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- PDB-7ljf: Cryo-EM structure of the Mpa hexamer in the presence of ATP and t... -

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Basic information

Entry
Database: PDB / ID: 7ljf
TitleCryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
ComponentsAAA ATPase forming ring-shaped complexes
KeywordsSTRUCTURAL PROTEIN / Mycobacterial proteasomal ATPase / Mycobacterium tuberculosis / structural biology / cryo-EM / ATP-Bound
Function / homology
Function and homology information


proteasome-activating nucleotidase complex / proteasomal protein catabolic process / modification-dependent protein catabolic process / ATP hydrolysis activity / ATP binding
Similarity search - Function
Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / : / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core ...Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / : / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / AAA ATPase forming ring-shaped complexes
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsYin, Y. / Li, H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Complementary and Integrative Health (NIH/NCCIH)AI070285 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)AI088075 United States
CitationJournal: J Biol Chem / Year: 2021
Title: The mycobacterial proteasomal ATPase Mpa forms a gapped ring to engage the 20S proteasome.
Authors: Yanting Yin / Amanda Kovach / Hao-Chi Hsu / K Heran Darwin / Huilin Li /
Abstract: Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. ...Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal proteasome-activating nucleotidase and eukaryotic proteasomal Rpt1-6 ATPases, such as the N-terminal coiled-coil domain, oligosaccharide-/oligonucleotide-binding domain, and ATPase domain. However, Mpa has a unique β-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle (CP). Thus, it is unclear how Mpa binds to proteasome CPs. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide-binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP hydrolysis-driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its β-grasp domains and activating C termini to facilitate engagement with proteasome CPs. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.
History
DepositionJan 29, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: AAA ATPase forming ring-shaped complexes
B: AAA ATPase forming ring-shaped complexes
C: AAA ATPase forming ring-shaped complexes
D: AAA ATPase forming ring-shaped complexes
E: AAA ATPase forming ring-shaped complexes
F: AAA ATPase forming ring-shaped complexes
hetero molecules


Theoretical massNumber of molelcules
Total (without water)400,93212
Polymers399,4986
Non-polymers1,4346
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
AAA ATPase forming ring-shaped complexes / ARC


Mass: 66582.969 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: arc, mpa, DSI38_15585, E5M52_17490, ERS007661_01151, ERS007665_00732, ERS007679_01634, ERS007681_02311, ERS007703_01049, ERS007720_01453, ERS007722_00998, ERS007741_01455, ERS023446_02559, ...Gene: arc, mpa, DSI38_15585, E5M52_17490, ERS007661_01151, ERS007665_00732, ERS007679_01634, ERS007681_02311, ERS007703_01049, ERS007720_01453, ERS007722_00998, ERS007741_01455, ERS023446_02559, ERS024276_00114, ERS027646_02035, ERS027659_02128, ERS027661_00811, ERS075361_03050, ERS094182_01863, F6W99_00704, FRD82_11355, SAMEA2683035_00457
Production host: Escherichia coli K-12 (bacteria) / References: UniProt: A0A045JPX7
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


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


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
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: Cryo-EM structure of the Mpa hexamer in the presence of ATP and the Pup-FabD substrate
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.39 MDa / Experimental value: YES
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 345023 / Symmetry type: POINT

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