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- PDB-8u7v: Human retinal variant phosphomimetic IMPDH1(546)-S477D filament b... -

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Basic information

Entry
Database: PDB / ID: 8u7v
TitleHuman retinal variant phosphomimetic IMPDH1(546)-S477D filament bound by GTP, ATP, IMP, and NAD+, interface-centered
ComponentsInosine-5'-monophosphate dehydrogenase 1
KeywordsOXIDOREDUCTASE / dehydrogenase / nucleotide synthesis / filament / phosphomimetic
Function / homology
Function and homology information


'de novo' XMP biosynthetic process / Purine ribonucleoside monophosphate biosynthesis / lymphocyte proliferation / IMP dehydrogenase / IMP dehydrogenase activity / GMP biosynthetic process / Azathioprine ADME / GTP biosynthetic process / azurophil granule lumen / secretory granule lumen ...'de novo' XMP biosynthetic process / Purine ribonucleoside monophosphate biosynthesis / lymphocyte proliferation / IMP dehydrogenase / IMP dehydrogenase activity / GMP biosynthetic process / Azathioprine ADME / GTP biosynthetic process / azurophil granule lumen / secretory granule lumen / ficolin-1-rich granule lumen / Potential therapeutics for SARS / nucleic acid binding / nucleotide binding / Neutrophil degranulation / DNA binding / RNA binding / extracellular region / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
IMP dehydrogenase / GMP reductase, conserved site / IMP dehydrogenase / GMP reductase signature. / Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase/GMP reductase / IMP dehydrogenase / GMP reductase domain / IMP dehydrogenase / GMP reductase domain / Domain in cystathionine beta-synthase and other proteins. / CBS domain / CBS domain / CBS domain profile. / Aldolase-type TIM barrel
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / INOSINIC ACID / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Inosine-5'-monophosphate dehydrogenase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsCalise, S.J. / Kollman, J.M.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM118396 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM149542 United States
National Institutes of Health/National Eye Institute (NIH/NEI)R21EY031546 United States
National Institutes of Health/Office of the DirectorS10OD032290 United States
Helen Hay Whitney Foundation United States
CitationJournal: J Cell Biol / Year: 2024
Title: Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.
Authors: S John Calise / Audrey G O'Neill / Anika L Burrell / Miles S Dickinson / Josephine Molfino / Charlie Clarke / Joel Quispe / David Sokolov / Rubén M Buey / Justin M Kollman /
Abstract: Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback ...Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.
History
DepositionSep 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Inosine-5'-monophosphate dehydrogenase 1
B: Inosine-5'-monophosphate dehydrogenase 1
C: Inosine-5'-monophosphate dehydrogenase 1
D: Inosine-5'-monophosphate dehydrogenase 1
E: Inosine-5'-monophosphate dehydrogenase 1
F: Inosine-5'-monophosphate dehydrogenase 1
G: Inosine-5'-monophosphate dehydrogenase 1
H: Inosine-5'-monophosphate dehydrogenase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)488,86548
Polymers468,3438
Non-polymers20,52140
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Inosine-5'-monophosphate dehydrogenase 1 / IMP dehydrogenase 1 / IMPD 1 / IMPDH 1 / IMPDH-I


Mass: 58542.906 Da / Num. of mol.: 8 / Mutation: S477D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IMPDH1, IMPD1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P20839, IMP dehydrogenase
#2: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#3: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
#4: Chemical
ChemComp-IMP / INOSINIC ACID


Mass: 348.206 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H13N4O8P / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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: single particle reconstruction

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Sample preparation

ComponentName: IMPDH1(546)-S477D filament bound by GTP, ATP, IMP, and NAD+
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.468 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 756

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
8ISOLDEmodel fitting
9Cootmodel fitting
11PHENIX1.20.1_4487:model refinement
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 434369
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149184 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00530216
ELECTRON MICROSCOPYf_angle_d0.80541120
ELECTRON MICROSCOPYf_dihedral_angle_d8.0824104
ELECTRON MICROSCOPYf_chiral_restr0.1144776
ELECTRON MICROSCOPYf_plane_restr0.0065056

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