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- PDB-7al9: human GTP cyclohydrolase I feedback regulatory protein (GFRP) in ... -

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Basic information

Entry
Database: PDB / ID: 7al9
Titlehuman GTP cyclohydrolase I feedback regulatory protein (GFRP) in complex with phenylalanine
ComponentsGTP cyclohydrolase 1 feedback regulatory protein
KeywordsPROTEIN BINDING / GTP cyclohydrolase 1 feedback regulatory protein / regulatory protein / L-phenylalanine binding (Phe) synthesis allosteric regulation
Function / homology
Function and homology information


GTP cyclohydrolase binding / negative regulation of biosynthetic process / regulation of nitric oxide biosynthetic process / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / melanosome / nuclear membrane / dendrite / nucleoplasm / nucleus / cytoplasm / cytosol
Similarity search - Function
GTP cyclohydrolase I, feedback regulatory protein / GFRP superfamily / GTP cyclohydrolase I feedback regulatory protein (GFRP)
Similarity search - Domain/homology
: / PHENYLALANINE / GTP cyclohydrolase 1 feedback regulatory protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.745 Å
AuthorsEbenhoch, R. / Nar, H.
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
DepositionOct 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: 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.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 7, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GTP cyclohydrolase 1 feedback regulatory protein
B: GTP cyclohydrolase 1 feedback regulatory protein
C: GTP cyclohydrolase 1 feedback regulatory protein
D: GTP cyclohydrolase 1 feedback regulatory protein
E: GTP cyclohydrolase 1 feedback regulatory protein
F: GTP cyclohydrolase 1 feedback regulatory protein
G: GTP cyclohydrolase 1 feedback regulatory protein
H: GTP cyclohydrolase 1 feedback regulatory protein
I: GTP cyclohydrolase 1 feedback regulatory protein
J: GTP cyclohydrolase 1 feedback regulatory protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,96830
Polymers99,92510
Non-polymers2,04320
Water6,287349
1
A: GTP cyclohydrolase 1 feedback regulatory protein
C: GTP cyclohydrolase 1 feedback regulatory protein
F: GTP cyclohydrolase 1 feedback regulatory protein
H: GTP cyclohydrolase 1 feedback regulatory protein
J: GTP cyclohydrolase 1 feedback regulatory protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,98415
Polymers49,9625
Non-polymers1,02110
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11230 Å2
ΔGint-68 kcal/mol
Surface area18760 Å2
MethodPISA
2
B: GTP cyclohydrolase 1 feedback regulatory protein
D: GTP cyclohydrolase 1 feedback regulatory protein
E: GTP cyclohydrolase 1 feedback regulatory protein
G: GTP cyclohydrolase 1 feedback regulatory protein
I: GTP cyclohydrolase 1 feedback regulatory protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,98415
Polymers49,9625
Non-polymers1,02110
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11390 Å2
ΔGint-65 kcal/mol
Surface area18770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)66.939, 98.123, 67.455
Angle α, β, γ (deg.)90, 102.53, 90
Int Tables number4
Space group name H-MP1211

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Components

#1: 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) / References: UniProt: P30047
#2: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-PHE / PHENYLALANINE


Type: L-peptide linking / Mass: 165.189 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: C9H11NO2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 349 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.2 Å3/Da / Density % sol: 44.2 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 5 / Details: 0.1 M NaCit pH 5.0; 20% w/v PEG 8000 (Proplex E11)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1.00004 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: May 17, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00004 Å / Relative weight: 1
ReflectionResolution: 1.745→54.68 Å / Num. obs: 56173 / % possible obs: 82.7 % / Redundancy: 3.4 % / CC1/2: 0.999 / Net I/σ(I): 9.1
Reflection shellResolution: 1.745→1.98 Å / Redundancy: 3.3 % / Num. unique obs: 16863 / CC1/2: 0.344 / % possible all: 76.3

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Processing

Software
NameVersionClassification
BUSTER2.11.7refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7ACC

7acc


Resolution: 1.745→54.68 Å / Cor.coef. Fo:Fc: 0.94 / Cor.coef. Fo:Fc free: 0.935 / SU R Cruickshank DPI: 0.269 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.263 / SU Rfree Blow DPI: 0.191 / SU Rfree Cruickshank DPI: 0.193
RfactorNum. reflection% reflectionSelection details
Rfree0.2346 2298 -RANDOM
Rwork0.2029 ---
obs0.2045 47515 55 %-
Displacement parametersBiso mean: 32.97 Å2
Baniso -1Baniso -2Baniso -3
1-2.9027 Å20 Å25.5349 Å2
2---2.0644 Å20 Å2
3----0.8383 Å2
Refine analyzeLuzzati coordinate error obs: 0.29 Å
Refinement stepCycle: LAST / Resolution: 1.745→54.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6700 0 130 349 7179
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0087070HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.99570HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2480SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes1170HARMONIC5
X-RAY DIFFRACTIONt_it6960HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion840SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact5729SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.52
X-RAY DIFFRACTIONt_other_torsion16.13
LS refinement shellResolution: 1.75→1.87 Å
RfactorNum. reflection% reflection
Rfree0.2787 44 -
Rwork0.2404 --
obs0.242 951 5.75 %
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.28850.69530.10811.5401-0.96331.6077-0.0156-0.07270.0698-0.0727-0.028-0.01310.0698-0.01310.04360.0343-0.00770.0794-0.08550.02940.0156-18.3698-2.234419.2536
22.7856-0.1514-0.72461.53650.90722.1994-0.17890.10350.13040.10350.0350.13470.13040.13470.14390.03230.02410.0946-0.09460.01020.043411.0682-2.514414.7364
31.8391.3183-0.91670.7605-0.12364.62770.17770.02850.03780.0285-0.14880.06250.03780.0625-0.02890.0376-0.03860.08050.02120.0556-0.1082-14.81739.602735.6762
41.255-0.508-1.211.09550.06073.5935-0.0576-0.01940.2118-0.01940.0342-0.15540.2118-0.15540.02330.058-0.02440.0813-0.0249-0.0367-0.0617.54128.2112-2.3986
52.64460.54360.46321.72860.74972.40360.2088-0.07-0.0683-0.07-0.03880.1249-0.06830.1249-0.1699-0.0115-0.05670.1276-0.1038-0.07410.049313.032829.915922.7127
63.1822-0.60970.1461.3615-0.07282.29920.25980.0809-0.11150.0809-0.0862-0.0896-0.1115-0.0896-0.1736-0.04010.05110.128-0.11130.0940.1054-20.129429.72269.1719
72.41621.2499-1.34342.7376-0.1472.07170.0904-0.1055-0.063-0.10550.08710.0423-0.0630.0423-0.17750.0258-0.01130.0732-0.04970.0727-0.02178.89528.38282.1702
82.0234-0.5333-0.92312.2895-0.49793.36450.16750.2252-0.34830.2252-0.0026-0.1672-0.3483-0.1672-0.16490.04840.01550.1158-0.0204-0.0818-0.0443-15.842529.456929.5919
93.8297-0.06760.81963.2404-0.67641.31570.07440.1823-0.00330.1823-0.05370.0214-0.00330.0214-0.02070.017-0.0140.03230.01450.0167-0.041214.53310.904730.1047
103.8688-0.06480.08312.4157-0.44891.602-0.0094-0.17080.0877-0.1708-0.0087-0.05950.0877-0.05950.01810.00790.00020.0341-0.0073-0.0357-0.0211-21.70210.17382.97
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A1 - 83
2X-RAY DIFFRACTION1{ A|* }A102
3X-RAY DIFFRACTION1{ A|* }A103 - 145
4X-RAY DIFFRACTION1{ A|* }A2101
5X-RAY DIFFRACTION2{ B|* }B1 - 83
6X-RAY DIFFRACTION2{ B|* }B102
7X-RAY DIFFRACTION2{ B|* }B103 - 138
8X-RAY DIFFRACTION2{ B|* }B2101
9X-RAY DIFFRACTION3{ C|* }C1 - 83
10X-RAY DIFFRACTION3{ C|* }C102
11X-RAY DIFFRACTION3{ C|* }C103 - 145
12X-RAY DIFFRACTION3{ C|* }C2101
13X-RAY DIFFRACTION4{ D|* }D1 - 83
14X-RAY DIFFRACTION4{ D|* }D102
15X-RAY DIFFRACTION4{ D|* }D103 - 137
16X-RAY DIFFRACTION4{ D|* }D2101
17X-RAY DIFFRACTION5{ E|* }E1 - 83
18X-RAY DIFFRACTION5{ E|* }E102
19X-RAY DIFFRACTION5{ E|* }E103 - 145
20X-RAY DIFFRACTION5{ E|* }E2101
21X-RAY DIFFRACTION6{ F|* }F1 - 83
22X-RAY DIFFRACTION6{ F|* }F102
23X-RAY DIFFRACTION6{ F|* }F103 - 140
24X-RAY DIFFRACTION6{ F|* }F2101
25X-RAY DIFFRACTION7{ G|* }G1 - 83
26X-RAY DIFFRACTION7{ G|* }G102
27X-RAY DIFFRACTION7{ G|* }G103 - 141
28X-RAY DIFFRACTION7{ G|* }G2101
29X-RAY DIFFRACTION8{ H|* }H1 - 83
30X-RAY DIFFRACTION8{ H|* }H102
31X-RAY DIFFRACTION8{ H|* }H103 - 137
32X-RAY DIFFRACTION8{ H|* }H2101
33X-RAY DIFFRACTION9{ I|* }I1 - 83
34X-RAY DIFFRACTION9{ I|* }I102
35X-RAY DIFFRACTION9{ I|* }I103 - 135
36X-RAY DIFFRACTION9{ I|* }I2101
37X-RAY DIFFRACTION10{ J|* }J1 - 83
38X-RAY DIFFRACTION10{ J|* }J102
39X-RAY DIFFRACTION10{ J|* }J103 - 139
40X-RAY DIFFRACTION10{ J|* }J2101

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