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- PDB-7pw8: Human SMG1-8-9 kinase complex bound to AMPPNP -

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Basic information

Entry
Database: PDB / ID: 7pw8
TitleHuman SMG1-8-9 kinase complex bound to AMPPNP
Components
  • Protein SMG8
  • Protein SMG9
  • SMG1,Serine/threonine-protein kinase SMG1,SMG1,Serine/threonine-protein kinase SMG1,SMG1
KeywordsTRANSFERASE / Complex
Function / homology
Function and homology information


diacylglycerol-dependent serine/threonine kinase activity / chromatoid body / eye development / regulation of telomere maintenance / regulation of protein kinase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / telomeric DNA binding / phosphatidylinositol phosphate biosynthetic process / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / mRNA export from nucleus ...diacylglycerol-dependent serine/threonine kinase activity / chromatoid body / eye development / regulation of telomere maintenance / regulation of protein kinase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / telomeric DNA binding / phosphatidylinositol phosphate biosynthetic process / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / mRNA export from nucleus / brain development / heart development / peptidyl-serine phosphorylation / in utero embryonic development / protein autophosphorylation / non-specific serine/threonine protein kinase / protein kinase activity / DNA repair / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / negative regulation of apoptotic process / RNA binding / nucleoplasm / ATP binding / metal ion binding / nucleus / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 / Serine/threonine-protein kinase SMG1 / Serine/threonine-protein kinase SMG1, N-terminal / SMG1, PIKK catalytic domain / Serine/threonine-protein kinase smg-1 / Serine/threonine-protein kinase SMG1 N-terminal / Rapamycin binding domain / FATC domain ...Nonsense-mediated mRNA decay factor SMG8/SMG9 / Smg8_Smg9 / Nonsense-mediated mRNA decay factor SMG9 / Serine/threonine-protein kinase SMG1 / Serine/threonine-protein kinase SMG1, N-terminal / SMG1, PIKK catalytic domain / Serine/threonine-protein kinase smg-1 / Serine/threonine-protein kinase SMG1 N-terminal / Rapamycin binding domain / FATC domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / Armadillo-type fold / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / ADENOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / Nonsense-mediated mRNA decay factor SMG8 / Serine/threonine-protein kinase SMG1 / Nonsense-mediated mRNA decay factor SMG9
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsLanger, L.M. / Conti, E.
Funding support Germany, 1items
OrganizationGrant numberCountry
Not funded Germany
CitationJournal: Elife / Year: 2021
Title: Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8.
Authors: Lukas M Langer / Fabien Bonneau / Yair Gat / Elena Conti /
Abstract: The PI3K-related kinase (PIKK) SMG1 monitors the progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 ...The PI3K-related kinase (PIKK) SMG1 monitors the progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analog at overall resolutions ranging from 2.8 to 3.6 Å. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al.,2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate-binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general.
History
DepositionOct 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: SMG1,Serine/threonine-protein kinase SMG1,SMG1,Serine/threonine-protein kinase SMG1,SMG1
B: Protein SMG8
C: Protein SMG9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)566,6367
Polymers564,9393
Non-polymers1,6984
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12370 Å2
ΔGint-84 kcal/mol
Surface area114920 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein SMG1,Serine/threonine-protein kinase SMG1,SMG1,Serine/threonine-protein kinase SMG1,SMG1 / SMG-1 / hSMG-1 / 61E3.4 / Lambda/iota protein kinase C-interacting protein / Lambda-interacting protein


Mass: 397395.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMG1, ATX, KIAA0421, LIP / Production host: Homo sapiens (human)
References: UniProt: Q96Q15, non-specific serine/threonine protein kinase
#2: Protein Protein SMG8 / Amplified in breast cancer gene 2 protein / Protein smg-8 homolog


Mass: 109825.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMG8, ABC2, C17orf71 / Production host: Homo sapiens (human) / References: UniProt: Q8ND04
#3: Protein Protein SMG9 / Protein SMG-9


Mass: 57717.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMG9, C19orf61 / Production host: Homo sapiens (human) / References: UniProt: Q9H0W8

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

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

ComponentName: SMG1-SMG8-SMG9 kinase complex bound to AMPPNP / Type: COMPLEX / Entity ID: #2-#3 / Source: RECOMBINANT
Molecular weightValue: 0.597 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 60.99 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 403079 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219310
ELECTRON MICROSCOPYf_angle_d0.58726375
ELECTRON MICROSCOPYf_dihedral_angle_d4.4932768
ELECTRON MICROSCOPYf_chiral_restr0.0393252
ELECTRON MICROSCOPYf_plane_restr0.0043274

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