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- PDB-9b0t: Cryo-EM structure of E227Q variant of uMtCK1 in complex with tran... -

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Basic information

Entry
Database: PDB / ID: 9b0t
TitleCryo-EM structure of E227Q variant of uMtCK1 in complex with transition state analog
ComponentsCreatine kinase U-type, mitochondrial
KeywordsTRANSFERASE / mitochondrial creatine kinase
Function / homology
Function and homology information


creatine kinase / Creatine metabolism / phosphocreatine biosynthetic process / creatine kinase activity / mitochondrial inner membrane / mitochondrion / ATP binding
Similarity search - Function
ATP:guanido phosphotransferase, N-terminal / ATP:guanido phosphotransferase, N-terminal domain superfamily / ATP:guanido phosphotransferase, N-terminal domain / Phosphagen kinase N-terminal domain profile. / ATP:guanido phosphotransferase active site / Phosphagen kinase active site signature. / ATP:guanido phosphotransferase / ATP:guanido phosphotransferase, catalytic domain / ATP:guanido phosphotransferase, C-terminal catalytic domain / Phosphagen kinase C-terminal domain profile. / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / N-[(E)-AMINO(IMINO)METHYL]-N-METHYLGLYCINE / NITRATE ION / Creatine kinase U-type, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsDemir, M. / Koepping, L. / Zhao, J. / Sergienko, E.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorOD026926 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA030199 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA251910 United States
CitationJournal: Structure / Year: 2025
Title: Structural basis for substrate binding, catalysis, and inhibition of cancer target mitochondrial creatine kinase by a covalent inhibitor.
Authors: Merve Demir / Laura Koepping / Ya Li / Lynn Fujimoto / Andrey Bobkov / Jianhua Zhao / Taro Hitosugi / Eduard Sergienko /
Abstract: Mitochondrial creatine kinases (MtCKs) are key players in maintaining energy homeostasis in cells that work with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. The ...Mitochondrial creatine kinases (MtCKs) are key players in maintaining energy homeostasis in cells that work with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. The inhibition of breast cancer growth by cyclocreatine targeting CKs indicates dependence of cancer cells on the "energy shuttle" for cell growth and survival. Hence, understanding key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present mutational and structural investigations on understudied ubiquitous MtCK that showed closure of the loop comprising His61 is specific to and relies on creatine binding and mechanism of phosphoryl transfer depends on electrostatics of active site. We demonstrate that previously identified pan-CK covalent inhibitor CKi inhibit breast cancer cell proliferation; however, our biochemical and structural data indicated that inhibition by CKi is highly dependent on covalent link formation and conformational changes upon creatine binding are not observed.
History
DepositionMar 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Apr 16, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Creatine kinase U-type, mitochondrial
B: Creatine kinase U-type, mitochondrial
C: Creatine kinase U-type, mitochondrial
D: Creatine kinase U-type, mitochondrial
E: Creatine kinase U-type, mitochondrial
F: Creatine kinase U-type, mitochondrial
G: Creatine kinase U-type, mitochondrial
H: Creatine kinase U-type, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)385,93940
Polymers380,7828
Non-polymers5,15732
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
Creatine kinase U-type, mitochondrial / Acidic-type mitochondrial creatine kinase / Mia-CK / Ubiquitous mitochondrial creatine kinase / U-MtCK


Mass: 47597.734 Da / Num. of mol.: 8 / Mutation: E227Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CKMT1A, CKMT, CKMT1B, CKMT / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P12532, creatine kinase
#2: Chemical
ChemComp-CRN / N-[(E)-AMINO(IMINO)METHYL]-N-METHYLGLYCINE / CREATINE


Mass: 131.133 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C4H9N3O2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: NO3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Octameric u-type mitochondrial creatine kinase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 10 mM creatine, 1 mM ADP, and 50 mM sodium nitrate
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris(hydroxymethyl)aminomethane1
2200 mMsodium chlorideNaCl1
350 mMmagnesium chlorideMgCl21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: 2100 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 34 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
7Cootmodel fitting
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3851095
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1053188 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322176
ELECTRON MICROSCOPYf_angle_d0.6230304
ELECTRON MICROSCOPYf_dihedral_angle_d9.2173272
ELECTRON MICROSCOPYf_chiral_restr0.0393424
ELECTRON MICROSCOPYf_plane_restr0.0043968

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