Human neuronal tryptophan hydroxylase

Target ID:

TPH2A

Deposition Date:

Thursday 11th September 2014

Families:

Miscellaneous

Related Diseases:

Neurobiology

Structure type:

protein-ligand

Download Coordinates:

4V06

Authors:

J.Kopec,A.Oberholzer,F.Fitzpatrick,J.Newman, C.Tallant,W.Kiyani,L.Shrestha,N.Burgess-Brown,F. von Delft,C.Arrowsmith,A.Edwards,C.Bountra,W.W.Yue

Site:

Oxford

4V06

tabs

Structure Details

Serotonin (also known as 5-hydroxytrptamine, 5-HT) is a monoamine neurotransmitter and hormone in the brain that regulates a diverse range of physiological processes, and has been implicated in several psychiatric disorders including schizophrenia, depression and obsessive-compulsive disorder. The biosynthesis of serotonin requires two enzymatic steps, the first of which is the rate-limiting hydroxylation of L-tryptophan into 5-hydroxy-L-tryptophan, a reaction catalysed by tryptophan hydroxylase (TPH, EC 1.14.16.4). 5-hydroxy-L-tryptophan is subsequently decarboxylated to form serotonin/5-HT by aromatic amino acid decarboxylase.
TPH belongs to the family of pterin-dependent, non-haem Fe(II) monooxygenase [2] which also includes as its members: phenylalanine hydroxylase (PAH, EC 1.14.16.1) whose dysfunction results in the metabolic disorder phenylketonuria, and tyrosine hydroxylase (TH, EC 1.14.16.2) that plays a role in catecholamine biosynthesis. All these enzymes share a common architecture and catalytic mechanism, requiring dioxygen and tetrahydrobiopterin (BH4) for the aromatic amino acid hydroxylation.
In humans there are two TPH isozymes. TPH1 is expressed in peripheral tissues to generate 5-hydroxy-L-tryptophan as precursor for melatonin biosynthesis. TPH2, the more recently discovered isoform, is expressed in the brain and plays the central role in serotonin-associated functions [3]. The two enzymes share 70% sequence identity, and adopt a three-domain architecture that comprises the N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerisation helix. Here we report the 2.63 Â crystal structure of a TPH2 segment (aa 148-490) containing the catalytic domain and tetramerisation helix.

Materials and Methods

TPH2

PDB:4V06

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:40083878
Entry Clone Source:SourceBioScience
SGC Clone Accession:
Tag:N-terminal, TEV protease cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMLEDVPWFPRKISELDKCSHRVLMYGSELDADHPGFKDNVYRQRRKYFVDVAMGYKYGQPIPRVEYTEEETKTWGVVFRELSKLYPTHACREYLKNFPLLTKYCGYREDNVPQLEDVSMFLKERSGFTVRPVAGYLSPRDFLAGLAYRVFHCTQYIRHGSDPLYTPEPDTCHELLGHVPLLADPKFAQFSQEIGLASLGASDEDVQKLATCYFFTIEFGLCKQEGQLRAYGAGLLSSIGELKHALSDKACVKAFDPKTTCLQECLITTFQEAYFVSESFEEAKEKMRDFAKSITRPFSVYFNPYTQSIEILKDTRSIENVVQDLRSDLNTVCDALNKMNQYLGI

MHHHHHHSSGVDLGTENLYFQ*SM is the purification tag plus TEV protease recognition site *.
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure

Extraction

Buffers
Procedure
Expression strain
BL21(DE3)-R3-pRARE2
A single colony was used to inoculate 50ml of TB media containing 50 ug/ml kanamycin and 50 ug/ml chloroamphenicol. The starter culture was incubated at 37 °C, 180 rpm and left to grow overnight. The following day the starter culture was used to inoculate 6L of TB media (7 ml starter culture per 1 L ) containing with 50 ug/ml kanamycin and grown at 37 °C, 180rpm.When the OD600 reached approximately 0.8 the temperature was reduced to 18°C and after a further 45 minutes the cells were induced by the addition of 0.1 mM IPTG. Expression was continued overnight.

Cell harvest
Cells were harvested by centrifugation at 4,500 g for 15 minutes after which the supernatant was discarded and the cell pellets were stored at -80 °C.

Buffers Used:
Binding/Lysis Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP
Wash Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5mM TCEP
Elution Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5mM TCEP
Gel Filtration Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 0.5mM TCEP
Anion Exchange Buffer A: 50 mM Hepes (pH 7.5), 5% Glycerol, 0.5mM TCEP
Anion Exchange Buffer B: 50 mM Hepes (pH 7.5), 2 M NaCl, 5% Glycerol, 0.5mM TCEP

Cell Lysis
Cell pellets (85g) were dissolved in approximately 240 ml lysis buffer containg EDTA-free protease inhibitors (diluted 1:1000) and lysed by passing through the constant cell disruptor at a constant pressure of 15KPa.The cell debris was pelleted at 35 000 g and the supernatant used for further purification.

Column 1
Ni-NTA (2 mL volume in a gravity-flow column).
The clarified cell extract was incubated with 4 mL pre-equilibriated 50% Ni-NTA resin suspension for 1 hour at 4°C with rotation. The suspension was centrifuged at 900g for 10 min. The supernatant was poured away and the beads were resuspended in lysis buffer and transferred onto a gravity column. The column was then washed with 20ml Binding Buffer (2 x 10ml) and 20 ml Wash Buffer (2 x10 ml). The protein was eluted with 20 ml of elution Buffer (5 x 4 ml fractions).

Column 2
Superdex s200 16/60 Gel Filtration.
Elution fractions (1-3) containing the target protein were applied to the size exclusion chromatography column, s200 (pre-equilibriated in GF buffer) at 1.0 ml/min. 1.0 ml fractions were collected.

Column 3
Pooled fractions from gel filtration were diluted 10 fold with anion exchange buffer A (50mM HEPES, pH 7.5, 5% glycerol, 0.5mM TCEP) and loaded onto 5 mL HiTrapQ column pre-equilibrated with anion exchange buffer A. The protein eluted in a linear gradient from 0 to 1M NaCl, using anion exchange buffer B.
Concentration:To set up crystallization plates the sample was concentrated to 11.6 mg/ml using a 10 kDa mwco concentrator.
Ligand
MassSpec:The mass was analysed by an electrospray time of flight instrument (MSD-TOF)Expected mass: 42116.9 Da Measured mass: 42116.7 Da
Crystallization:Crystals were grown by vapour diffusion in sitting drop at 20°C. A sitting drop consisting of 100 nl protein pre-incubated with 5 mM DL-Tryptophan and 50 nl well solution was equilibrated against a well solution containing 0.20M sodium acetate, 0.1M BisTris-Propane pH 6.5, 20.0% PEG 3350 and 10.0% ethylene glycol
NMR Spectroscopy:
Data Collection:Resolution: 2.63 Å X-ray source: Diamond Light Source beamline IO3
Data Processing:

References

[1] Markus CR (2008). Dietary amino acids and brain serotonin function; implications for stress-related affective changes. Neuromolecular Med. 10:247-58.

[2] Ulrich R and Hofrichter M. (2007) Enzymatic hydroxylation of aromatic compounds. Cell Mol Life Sci 64:271-293.

[3] Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG (2004). Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217.