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- EMDB-29657: Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Ace... -

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Entry
Database: EMDB / ID: EMD-29657
TitleSemi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies
Map data
Sample
  • Complex: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
    • Protein or peptide: N-alpha-acetyltransferase 20
    • Protein or peptide: N-alpha-acetyltransferase 25, NatB auxiliary subunit
    • Protein or peptide: Alpha-synuclein
  • Ligand: CARBOXYMETHYL COENZYME *A
KeywordsN-terminal acetyltransferase / TRANSFERASE
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 / : / neutral lipid metabolic process / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / regulation of synaptic vesicle recycling / regulation of reactive oxygen species biosynthetic process / negative regulation of platelet-derived growth factor receptor signaling pathway / positive regulation of protein localization to cell periphery / negative regulation of exocytosis / regulation of glutamate secretion / response to iron(II) ion / SNARE complex assembly / positive regulation of neurotransmitter secretion / dopamine biosynthetic process / regulation of norepinephrine uptake / transporter regulator activity / regulation of locomotion / synaptic vesicle priming / mitochondrial ATP synthesis coupled electron transport / regulation of macrophage activation / positive regulation of inositol phosphate biosynthetic process / negative regulation of microtubule polymerization / synaptic vesicle transport / positive regulation of receptor recycling / dopamine uptake involved in synaptic transmission / protein kinase inhibitor activity / dynein complex binding / regulation of dopamine secretion / negative regulation of thrombin-activated receptor signaling pathway / cuprous ion binding / positive regulation of endocytosis / positive regulation of exocytosis / response to magnesium ion / synaptic vesicle exocytosis / enzyme inhibitor activity / kinesin binding / synaptic vesicle endocytosis / regulation of presynapse assembly / response to type II interferon / cysteine-type endopeptidase inhibitor activity / negative regulation of serotonin uptake / alpha-tubulin binding / supramolecular fiber organization / inclusion body / phospholipid metabolic process / cellular response to copper ion / axon terminus / cellular response to epinephrine stimulus / cytoskeleton organization / Hsp70 protein binding / response to interleukin-1 / regulation of microtubule cytoskeleton organization / SNARE binding / positive regulation of release of sequestered calcium ion into cytosol / adult locomotory behavior / negative regulation of protein kinase activity / excitatory postsynaptic potential / fatty acid metabolic process / regulation of actin cytoskeleton organization / phosphoprotein binding / protein tetramerization / microglial cell activation / regulation of long-term neuronal synaptic plasticity / synapse organization / ferrous iron binding / protein destabilization / PKR-mediated signaling / phospholipid binding / receptor internalization / tau protein binding / long-term synaptic potentiation / synaptic vesicle membrane / positive regulation of inflammatory response / actin cytoskeleton / actin binding / growth cone / cell cortex / cellular response to oxidative stress / neuron apoptotic process / chemical synaptic transmission / molecular adaptor activity / negative regulation of neuron apoptotic process / response to lipopolysaccharide / histone binding
Similarity search - Function
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. ...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 / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Alpha-synuclein / N-alpha-acetyltransferase 20 / N-alpha-acetyltransferase 25, NatB auxiliary subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsGardner SM / Marmorstein R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118090-07 United States
CitationJournal: 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, 2023-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29657.png

Downloads & links

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Map

FileDownload / File: emd_29657.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 300 pix.
= 258. Å		0.86 Å/pix.
x 300 pix.
= 258. Å		0.86 Å/pix.
x 300 pix.
= 258. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.24982624 - 0.78872156
Average (Standard dev.)0.0017144546 (±0.028970562)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 258.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29657_half_map_1.map
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Histogram (log scale)

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Half map: #2

Fileemd_29657_half_map_2.map
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Sample components

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Entire : Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein

EntireName: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
Components
  • Complex: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
    • Protein or peptide: N-alpha-acetyltransferase 20
    • Protein or peptide: N-alpha-acetyltransferase 25, NatB auxiliary subunit
    • Protein or peptide: Alpha-synuclein
  • Ligand: CARBOXYMETHYL COENZYME *A

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Supramolecule #1: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein

SupramoleculeName: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: N-alpha-acetyltransferase 20

MacromoleculeName: N-alpha-acetyltransferase 20 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: N-terminal methionine Nalpha-acetyltransferase NatB
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.390133 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MTTLRAFTCD DLFRFNNINL DPLTETYGIP FYLQYLAHWP EYFIVAEAPG GELMGYIMGK AEGSVAREEW HGHVTALSVA PEFRRLGLA AKLMELLEEI SERKGGFFVD LFVRVSNQVA VNMYKQLGYS VYRTVIEYYS ASNGEPDEDA YDMRKALSRD T EKKSIIPL PHPVRPEDIE

UniProtKB: N-alpha-acetyltransferase 20

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Macromolecule #2: N-alpha-acetyltransferase 25, NatB auxiliary subunit

MacromoleculeName: N-alpha-acetyltransferase 25, NatB auxiliary subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 112.444258 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MATRGHVQDP NDRRLRPIYD YLDNGNNKMA IQQADKLLKK HKDLHCAKVL KAIGLQRTGK QEEAFTLAQE VAALEPTDDN SLQALTILY REMHRPELVT KLYEAAVKKV PNSEEYHSHL FMAYARVGEY KKMQQAGMAL YKIVPKNPYY FWSVMSLIMQ S ISAQDENL ...String:
MATRGHVQDP NDRRLRPIYD YLDNGNNKMA IQQADKLLKK HKDLHCAKVL KAIGLQRTGK QEEAFTLAQE VAALEPTDDN SLQALTILY REMHRPELVT KLYEAAVKKV PNSEEYHSHL FMAYARVGEY KKMQQAGMAL YKIVPKNPYY FWSVMSLIMQ S ISAQDENL SKTMFLPLAE RMVEKMVKED KIEAEAEVEL YYMILERLGK YQEALDVIRG KLGEKLTSEI QSRENKCMAM YK KLSRWPE CNALSRRLLL KNSDDWQFYL TYFDSVFRLI EEAWSPPAEG EHSLEGEVHY SAEKAVKFIE DRITEESKSS RHL RGPHLA KLELIRRLRS QGCNDEYKLG DPEELMFQYF KKFGDKPCCF TDLKVFVDLL PATQCTKFIN QLLGVVPLST PTED KLALP ADIRALQQHL CVVQLTRLLG LYHTMDKNQK LSVVRELMLR YQHGLEFGKT CLKTELQFSD YYCLLAVHAL IDVWR ETGD ETTVWQALTL LEEGLTHSPS NAQFKLLLVR IYCMLGAFEP VVDLYSSLDA KHIQHDTIGY LLTRYAESLG QYAAAS QSC NFALRFFHSN QKDTSEYIIQ AYKYGAFEKI PEFIAFRNRL NNSLHFAQVR TERMLLDLLL EANISTSLAE SIKSMNL RP EEDDIPWEDL RDNRDLNVFF SWDPKDRDVS EEHKKLSLEE ETLWLRIRSL TLRLISGLPS LNHPVEPKNS EKTAENGV S SRIDILRLLL QQLEATLETG KRFIEKDIQY PFLGPVPTRM GGFFNSGCSQ CQISSFYLVN DIYELDTSGL EDTMEIQER IENSFKSLLD QLKDVFSKCK GDLLEVKDGN LKTHPTLLEN LVFFVETISV ILWVSSYCES VLRPYKLNLQ KKKKKKKETS IIMPPVFTS FQDYVTGLQT LISNVVDHIK GLETHLIALK LEELILEDTS LSPEERKFSK TVQGKVQSSY LHSLLEMGEL L KKRLETTK KLKI

UniProtKB: N-alpha-acetyltransferase 25, NatB auxiliary subunit

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Macromolecule #3: Alpha-synuclein

MacromoleculeName: Alpha-synuclein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 641.799 Da
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MDVFM

UniProtKB: Alpha-synuclein

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Macromolecule #4: CARBOXYMETHYL COENZYME *A

MacromoleculeName: CARBOXYMETHYL COENZYME *A / type: ligand / ID: 4 / Number of copies: 1 / Formula: CMC
Molecular weightTheoretical: 825.57 Da
Chemical component information

ChemComp-CMC:
CARBOXYMETHYL COENZYME *A

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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

Concentration4 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5470 / Average electron dose: 43.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6vp9

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 192518
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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