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- EMDB-45856: CryoEM Structure of the C-terminally truncated form of human NAD ... -

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Basic information

Entry
Database: EMDB / ID: EMD-45856
TitleCryoEM Structure of the C-terminally truncated form of human NAD Kinase bound to NAD
Map data
Sample
  • Complex: human NAD Kinase
    • Protein or peptide: NAD kinase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Keywordscatalyzes the reaction of ATP + NAD+ = ADP + H+ + NADP+ / SIGNALING PROTEIN
Function / homology
Function and homology information


NAD+ kinase / NAD+ kinase activity / NADP biosynthetic process / Nicotinate metabolism / NAD metabolic process / positive regulation of insulin secretion involved in cellular response to glucose stimulus / ATP metabolic process / phosphorylation / ATP binding / metal ion binding / cytosol
Similarity search - Function
ATP-NAD kinase C-terminal domain / NAD kinase / ATP-NAD kinase, PpnK-type, C-terminal / ATP-NAD kinase N-terminal domain / NAD kinase/diacylglycerol kinase-like domain superfamily / Inorganic polyphosphate/ATP-NAD kinase, N-terminal
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.34 Å
AuthorsLi Y / Chen Z / Mary C / Labesse G / Hoxhaj G
Funding support United States, France, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01GM143236 United States
Welch FoundationI-2067-20210327 United States
Welch FoundationI-2067-20240404 United States
Agence Nationale de la Recherche (ANR)ANR-20-CE18-0025 France
Fondation pour la Recherche Medicale (FRM)EQU202303016265 France
CitationJournal: Sci Adv / Year: 2025
Title: Cryo-EM structure and regulation of human NAD kinase.
Authors: Prakash P Praharaj / Yang Li / Charline Mary / Mona H Soflaee / Kevin Ryu / Dohun Kim / Diem H Tran / Trishna Dey / Harrison J Tom / Halie Rion / Muriel Gelin / Andrew Lemoff / Lauren G ...Authors: Prakash P Praharaj / Yang Li / Charline Mary / Mona H Soflaee / Kevin Ryu / Dohun Kim / Diem H Tran / Trishna Dey / Harrison J Tom / Halie Rion / Muriel Gelin / Andrew Lemoff / Lauren G Zacharias / João S Patricio / Thomas P Mathews / Zhe Chen / Corinne Lionne / Gerta Hoxhaj / Gilles Labesse /
Abstract: Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial reducing cofactor for reductive biosynthesis and protection from oxidative stress. To fulfill their heightened anabolic and ...Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial reducing cofactor for reductive biosynthesis and protection from oxidative stress. To fulfill their heightened anabolic and reductive power demands, cancer cells must boost their NADPH production. Progrowth and mitogenic protein kinases promote the activity of cytosolic NAD kinase (NADK), which produces NADP, a limiting NADPH precursor. However, the molecular architecture and mechanistic regulation of human NADK remain undescribed. Here, we report the cryo-electron microscopy structure of human NADK, both in its apo-form and in complex with its substrate NAD (nicotinamide adenine dinucleotide), revealing a tetrameric organization with distinct structural features. We discover that the amino (N)- and carboxyl (C)-terminal tails of NADK have opposing effects on its enzymatic activity and cellular NADP(H) levels. Specifically, the C-terminal region is critical for NADK activity, whereas the N-terminal region exhibits an inhibitory role. This study highlights molecular insights into the regulation of a vital enzyme governing NADP(H) production.
History
DepositionJul 21, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45856.png

Downloads & links

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Map

FileDownload / File: emd_45856.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 240 pix.
= 250.92 Å		1.05 Å/pix.
x 240 pix.
= 250.92 Å		1.05 Å/pix.
x 240 pix.
= 250.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0455 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.0017541468 - 1.6199858
Average (Standard dev.)0.0011847111 (±0.024737336)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 250.92001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_45856_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45856_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : human NAD Kinase

EntireName: human NAD Kinase
Components
  • Complex: human NAD Kinase
    • Protein or peptide: NAD kinase
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: human NAD Kinase

SupramoleculeName: human NAD Kinase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Tetramer of c-terminally truncated form of human NAD Kinase bound to NAD
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49 kDa/nm

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Macromolecule #1: NAD kinase

MacromoleculeName: NAD kinase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: NAD+ kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.06991 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPSPVTTFGP KATVETQDPA SVRLTWNKSP KSVLVIKKMR DASLLQPFKE LCTHLMEENM IVYVEKKVLE DPAIASDESF GAVKKKFTT FREDYDDISN QIDFIICLGG DGTLLYASSL FQGSVPPVMA FHLGSLGFLT PFSFENFQSQ VTQVIEGNAA V VLRSRLKV ...String:
MPSPVTTFGP KATVETQDPA SVRLTWNKSP KSVLVIKKMR DASLLQPFKE LCTHLMEENM IVYVEKKVLE DPAIASDESF GAVKKKFTT FREDYDDISN QIDFIICLGG DGTLLYASSL FQGSVPPVMA FHLGSLGFLT PFSFENFQSQ VTQVIEGNAA V VLRSRLKV RVVKELRGKK TAVHNGLGEN GSQAAGLDMD VGKQAMQYQV LNEVVIDRGP SSYLSNVDVY LDGHLITTVQ GD GVIVSTP TGSTAYAAAA GASMIHPNVP AIMITPICPH SLSFRPIVVP AGVELKIMLS PEARNTAWVS FDGRKRQEIR HGD SISITT STYPLPSICV RDPVSDWFES LAQCLHWNVR KKQAHFELGL EHHHHHH

UniProtKB: NAD kinase

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Macromolecule #2: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 4 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.8 / Component:
ConcentrationName
50.0 mMTRIS
50.0 mMNaCl
2.0 mMDTT
/ Details: 50 mM Tris (pH 7.8), 50 mM NaCl, and 2 mM DTT.
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV
DetailsPurified by FPLC. Monodispersed.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 9105 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1716164 / Details: template based picking using a low resolution map
Startup modelType of model: EMDB MAP
EMDB ID:

45832

Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 251813
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-9cra:
CryoEM Structure of the C-terminally truncated form of human NAD Kinase bound to NAD

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