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- PDB-8t0z: Human liver-type glutaminase (K253A) with L-Gln, filamentous form -

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Basic information

Entry
Database: PDB / ID: 8t0z
TitleHuman liver-type glutaminase (K253A) with L-Gln, filamentous form
ComponentsGlutaminase liver isoform, mitochondrial
KeywordsHYDROLASE / Metabolic / Cancer
Function / homology
Function and homology information


L-glutamine catabolic process / glutamate biosynthetic process / Glutamate and glutamine metabolism / glutaminase / Glutamate Neurotransmitter Release Cycle / glutaminase activity / amino acid metabolic process / reactive oxygen species metabolic process / TP53 Regulates Metabolic Genes / regulation of apoptotic process ...L-glutamine catabolic process / glutamate biosynthetic process / Glutamate and glutamine metabolism / glutaminase / Glutamate Neurotransmitter Release Cycle / glutaminase activity / amino acid metabolic process / reactive oxygen species metabolic process / TP53 Regulates Metabolic Genes / regulation of apoptotic process / mitochondrial matrix / mitochondrion
Similarity search - Function
Glutaminase, EF-hand domain / EF-hand domain / Glutaminase / Glutaminase / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / Beta-lactamase/transpeptidase-like / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
GLUTAMINE / Glutaminase liver isoform, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsFeng, S. / Aplin, C. / Nguyen, T.-T.T. / Milano, S.K. / Cerione, R.A.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122575 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA201402 United States
National Science Foundation (NSF, United States)DMR-1719875 United States
National Institutes of Health/Office of the DirectorS10OD030470-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129539 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA223534 United States
CitationJournal: Nat Commun / Year: 2024
Title: Filament formation drives catalysis by glutaminase enzymes important in cancer progression.
Authors: Shi Feng / Cody Aplin / Thuy-Tien T Nguyen / Shawn K Milano / Richard A Cerione /
Abstract: The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the 'glutamine addiction' of cancer cells. They are the targets of anti-cancer drugs; however, their ...The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the 'glutamine addiction' of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an 'activation loop' to assume a specific conformation that works together with a 'lid' to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.
History
DepositionJun 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutaminase liver isoform, mitochondrial
B: Glutaminase liver isoform, mitochondrial
C: Glutaminase liver isoform, mitochondrial
D: Glutaminase liver isoform, mitochondrial
E: Glutaminase liver isoform, mitochondrial
G: Glutaminase liver isoform, mitochondrial
I: Glutaminase liver isoform, mitochondrial
K: Glutaminase liver isoform, mitochondrial
F: Glutaminase liver isoform, mitochondrial
H: Glutaminase liver isoform, mitochondrial
J: Glutaminase liver isoform, mitochondrial
L: Glutaminase liver isoform, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)797,97324
Polymers796,21912
Non-polymers1,75412
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Glutaminase liver isoform, mitochondrial / GLS / L-glutaminase / L-glutamine amidohydrolase


Mass: 66351.617 Da / Num. of mol.: 12 / Mutation: K253A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GLS2, GA / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UI32, glutaminase
#2: Chemical
ChemComp-GLN / GLUTAMINE


Type: L-peptide linking / Mass: 146.144 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C5H10N2O3 / 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: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Human liver-type glutaminase (K253A) with L-Gln, filamentous form
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: 48 ° / Axial rise/subunit: 66 Å / Axial symmetry: C1
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48000 / Symmetry type: HELICAL

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