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Yorodumi- PDB-8g0l: Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Ace... -
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-Basic information
Entry | Database: PDB / ID: 8g0l | ||||||
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Title | Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies | ||||||
Components |
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Keywords | TRANSFERASE / N-terminal acetyltransferase | ||||||
Function / homology | Function and homology information N-terminal peptidyl-glutamine acetylation / N-terminal methionine Nalpha-acetyltransferase NatB / N-terminal peptidyl-aspartic acid acetylation / N-terminal peptidyl-glutamic acid acetylation / NatB complex / N-terminal protein amino acid acetylation / peptide alpha-N-acetyltransferase activity / negative regulation of mitochondrial electron transport, NADH to ubiquinone / neutral lipid metabolic process / regulation of phospholipase activity ...N-terminal peptidyl-glutamine acetylation / N-terminal methionine Nalpha-acetyltransferase NatB / N-terminal peptidyl-aspartic acid acetylation / N-terminal peptidyl-glutamic acid acetylation / NatB complex / N-terminal protein amino acid acetylation / peptide alpha-N-acetyltransferase activity / negative regulation of mitochondrial electron transport, NADH to ubiquinone / neutral lipid metabolic process / regulation of phospholipase activity / negative regulation of monooxygenase activity / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of glutathione peroxidase activity / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / negative regulation of transporter activity / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / positive regulation of protein localization to cell periphery / regulation of synaptic vesicle recycling / negative regulation of platelet-derived growth factor receptor signaling pathway / negative regulation of exocytosis / regulation of glutamate secretion / response to iron(II) ion / regulation of norepinephrine uptake / SNARE complex assembly / positive regulation of neurotransmitter secretion / dopamine biosynthetic process / regulation of locomotion / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle priming / regulation of macrophage activation / negative regulation of microtubule polymerization / synaptic vesicle transport / dopamine uptake involved in synaptic transmission / dynein complex binding / regulation of dopamine secretion / positive regulation of receptor recycling / protein kinase inhibitor activity / negative regulation of thrombin-activated receptor signaling pathway / response to type II interferon / cuprous ion binding / positive regulation of exocytosis / synaptic vesicle exocytosis / positive regulation of endocytosis / kinesin binding / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / response to magnesium ion / synaptic vesicle endocytosis / regulation of presynapse assembly / negative regulation of serotonin uptake / alpha-tubulin binding / phospholipid metabolic process / supramolecular fiber organization / axon terminus / mitochondrial ATP synthesis coupled electron transport / inclusion body / fatty acid metabolic process / cellular response to copper ion / cellular response to epinephrine stimulus / Hsp70 protein binding / response to interleukin-1 / adult locomotory behavior / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of release of sequestered calcium ion into cytosol / SNARE binding / excitatory postsynaptic potential / long-term synaptic potentiation / phosphoprotein binding / protein tetramerization / regulation of transmembrane transporter activity / synapse organization / microglial cell activation / negative regulation of protein kinase activity / regulation of long-term neuronal synaptic plasticity / protein destabilization / ferrous iron binding / tau protein binding / positive regulation of protein serine/threonine kinase activity / PKR-mediated signaling / receptor internalization / phospholipid binding / activation of cysteine-type endopeptidase activity involved in apoptotic process / synaptic vesicle membrane / positive regulation of inflammatory response / positive regulation of peptidyl-serine phosphorylation / actin cytoskeleton / actin binding / cellular response to oxidative stress / histone binding / cell cortex / growth cone / chemical synaptic transmission / neuron apoptotic process / negative regulation of neuron apoptotic process / transcription cis-regulatory region binding Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å | ||||||
Authors | Gardner, S.M. / Marmorstein, R. | ||||||
Funding support | United States, 1items
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Citation | Journal: bioRxiv / Year: 2023 Title: Semi-synthetic CoA-α-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies. Authors: Buyan Pan / Sarah Gardner / Kollin Schultz / Ryann M Perez / Sunbin Deng / Marie Shimogawa / Kohei Sato / Elizabeth Rhoades / Ronen Marmorstein / E James Petersson Abstract: N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including α- ...N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of αS have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human αS, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, αS remains disordered when in complex with hNatB. We further probe changes in the αS conformation by single molecule Förster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes and their implications for hNatB substrate recognition and specific inhibition of the interaction with αS. Beyond the study of αS and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8g0l.cif.gz | 199.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8g0l.ent.gz | 161.7 KB | Display | PDB format |
PDBx/mmJSON format | 8g0l.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8g0l_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 8g0l_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 8g0l_validation.xml.gz | 44.4 KB | Display | |
Data in CIF | 8g0l_validation.cif.gz | 65.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g0/8g0l ftp://data.pdbj.org/pub/pdb/validation_reports/g0/8g0l | HTTPS FTP |
-Related structure data
Related structure data | 29657MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 20390.133 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: NAA20, NAT5 / Production host: Spodoptera frugiperda (fall armyworm) References: UniProt: P61599, N-terminal methionine Nalpha-acetyltransferase NatB |
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#2: Protein | Mass: 112444.258 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: NAA25, C12orf30, MDM20, NAP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14CX7 |
#3: Protein/peptide | Mass: 641.799 Da / Num. of mol.: 1 / Fragment: UNP residues 1-5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: SNCA, NACP, PARK1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P37840 |
#4: Chemical | ChemComp-CMC / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) / Strain: sf9 |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE / Humidity: 100 % |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 43.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5470 |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192518 / Symmetry type: POINT | ||||||||||||||||||||||||
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