation of cholesterol biosynthetic process / ADP binding / response to estrogen / AMPK-induced ERAD and lysosome mediated degradation of PD-L1(CD274) / tau protein binding / regulation of circadian rhythm / positive regulation of T cell activation / cellular response to hydrogen peroxide / glucose homeostasis / late endosome / positive regulation of cold-induced thermogenesis / outer membrane-bounded periplasmic space / ciliary basal body / cellular response to oxidative stress / spermatogenesis / Regulation of TP53 Activity through Phosphorylation / response to hypoxia / regulation of cell cycle / cellular response to hypoxia / protein kinase activity / protein phosphorylation / non-specific serine/threonine protein kinase / nuclear speck / apical plasma membrane / endoplasmic reticulum lumen / negative regulation of gene expression / protein serine kinase activity / axon / protein serine/threonine kinase activity / neuronal cell body / chromatin binding / positive regulation of cell population proliferation
Similarity search - Function
PRKAA1, UBA-like autoinhibitory domain / 5'-AMP-activated protein kinase alpha 1 catalytic subunit, C-terminal / : / AMP-activated protein kinase, alpha subunit, autoinhibitory domain / : / Association with the SNF1 complex (ASC) domain / ASC domain superfamily / : / 5'-AMP-activated protein kinase beta subunit, interaction domain / 5'-AMP-activated protein kinase beta subunit, interation domain ...PRKAA1, UBA-like autoinhibitory domain / 5'-AMP-activated protein kinase alpha 1 catalytic subunit, C-terminal / : / AMP-activated protein kinase, alpha subunit, autoinhibitory domain / : / Association with the SNF1 complex (ASC) domain / ASC domain superfamily / : / 5'-AMP-activated protein kinase beta subunit, interaction domain / 5'-AMP-activated protein kinase beta subunit, interation domain / AMPK, C-terminal adenylate sensor domain / Adenylate sensor of SNF1-like protein kinase / AMP-activated protein kinase, glycogen-binding domain / Glycogen recognition site of AMP-activated protein kinase / KA1 domain/Ssp2, C-terminal / Domain in cystathionine beta-synthase and other proteins. / CBS domain superfamily / CBS domain / CBS domain / CBS domain profile. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Immunoglobulin E-set / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Immunoglobulins / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
alpha-maltose / ADENOSINE-5'-DIPHOSPHATE / ADENOSINE MONOPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Chem-TAK / Maltodextrin-binding protein / 5'-AMP-activated protein kinase subunit beta-2 / 5'-AMP-activated protein kinase subunit gamma-1 / 5'-AMP-activated protein kinase catalytic subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli K-12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsYan, Y. / Murkherjee, S. / Zhou, X.E. / Xu, T.H. / Xu, H.E. / Kossiakoff, A.A. / Melcher, K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM117372 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM129436 United States
CitationJournal: Science / Year: 2021
Title: Structure of an AMPK complex in an inactive, ATP-bound state.
Authors: Yan Yan / Somnath Mukherjee / Kaleeckal G Harikumar / Timothy S Strutzenberg / X Edward Zhou / Kelly Suino-Powell / Ting-Hai Xu / Ryan D Sheldon / Jared Lamp / Joseph S Brunzelle / Katarzyna ...Authors: Yan Yan / Somnath Mukherjee / Kaleeckal G Harikumar / Timothy S Strutzenberg / X Edward Zhou / Kelly Suino-Powell / Ting-Hai Xu / Ryan D Sheldon / Jared Lamp / Joseph S Brunzelle / Katarzyna Radziwon / Abigail Ellis / Scott J Novick / Irving E Vega / Russell G Jones / Laurence J Miller / H Eric Xu / Patrick R Griffin / Anthony A Kossiakoff / Karsten Melcher /
Abstract: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in ...Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.
History
DepositionJul 20, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 21, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 15, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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 / _citation_author.identifier_ORCID / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Nov 6, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: 5'-AMP-activated protein kinase catalytic subunit alpha-1
B: 5'-AMP-activated protein kinase subunit beta-2
G: 5'-AMP-activated protein kinase subunit gamma-1
M: Maltodextrin-binding protein
L: Fab light chain
H: Fab heavy chain
N: Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)222,18412
Polymers220,1607
Non-polymers2,0235
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Fab and nanobody were prepared individually. Purified AMPK was incubated with CpdC and Fab and nanobody.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22610 Å2
ΔGint-106 kcal/mol
Surface area75760 Å2

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Components

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Protein , 2 types, 2 molecules AM

#1: Protein 5'-AMP-activated protein kinase catalytic subunit alpha-1 / AMPK subunit alpha-1 / Acetyl-CoA carboxylase kinase / ACACA kinase / Hydroxymethylglutaryl-CoA ...AMPK subunit alpha-1 / Acetyl-CoA carboxylase kinase / ACACA kinase / Hydroxymethylglutaryl-CoA reductase kinase / HMGCR kinase / Tau-protein kinase PRKAA1


Mass: 56004.395 Da / Num. of mol.: 1 / Fragment: UNP residues 22-480,535-559
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRKAA1, AMPK1 / Production host: Escherichia coli K-12 (bacteria)
References: UniProt: Q13131, non-specific serine/threonine protein kinase, EC: 2.7.11.27, [hydroxymethylglutaryl-CoA reductase (NADPH)] kinase, tau-protein kinase
#4: Protein Maltodextrin-binding protein


Mass: 40827.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: malE / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: C3SHQ8

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5'-AMP-activated protein kinase subunit ... , 2 types, 2 molecules BG

#2: Protein 5'-AMP-activated protein kinase subunit beta-2 / AMPK subunit beta-2


Mass: 22384.650 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRKAB2 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: O43741
#3: Protein 5'-AMP-activated protein kinase subunit gamma-1 / AMPKg


Mass: 34833.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRKAG1 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P54619

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Antibody , 3 types, 3 molecules LHN

#5: Antibody Fab light chain


Mass: 23212.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli K-12 (bacteria)
#6: Antibody Fab heavy chain


Mass: 25483.488 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli K-12 (bacteria)
#7: Antibody Nanobody


Mass: 17414.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli K-12 (bacteria)

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Sugars , 1 types, 1 molecules

#8: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1a_1-5]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 4 molecules

#9: Chemical ChemComp-TAK / 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine / Dorsomorphin


Mass: 399.488 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H25N5O / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#11: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#12: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Comment: AMP*YM

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Details

Has ligand of interestY
Has protein modificationY

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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 ATP-bound fully inactive AMPK in complex with Dorsomorphin (Compound C) and Fab-nanobodyCOMPLEX#1-#70MULTIPLE SOURCES
2ATP-bound fully inactive AMPK in complex with Dorsomorphin (Compound C)COMPLEX#1-#41RECOMBINANT
3Fab-nanobodyCOMPLEX#5-#71RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli K-12 (bacteria)83333
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 83 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 6157

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1.10CTF correction
7UCSF Chimeramodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 943787
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 286895 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
14RER

4rer

14RER1PDBexperimental model
26CMO

6cmo

16CMO2PDBexperimental model
36H16

6h16

16H163PDBexperimental model

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