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- PDB-8g0l: Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Ace... -

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Basic information

Entry
Database: PDB / ID: 8g0l
TitleSemi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies
Components
  • Alpha-synuclein
  • N-alpha-acetyltransferase 20
  • N-alpha-acetyltransferase 25, NatB auxiliary subunit
KeywordsTRANSFERASE / 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 / protein N-terminal-methionine acetyltransferase activity / protein-N-terminal amino-acid acetyltransferase activity / acetyltransferase activator activity / negative regulation of mitochondrial electron transport, NADH to ubiquinone ...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 / protein N-terminal-methionine acetyltransferase activity / protein-N-terminal amino-acid acetyltransferase activity / acetyltransferase activator activity / negative regulation of mitochondrial electron transport, NADH to ubiquinone / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / response to desipramine / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / regulation of synaptic vesicle recycling / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / positive regulation of protein localization to cell periphery / negative regulation of exocytosis / dopamine biosynthetic process / dopamine uptake involved in synaptic transmission / response to iron(II) ion / negative regulation of dopamine metabolic process / negative regulation of platelet-derived growth factor receptor signaling pathway / SNARE complex assembly / negative regulation of thrombin-activated receptor signaling pathway / Lewy body / negative regulation of microtubule polymerization / synaptic vesicle priming / synaptic vesicle transport / regulation of norepinephrine uptake / transporter regulator activity / protein kinase inhibitor activity / positive regulation of inositol phosphate biosynthetic process / regulation of dopamine secretion / positive regulation of receptor recycling / cuprous ion binding / positive regulation of exocytosis / nuclear outer membrane / dynein complex binding / synaptic transmission, dopaminergic / synaptic vesicle exocytosis / response to magnesium ion / positive regulation of endocytosis / negative regulation of serotonin uptake / kinesin binding / cysteine-type endopeptidase inhibitor activity / regulation of presynapse assembly / synaptic vesicle endocytosis / alpha-tubulin binding / beta-tubulin binding / phospholipase binding / cytoskeleton organization / behavioral response to cocaine / cellular response to fibroblast growth factor stimulus / supramolecular fiber organization / response to type II interferon / cellular response to epinephrine stimulus / inclusion body / response to interleukin-1 / Hsp70 protein binding / positive regulation of release of sequestered calcium ion into cytosol / cellular response to copper ion / axon terminus / enzyme inhibitor activity / glutathione metabolic process / regulation of actin cytoskeleton organization / regulation of microtubule cytoskeleton organization / SNARE binding / protein tetramerization / protein sequestering activity / receptor internalization / phosphoprotein binding / microglial cell activation / tubulin binding / protein destabilization / ferrous iron binding / synapse organization / phospholipid binding / PKR-mediated signaling / tau protein binding / enzyme activator activity / positive regulation of inflammatory response / terminal bouton / actin cytoskeleton / synaptic vesicle membrane / growth cone / response to lipopolysaccharide / cellular response to oxidative stress / actin binding / histone binding / cell cortex / negative regulation of neuron apoptotic process / microtubule binding / amyloid fibril formation / mitochondrial outer membrane / lysosome / oxidoreductase activity
Similarity search - Function
Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat - #1040 / N-acetyltransferase B complex, non-catalytic subunit / N-acetyltransferase B complex (NatB) non catalytic subunit / : / Synuclein / Alpha-synuclein / Synuclein / Acetyltransferase (GNAT) family / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain ...Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat - #1040 / N-acetyltransferase B complex, non-catalytic subunit / N-acetyltransferase B complex (NatB) non catalytic subunit / : / Synuclein / Alpha-synuclein / Synuclein / Acetyltransferase (GNAT) family / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain / TPR repeat region circular profile. / Acyl-CoA N-acyltransferase / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Tetratricopeptide-like helical domain superfamily / Mainly Alpha
Similarity search - Domain/homology
CARBOXYMETHYL COENZYME *A / Alpha-synuclein / N-alpha-acetyltransferase 20 / N-alpha-acetyltransferase 25, NatB auxiliary subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsGardner, S.M. / Marmorstein, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118090-07 United States
Citation
Journal: J Am Chem Soc / Year: 2023
Title: Semi-Synthetic CoA-α-Synuclein Constructs Trap N-Terminal Acetyltransferase NatB for Binding Mechanism Studies.
Authors: Buyan Pan / Sarah M 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. A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein ...N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases. 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 as well as 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.
#1: 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
DepositionJan 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: N-alpha-acetyltransferase 20
B: N-alpha-acetyltransferase 25, NatB auxiliary subunit
C: Alpha-synuclein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,3024
Polymers133,4763
Non-polymers8261
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein N-alpha-acetyltransferase 20 / Methionine N-acetyltransferase / N-acetyltransferase 5 / N-terminal acetyltransferase B complex ...Methionine N-acetyltransferase / N-acetyltransferase 5 / N-terminal acetyltransferase B complex catalytic subunit NAA20 / N-terminal acetyltransferase B complex catalytic subunit NAT5 / NatB complex subunit NAT5 / NatB catalytic subunit


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
#2: Protein N-alpha-acetyltransferase 25, NatB auxiliary subunit / Mitochondrial distribution and morphology protein 20 / N-terminal acetyltransferase B complex ...Mitochondrial distribution and morphology protein 20 / N-terminal acetyltransferase B complex subunit MDM20 / NatB complex subunit MDM20 / N-terminal acetyltransferase B complex subunit NAA25 / p120


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 Alpha-synuclein / Non-A beta component of AD amyloid / Non-A4 component of amyloid precursor / NACP


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 / CARBOXYMETHYL COENZYME *A


Mass: 825.570 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H38N7O18P3S / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: sf9
Buffer solutionpH: 7.5
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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 / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 43.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5470

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192518 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029051
ELECTRON MICROSCOPYf_angle_d0.45712214
ELECTRON MICROSCOPYf_dihedral_angle_d3.7661198
ELECTRON MICROSCOPYf_chiral_restr0.0341350
ELECTRON MICROSCOPYf_plane_restr0.0031555

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