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- PDB-7qri: Regulatory domain dimer of tryptophan hydroxylase 2 in complex wi... -

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Basic information

Entry
Database: PDB / ID: 7qri
TitleRegulatory domain dimer of tryptophan hydroxylase 2 in complex with L-Phe
ComponentsTryptophan 5-hydroxylase 2
KeywordsOXIDOREDUCTASE / Tryptophan hydroxylase 2 / serotonin biosynthesis / aromatic amino acid hydroxylase
Function / homology
Function and homology information


tryptophan 5-monooxygenase / tryptophan 5-monooxygenase activity / Serotonin and melatonin biosynthesis / aromatic amino acid metabolic process / serotonin biosynthetic process / neuron projection / iron ion binding / cytosol
Similarity search - Function
Tryptophan 5-monooxygenase / Tryptophan 5-hydroxylase, catalytic domain / Tyrosine 3-monooxygenase-like / Aromatic amino acid hydroxylase, iron/copper binding site / Biopterin-dependent aromatic amino acid hydroxylases signature. / Aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase, C-terminal / Aromatic amino acid monoxygenase, C-terminal domain superfamily / Aromatic amino acid hydroxylase superfamily / Biopterin-dependent aromatic amino acid hydroxylase ...Tryptophan 5-monooxygenase / Tryptophan 5-hydroxylase, catalytic domain / Tyrosine 3-monooxygenase-like / Aromatic amino acid hydroxylase, iron/copper binding site / Biopterin-dependent aromatic amino acid hydroxylases signature. / Aromatic amino acid hydroxylase / Aromatic amino acid hydroxylase, C-terminal / Aromatic amino acid monoxygenase, C-terminal domain superfamily / Aromatic amino acid hydroxylase superfamily / Biopterin-dependent aromatic amino acid hydroxylase / Biopterin-dependent aromatic amino acid hydroxylase family profile. / ACT domain profile. / ACT domain / ACT-like domain
Similarity search - Domain/homology
PHENYLALANINE / Tryptophan 5-hydroxylase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsVedel, I.M. / Prestel, A. / Harris, P. / Peters, G.H.J. / Kragelund, B.B.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk Foundation#NNF18OC0032996 Denmark
CitationJournal: Structure / Year: 2023
Title: Structural characterization of human tryptophan hydroxylase 2 reveals that L-Phe is superior to L-Trp as the regulatory domain ligand.
Authors: Ida M Vedel / Andreas Prestel / Zhenwei Zhang / Natalia T Skawinska / Holger Stark / Pernille Harris / Birthe B Kragelund / Günther H J Peters /
Abstract: Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the brain. Consequently, regulation of TPH2 is relevant for serotonin-related diseases, yet the ...Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the brain. Consequently, regulation of TPH2 is relevant for serotonin-related diseases, yet the regulatory mechanism of TPH2 is poorly understood and structural and dynamical insights are missing. We use NMR spectroscopy to determine the structure of a 47 N-terminally truncated variant of the regulatory domain (RD) dimer of human TPH2 in complex with L-Phe, and show that L-Phe is the superior RD ligand compared with the natural substrate, L-Trp. Using cryo-EM, we obtain a low-resolution structure of a similarly truncated variant of the complete tetrameric enzyme with dimerized RDs. The cryo-EM two-dimensional (2D) class averages additionally indicate that the RDs are dynamic in the tetramer and likely exist in a monomer-dimer equilibrium. Our results provide structural information on the RD as an isolated domain and in the TPH2 tetramer, which will facilitate future elucidation of TPH2's regulatory mechanism.
History
DepositionJan 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1May 10, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 14, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jun 19, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tryptophan 5-hydroxylase 2
B: Tryptophan 5-hydroxylase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,8984
Polymers22,5682
Non-polymers3302
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, gel filtration, NMR Distance Restraints
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Tryptophan 5-hydroxylase 2 / Neuronal tryptophan hydroxylase / Tryptophan 5-monooxygenase 2


Mass: 11283.862 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TPH2, NTPH / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8IWU9, tryptophan 5-monooxygenase
#2: Chemical ChemComp-PHE / PHENYLALANINE


Type: L-peptide linking / Mass: 165.189 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H11NO2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
171isotropic22D 1H-15N HSQC
111isotropic23D HN(CA)CB
121isotropic23D HN(COCA)CB
131isotropic23D HNCO
141isotropic23D HN(CA)CO
151isotropic22D 1H-13C HSQC aliphatic
1121isotropic22D 1H-13C HSQC aromatic
161isotropic13D 1H-13C NOESY aliphatic
181isotropic13D 1H-15N NOESY
191isotropic13D HNCA
1101isotropic13D 1H-13C NOESY aliphatic 13C/15N filtered
1111isotropic13D 1H-15N NOESY 13C/15N filtered
1191isotropic13D (H)CCH-TOCSY
1181isotropic22D 1H-13C HSQC aliphatic amino acid specific
1162isotropic12D 1H-13C HSQC aromatic
1172isotropic12D 1H-13C HSQC aliphatic

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution11.1 mM [U-99% 13C; U-99% 15N] Tryptophan hydroxylase 2 (GP+ 48 to 145), 10 mM L-Phe, 20 mM sodium phosphate, 100 mM ammonium sulfate, 125 uM DSS, 95% H2O/5% D2OProtein95% H2O/5% D2O
solution20.45 mM Tryptophan hydroxylase 2 (GP+ 48 to 145), 2.6 mM [U-13C; U-15N] L-Phe, 20 mM sodium phosphate, 100 mM ammonium sulfate, 125 uM DSS, 95% H2O/5% D2OL-Phe95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.1 mMTryptophan hydroxylase 2 (GP+ 48 to 145)[U-99% 13C; U-99% 15N]1
10 mML-Phenone1
20 mMsodium phosphatenone1
100 mMammonium sulfatenone1
125 uMDSSnone1
0.45 mMTryptophan hydroxylase 2 (GP+ 48 to 145)none2
2.6 mML-Phe[U-13C; U-15N]2
20 mMsodium phosphatenone2
100 mMammonium sulfatenone2
125 uMDSSnone2
Sample conditionsIonic strength: 0.344 M / Label: Standard / pH: 7.0 / Pressure: 1 atm / Temperature: 303 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIIBrukerAVANCE III7501
Bruker AVANCE IIIBrukerAVANCE III6002

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Processing

NMR software
NameVersionDeveloperClassification
X-PLOR NIH2.44Schwieters, Kuszewski, Tjandra and Clorerefinement
CYANA3.98.5Guntert, Mumenthaler and Wuthrichstructure calculation
CcpNmr Analysis2.4.2CCPNchemical shift assignment
CcpNmr Analysis2.4.2CCPNpeak picking
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
TopSpin3.5Bruker Biospinprocessing
qMDDKazimierczuk, Orekhovprocessing
TALOSCornilescu, Delaglio and Baxdata analysis
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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