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- EMDB-40727: Cryo-EM structure of TRPM2 chanzyme in the presence of Magnesium ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40727
TitleCryo-EM structure of TRPM2 chanzyme in the presence of Magnesium and ADP-ribose, open state
Map dataSharpened composite map.
Sample
  • Complex: TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; ADP-ribose intact; open state.
    • Protein or peptide: TRPM2 chanzyme
  • Ligand: ADENOSINE-5-DIPHOSPHORIBOSE
  • Ligand: MAGNESIUM ION
  • Ligand: CHOLESTEROL
  • Ligand: water
KeywordsTRPM2 Chanzyme / Channel-enzyme / MEMBRANE PROTEIN
Function / homology
Function and homology information


calcium channel activity / plasma membrane
Similarity search - Function
TRPM, SLOG domain / : / SLOG in TRPM / Nudix hydrolase domain profile. / NUDIX hydrolase domain / Voltage-dependent channel domain superfamily / NUDIX hydrolase-like domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Biological speciesSalpingoeca rosetta (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsHuang Y / Kumar S / Lu W / Du J
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL153219 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS112363 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS111031 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.
Authors: Yihe Huang / Sushant Kumar / Junuk Lee / Wei Lü / Juan Du /
Abstract: Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that ...Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species.
History
DepositionMay 5, 2023-
Header (metadata) releaseMay 15, 2024-
Map releaseMay 15, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40727.png

Downloads & links

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Map

FileDownload / File: emd_40727.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened composite map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 440 pix.
= 363.44 Å		0.83 Å/pix.
x 440 pix.
= 363.44 Å		0.83 Å/pix.
x 440 pix.
= 363.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 8.0
Minimum - Maximum-21.298155000000001 - 38.804188000000003
Average (Standard dev.)0.07635498 (±1.0114208)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 363.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened composite map.

Fileemd_40727_additional_1.map
AnnotationUnsharpened composite map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened consensus map.

Fileemd_40727_additional_2.map
AnnotationSharpened consensus map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened consensus map.

Fileemd_40727_additional_3.map
AnnotationUnsharpened consensus map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened NUDT9-H focused map.

Fileemd_40727_additional_4.map
AnnotationSharpened NUDT9-H focused map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened NUDT9-H focused map.

Fileemd_40727_additional_5.map
AnnotationUnsharpened NUDT9-H focused map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; A...

EntireName: TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; ADP-ribose intact; open state.
Components
  • Complex: TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; ADP-ribose intact; open state.
    • Protein or peptide: TRPM2 chanzyme
  • Ligand: ADENOSINE-5-DIPHOSPHORIBOSE
  • Ligand: MAGNESIUM ION
  • Ligand: CHOLESTEROL
  • Ligand: water

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Supramolecule #1: TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; A...

SupramoleculeName: TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; ADP-ribose intact; open state.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Salpingoeca rosetta (eukaryote)

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Macromolecule #1: TRPM2 chanzyme

MacromoleculeName: TRPM2 chanzyme / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Salpingoeca rosetta (eukaryote)
Molecular weightTheoretical: 168.252484 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MQRARPGELV EVIMFRPTGK ARVSNLDESM AMEFTDLRTR AMSSAAMIRQ SVAAKTLLIE NEDGKGSTRM EVQDFMKRFH MHASEDDKT GSPSTAWGTL RFPTKEATAP YLRLSVNDDP EDALLFVKAM LAQKYGETYD RPSLILSVTG GARNFTLPPR L ETAIAKGL ...String:
MQRARPGELV EVIMFRPTGK ARVSNLDESM AMEFTDLRTR AMSSAAMIRQ SVAAKTLLIE NEDGKGSTRM EVQDFMKRFH MHASEDDKT GSPSTAWGTL RFPTKEATAP YLRLSVNDDP EDALLFVKAM LAQKYGETYD RPSLILSVTG GARNFTLPPR L ETAIAKGL RLAAQRTNAW VVTGGTNTGV MKLTGQIMEA LSKTQSHFIP PTIGIATYGV IIGGDDMTRG EPPKIGLEYE MH KKDPPKT TPLDDNHNLF LLVDDGSTNK FGKEIKFRAA FENAAGQAFA APVVTIVVQG GPGTLGTALQ AVRQGTPIVV VDG SGLAAD VLAYAYNFMH NPLTRFKSYT IDDLRQKVAQ TFNPKSSQQL TNLLDSALEC VQDPNLVVVY SLQESGIDEF DDCI LKAIF SSQGKLGNKL KQAMYFDQLD VAKRALSEAS KNGQHNEIAA CINDNLMAAM MHNKPHFVEL YLGFDAKIYE LKPSE EVAK TNITALDELP SFALAIEELY KREAKKPHSH VQRLVSLSNT DVLGRHYRVS TQRGDGTTRR IGRDLANTRA YNVLRM DQI FARLVSKDFS VNRDFTIYDS KYDKVPGIQF RRTAQASHML FLWAICLDRF RMARHFWLIG DQSIINALVA SRILERL ST HRALQGPHLA EERAKMQHNA KKFEELAVGV LGECHGSDSH MASEMLHSKN DMFNKKNAIN IAYDAKSLAF LSHPATQS V INADWYGHLK SVTSFWAVLF AFFFPFFVLP FINFSEDHAE QQVEAPRDFF TDAPRSSHSA NSTTSGAHRL RRKFAKFYS APYTRFISDL LSHFVLCVVT SYFVLDKLED TISAIEWILL VWFVALLLEE LRQMIFCDGI AEYISDTWNR LDLIMITLFF VGFFTHASD PSNQDSKVVS KGIHAFLVVV LWLRFMRYYA LSKNLGPKLI MMMEMMKDVS TFVFLLLIFL IGYGVAAQSL L SPDEDFSS RTFIGVLFRP YFQIYGELFL DDLNSEANCL GDTPFTECSR ETVRMVPFFL AVYILGSNVL LVNLLIAMFN DT YMKVQEA AEDLWRKQNY ELCAEYKDRP FLPAPFILLA HVHMLFMRLL RLCGVHTQEH EKIQDDETKR KITTFEELNT DKF LRRWER ERQEMLEARV KMTNDNVVQA MGMMDQLLEH MISFRFSLDQ QATKIKQEIR DDGLPSTEPT GLVSRTPSQP INRL NSAVA VHGHTAEAAE WYVPPEEYPK SGGVKRYLID ASMVPLSIMC PSYDPVEYTH PSVAAQPVWA DPADPRKIKF NVKDE VNGK VVDRTSCHPS GISIDSNTGR PINPWGRTGM TGRGLLGKWG VNQAADTVVT RWKRSPDGSI LERDGKKVLE FVAIQR QDN KMWAIPGGFV DNGEDVALTS GREFMEEALG MGTSADLMSA ESKDSLAALF SSGTIVARIY CEDPRNTDNA WVETTCV NF HDESGRHAAR LKLQGGDDAE HARWMMVHGG LNLFASHRTL LQHVTSALNA YF

UniProtKB: Nudt9 protein

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Macromolecule #2: ADENOSINE-5-DIPHOSPHORIBOSE

MacromoleculeName: ADENOSINE-5-DIPHOSPHORIBOSE / type: ligand / ID: 2 / Number of copies: 8 / Formula: APR
Molecular weightTheoretical: 559.316 Da
Chemical component information

ChemComp-APR:
ADENOSINE-5-DIPHOSPHORIBOSE

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 21 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 4 / Number of copies: 12 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 9 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration8.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 0.02 sec. / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.9 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 35103
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8srd:
Cryo-EM structure of TRPM2 chanzyme in the presence of Magnesium and ADP-ribose, open state

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