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- PDB-8g7l: ATP-bound mtHsp60 V72I -

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Basic information

Entry
Database: PDB / ID: 8g7l
TitleATP-bound mtHsp60 V72I
Components60 kDa heat shock protein, mitochondrial
KeywordsCHAPERONE / chaperonin / ATPase / foldase
Function / homology
Function and homology information


coated vesicle / isotype switching to IgG isotypes / mitochondrial unfolded protein response / lipopolysaccharide receptor complex / apolipoprotein A-I binding / TFAP2A acts as a transcriptional repressor during retinoic acid induced cell differentiation / protein import into mitochondrial intermembrane space / migrasome / high-density lipoprotein particle binding / positive regulation of T cell mediated immune response to tumor cell ...coated vesicle / isotype switching to IgG isotypes / mitochondrial unfolded protein response / lipopolysaccharide receptor complex / apolipoprotein A-I binding / TFAP2A acts as a transcriptional repressor during retinoic acid induced cell differentiation / protein import into mitochondrial intermembrane space / migrasome / high-density lipoprotein particle binding / positive regulation of T cell mediated immune response to tumor cell / chaperonin ATPase / Mitochondrial protein import / positive regulation of macrophage activation / cellular response to interleukin-7 / biological process involved in interaction with symbiont / MyD88-dependent toll-like receptor signaling pathway / 'de novo' protein folding / sperm plasma membrane / B cell activation / DNA replication origin binding / B cell proliferation / apoptotic mitochondrial changes / positive regulation of interferon-alpha production / apolipoprotein binding / positive regulation of interleukin-10 production / protein maturation / response to unfolded protein / chaperone-mediated protein complex assembly / clathrin-coated pit / isomerase activity / sperm midpiece / response to cold / positive regulation of interleukin-12 production / T cell activation / secretory granule / lipopolysaccharide binding / ATP-dependent protein folding chaperone / positive regulation of interleukin-6 production / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of T cell activation / unfolded protein binding / positive regulation of type II interferon production / p53 binding / protein folding / single-stranded DNA binding / double-stranded RNA binding / protein-folding chaperone binding / protein refolding / mitochondrial inner membrane / protein stabilization / early endosome / mitochondrial matrix / positive regulation of apoptotic process / ubiquitin protein ligase binding / negative regulation of apoptotic process / enzyme binding / cell surface / ATP hydrolysis activity / protein-containing complex / mitochondrion / extracellular space / RNA binding / extracellular exosome / ATP binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / : / 60 kDa heat shock protein, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsBraxton, J.R. / Shao, H. / Tse, E. / Gestwicki, J.E. / Southworth, D.R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: bioRxiv / Year: 2023
Title: Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly.
Authors: Julian R Braxton / Hao Shao / Eric Tse / Jason E Gestwicki / Daniel R Southworth /
Abstract: The mitochondrial chaperonin, mtHsp60, promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its ...The mitochondrial chaperonin, mtHsp60, promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin binds to clients and progresses through its ATP-dependent reaction cycle are not clear. Here, we determined cryo-electron microscopy (cryo-EM) structures of a hyperstable disease-associated mtHsp60 mutant, V72I, at three stages in this cycle. Unexpectedly, client density is identified in all states, revealing interactions with mtHsp60's apical domains and C-termini that coordinate client positioning in the folding chamber. We further identify a striking asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60/mtHsp10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify a new role for the apical domains in coordinating client capture and progression through the cycle, and suggest a conserved mechanism of group I chaperonin function.
History
DepositionFeb 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 60 kDa heat shock protein, mitochondrial
G: 60 kDa heat shock protein, mitochondrial
F: 60 kDa heat shock protein, mitochondrial
E: 60 kDa heat shock protein, mitochondrial
D: 60 kDa heat shock protein, mitochondrial
C: 60 kDa heat shock protein, mitochondrial
B: 60 kDa heat shock protein, mitochondrial
K: 60 kDa heat shock protein, mitochondrial
J: 60 kDa heat shock protein, mitochondrial
I: 60 kDa heat shock protein, mitochondrial
H: 60 kDa heat shock protein, mitochondrial
N: 60 kDa heat shock protein, mitochondrial
M: 60 kDa heat shock protein, mitochondrial
L: 60 kDa heat shock protein, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)824,48256
Polymers816,49414
Non-polymers7,98842
Water0
1


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

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Components

#1: Protein
60 kDa heat shock protein, mitochondrial / 60 kDa chaperonin / Chaperonin 60 / CPN60 / Heat shock protein 60 / HSP-60 / Hsp60 / HuCHA60 / ...60 kDa chaperonin / Chaperonin 60 / CPN60 / Heat shock protein 60 / HSP-60 / Hsp60 / HuCHA60 / Mitochondrial matrix protein P1 / P60 lymphocyte protein


Mass: 58321.000 Da / Num. of mol.: 14 / Mutation: V72I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HSPD1, HSP60 / Production host: Escherichia coli (E. coli) / References: UniProt: P10809, chaperonin ATPase
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
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: ATP-bound Hsp60 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco easiGlow, 15 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: Wait time 10 sec, blot time 3 sec, blot force 0

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Calibrated magnification: 53937 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 66 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 15900

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 405263 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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