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- PDB-8qa6: MTHFR + SAM inhibited state -

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Basic information

Entry
Database: PDB / ID: 8qa6
TitleMTHFR + SAM inhibited state
ComponentsMethylenetetrahydrofolate reductase (NADPH)
KeywordsFLAVOPROTEIN / Inhibited / allosteric / folate / S-Adenosylmethionin
Function / homology
Function and homology information


methylenetetrahydrofolate reductase (NADPH) / response to vitamin B2 / methylenetetrahydrofolate reductase (NADPH) activity / methylenetetrahydrofolate reductase [NAD(P)H] activity / methionine metabolic process / modified amino acid binding / heterochromatin organization / homocysteine metabolic process / S-adenosylmethionine metabolic process / response to folic acid ...methylenetetrahydrofolate reductase (NADPH) / response to vitamin B2 / methylenetetrahydrofolate reductase (NADPH) activity / methylenetetrahydrofolate reductase [NAD(P)H] activity / methionine metabolic process / modified amino acid binding / heterochromatin organization / homocysteine metabolic process / S-adenosylmethionine metabolic process / response to folic acid / Metabolism of folate and pterines / methionine biosynthetic process / tetrahydrofolate interconversion / response to amino acid / FAD binding / response to interleukin-1 / neural tube closure / NADP binding / flavin adenine dinucleotide binding / response to hypoxia / response to xenobiotic stimulus / protein-containing complex binding / cytosol
Similarity search - Function
Eukaryotic-type methylenetetrahydrofolate reductase / : / MTHFR, SAM-binding regulatory domain / Methylenetetrahydrofolate reductase-like / Methylenetetrahydrofolate reductase / FAD-linked oxidoreductase-like
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / S-ADENOSYLMETHIONINE / Methylenetetrahydrofolate reductase (NADPH)
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsBlomgren, L.K.M. / Yue, W.W. / Froese, D.S. / McCorvie, T.J.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_192505 Switzerland
CitationJournal: Nat Commun / Year: 2024
Title: Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase.
Authors: Linnea K M Blomgren / Melanie Huber / Sabrina R Mackinnon / Céline Bürer / Arnaud Baslé / Wyatt W Yue / D Sean Froese / Thomas J McCorvie /
Abstract: 5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved ...5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product S-adenosyl-L-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.
History
DepositionAug 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _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: Methylenetetrahydrofolate reductase (NADPH)
B: Methylenetetrahydrofolate reductase (NADPH)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)154,0878
Polymers150,9222
Non-polymers3,1656
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 Methylenetetrahydrofolate reductase (NADPH)


Mass: 75461.195 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTHFR / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P42898
#2: Chemical
ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
Has ligand of interestY

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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: Human 5,10-methylenetetrahydrofolate reductase in complex with S-Adenosyl-L-Methionine
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM S-Adenosyl-L-Methionine, filter sterilised
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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: 240000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6.17 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 2394

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
4cryoSPARC4.2.1CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
11cryoSPARC4.2.1classification
12cryoSPARC4.2.13D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 882000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135192 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 105.2 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coeficient
Atomic model building
ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameTypeDetails
11P461511AlphaFoldin silico model
21SwissModelin silico modelHomology modeling of human MTHFR using the AlphaFold models as a template
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039364
ELECTRON MICROSCOPYf_angle_d0.62512734
ELECTRON MICROSCOPYf_dihedral_angle_d6.2351204
ELECTRON MICROSCOPYf_chiral_restr0.0421372
ELECTRON MICROSCOPYf_plane_restr0.0061614

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