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- PDB-6z80: stimulatory human GTP cyclohydrolase I - GFRP complex -

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Basic information

Entry
Database: PDB / ID: 6z80
Titlestimulatory human GTP cyclohydrolase I - GFRP complex
Components
  • GTP cyclohydrolase 1
  • GTP cyclohydrolase 1 feedback regulatory protein
KeywordsHYDROLASE / GTP cyclohydrolase GFTP / I / EC:3.5.4.16 / Tetrahydrobiopterin (BH4) synthesis / Cytosol / Zinc Ion Binding / Hydrolase Activity / Metal Ion Binding / Nucleotide Binding / allosteric enzyme / substrate analogue bound
Function / homology
Function and homology information


GTP cyclohydrolase binding / GTP cyclohydrolase I regulator activity / negative regulation of small molecule metabolic process / pteridine-containing compound biosynthetic process / negative regulation of biosynthetic process / regulation of lung blood pressure / GTP cyclohydrolase I / GTP cyclohydrolase I activity / neuromuscular process controlling posture / regulation of removal of superoxide radicals ...GTP cyclohydrolase binding / GTP cyclohydrolase I regulator activity / negative regulation of small molecule metabolic process / pteridine-containing compound biosynthetic process / negative regulation of biosynthetic process / regulation of lung blood pressure / GTP cyclohydrolase I / GTP cyclohydrolase I activity / neuromuscular process controlling posture / regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / neuron projection terminus / regulation of nitric oxide biosynthetic process / mitogen-activated protein kinase binding / response to pain / dopamine biosynthetic process / response to tumor necrosis factor / enzyme inhibitor activity / response to type II interferon / tetrahydrofolate biosynthetic process / positive regulation of heart rate / positive regulation of nitric-oxide synthase activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / nitric oxide biosynthetic process / regulation of blood pressure / melanosome / cytoplasmic vesicle / nuclear membrane / response to lipopolysaccharide / GTPase activity / dendrite / GTP binding / protein homodimerization activity / protein-containing complex / zinc ion binding / nucleoplasm / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
GTP cyclohydrolase I, feedback regulatory protein / GFRP superfamily / GTP cyclohydrolase I feedback regulatory protein (GFRP) / GTP cyclohydrolase I signature 2. / GTP cyclohydrolase I / GTP cyclohydrolase I, conserved site / GTP cyclohydrolase I domain / GTP cyclohydrolase I, N-terminal domain / GTP cyclohydrolase I / GTP cyclohydrolase I signature 1. / GTP cyclohydrolase I, C-terminal/NADPH-dependent 7-cyano-7-deazaguanine reductase
Similarity search - Domain/homology
8-OXO-GUANOSINE-5'-TRIPHOSPHATE / PHENYLALANINE / GTP cyclohydrolase 1 feedback regulatory protein / GTP cyclohydrolase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsEbenhoch, R. / Nar, H. / Vonck, J.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I.
Authors: Rebecca Ebenhoch / Simone Prinz / Susann Kaltwasser / Deryck J Mills / Robert Meinecke / Martin Rübbelke / Dirk Reinert / Margit Bauer / Lisa Weixler / Markus Zeeb / Janet Vonck / Herbert Nar /
Abstract: Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). ...Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
History
DepositionJun 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2May 22, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Jul 9, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: GTP cyclohydrolase 1
L: GTP cyclohydrolase 1 feedback regulatory protein
B: GTP cyclohydrolase 1
C: GTP cyclohydrolase 1
E: GTP cyclohydrolase 1
G: GTP cyclohydrolase 1
H: GTP cyclohydrolase 1
I: GTP cyclohydrolase 1
J: GTP cyclohydrolase 1
F: GTP cyclohydrolase 1
D: GTP cyclohydrolase 1
K: GTP cyclohydrolase 1 feedback regulatory protein
N: GTP cyclohydrolase 1 feedback regulatory protein
M: GTP cyclohydrolase 1 feedback regulatory protein
O: GTP cyclohydrolase 1 feedback regulatory protein
P: GTP cyclohydrolase 1 feedback regulatory protein
Q: GTP cyclohydrolase 1 feedback regulatory protein
R: GTP cyclohydrolase 1 feedback regulatory protein
S: GTP cyclohydrolase 1 feedback regulatory protein
T: GTP cyclohydrolase 1 feedback regulatory protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)360,87250
Polymers353,17420
Non-polymers7,69830
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area86470 Å2
ΔGint-811 kcal/mol
Surface area87700 Å2
MethodPISA

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Components

#1: Protein
GTP cyclohydrolase 1 / GTP cyclohydrolase I / GTP-CH-I


Mass: 25324.920 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GCH1, DYT5, GCH / Production host: Escherichia coli (E. coli) / Strain (production host): BL21Star (DE3) / References: UniProt: P30793, GTP cyclohydrolase I
#2: Protein
GTP cyclohydrolase 1 feedback regulatory protein / GFRP / GTP cyclohydrolase I feedback regulatory protein / p35


Mass: 9992.483 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GCHFR, GFRP / Production host: Escherichia coli (E. coli) / Strain (production host): BL21Star (DE3) / References: UniProt: P30047
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-PHE / PHENYLALANINE


Type: L-peptide linking / Mass: 165.189 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C9H11NO2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-8GT / 8-OXO-GUANOSINE-5'-TRIPHOSPHATE


Mass: 539.180 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H16N5O15P3 / 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: stimulatory GCH1-GFRP complex (Phenylalanine and 8-oxo-GTP bound)
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.353 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium phosphateNa2HPO41
220 mMsodium chlorideNaCl1
320 mMphenylalanineC9H11NO21
40.1 mM8-Oxo-guanosine-5'-triphosphate, Sodium saltC10H16N5O15P31
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3121
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: (1.13_2998:phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
2RELION3particle selection
3EPUimage acquisition
5CTFFIND4.1.10.CTF correction
9PHENIXmodel refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1272592
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122310 / Symmetry type: POINT
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 0 Å2 / Biso mean: 0 Å2 / Biso min: 0 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00722060
ELECTRON MICROSCOPYf_angle_d0.75129930
ELECTRON MICROSCOPYf_dihedral_angle_d14.81913490
ELECTRON MICROSCOPYf_chiral_restr0.0483360
ELECTRON MICROSCOPYf_plane_restr0.0043900

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