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- PDB-9z2d: Mitochondrial Creatine Kinase in complex with ADP, creatine, and ... -

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Basic information

Entry
Database: PDB / ID: 9z2d
TitleMitochondrial Creatine Kinase in complex with ADP, creatine, and uncompetitive inhibitor uci
ComponentsCreatine kinase U-type, mitochondrial
KeywordsTRANSFERASE/INHIBITOR / Kinase / inhibitor / TRANSFERASE-INHIBITOR complex / transition state analog
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 / Creatine kinase U-type, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsDemir, M. / Zhao, J. / Sergienko, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA251910 United States
CitationJournal: bioRxiv / Year: 2026
Title: Uncompetitive Allosteric Inhibitor of Mitochondrial Creatine Kinase Prevents Binding and Release of Creatine by Stabilization of Loop Closure.
Abstract: Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in ...Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in many cancer cells to meet elevated energy demands, which is associated with poor prognosis. This suggests that MtCK may be a promising target for cancer therapeutics. We sought to discover first-in-class selective inhibitors of MtCK with diverse mechanisms of action using high-throughput screening, biochemical characterization and cryo-EM studies. Through these studies, we identified diverse types of compounds that modulate activity of MtCK , including fast-equilibrium and time-dependent orthosteric and allosteric inhibitors. Select hits were subjected to enzymatic and binding assays to assess MtCK inhibition and binding. A subset of inhibitors was advanced into structural studies using cryo-EM resulting in the molecular structure of MtCK with and without bound substrates in complex with an allosteric uncompetitive inhibitor that we discovered. These studies identified compound's unique binding pocket on MtCK and established the molecular steps manifesting in the apparent uncompetitive mode of inhibition. We demonstrate that the compound stabilizes an active site loop in a closed conformation restricting access of the creatine substrate to and the release of phosphocreatine product from its binding pocket. These findings establish chemical tools suitable for validation of MtCK as a promising breast cancer target with high therapeutic potential and build a foundation for future structure-guided optimization of the hit compounds of MtCK we identified and de novo rational design of novel MtCK inhibitors.
History
DepositionNov 4, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 28, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 28, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)388,47940
Polymers380,7908
Non-polymers7,68932
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: 47598.719 Da / Num. of mol.: 8
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-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
#3: 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
#4: Chemical
ChemComp-A1CZQ / [4-(2-amino-6-methylpyrimidin-4-yl)piperazin-1-yl](3,5,7-trimethyl-1H-indol-2-yl)methanone


Mass: 378.471 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C21H26N6O / 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: Mitochondrial Creatine Kinase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS 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
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1083440 / Symmetry type: POINT
RefinementHighest resolution: 2.6 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00423464
ELECTRON MICROSCOPYf_angle_d0.55732008
ELECTRON MICROSCOPYf_dihedral_angle_d6.6553432
ELECTRON MICROSCOPYf_chiral_restr0.0393512
ELECTRON MICROSCOPYf_plane_restr0.0044176

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