Human phosphoserine aminotransferase in complex with PLP

Target ID:

SERCA

Aliases:

EPIPMGC1460PSAPSATPSAT1

Deposition Date:

Monday 18th August 2008

Families:

AA metabolic enzymes

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3E77

Authors:

LEHTIO,L., KARLBERG,T., ANDERSSON,J., ARROWSMITH,C.H., BERGLUND,H., BOUNTRA, C., COLLINS,R., DAHLGREN,L.G., EDWARDS,A.M., FLODIN,S., FLORES,A., GRASLUND,S., HAMMARSTROM,M., JOHANSSON,A., JOHANSSON,I., KOTENYOVA,T., MOCHE,M., NILSSON,M.E., NORDLUND,P., NYMAN,T., OLESEN,K., PERSSON,C., SAGEMARK,J., THORSELL,A.G., TRESAUGUES,L., VAN DEN BERG,S., WELIN,M., WIKSTROM,M., WISNIEWSKA,M., WEIGELT, J., SCHUELER,H.

Site:

Stockholm

3E77

tabs

Structure Details

L-phosphoserine aminotransferase (SERC) is a member of the Class V pyridoxal phosphate dependent aminotransferase family. It catalyses the second of three steps in L-serine production from 3-phosphoglycerate, the conversion of 3-phosphohydroxypyruvate to 3-phosphoserine, with the consumption of an L-glutamate. Besides its role in protein synthesis, L-serine can play a part in the biosynthesis of small amino acids, serine phospholipids, sphingomyelins and cerebrosides. It is also required for the synthesis of nucleotide precursors, such as purines and thymidine, which is directly linked to cellular replication [1].

SERC activity is found in a number of organs but is increased in tissues with a high rate of cell turnover. SERC is expressed at high levels in the brain, liver, kidney and pancreas, and at low levels in the thymus prostate, testis and colon [2]. Two isoforms of SERC, α and β, exist in human cells. The β isoform, which contains a 46-amino-acid insert between Val290 and Ser337, is the physiologically relevant one [1]. SERC deficiency is characterized biochemically by low concentrations of serine and glycine in plasma and cerebrospinal fluid (CSF), and clinically by intractable seizures, acquired microcephaly, hypertonia, and psychomotor retardation. Prognosis is poor once the individual becomes symptomatic, but treatment with serine and glycine supplementation from birth can lead to normal development [3]. SERC is overexpressed in colon adenocarcinoma [2] and increases with tumour stage in colon cancer [4]. SERC also stimulates the proliferation of colorectal cancer cells and modulates their chemotherapy sensitivity both in vitro and in vivo [5].

Here we present the crystal structure of SERC (residues Leu17 to Leu370) in complex with pyridoxal phosphate (PLP) at a resolution of 2.5Å. Similar to the structure of other members of this family of aminotransferases the protein consists of a homodimer with both monomers contributing to the PLP-containing active site at their interface. The individual monomers can be divided into two domains: The larger domain (residues Leu17 to Gly263) consists of six alpha helices and a seven stranded mostly parallel β-sheet, and binds PLP covalently through Lys200. The smaller domain consists of a two-stranded antiparallel β-sheet and three alpha helices. PLP is modelled into two of the monomers in the asymmetric unit while the third one lacks density for modelling of the cofactor. The presence of PLP does not appear to cause any significant structural changes. An active site loop (residues Met1 - Lys16) was not included in this protein construct; however similar residues from the affinity purification tag were found to rest in an orientation reminiscent of those of the active site loop of the MR model (E. coli serC PDB entry 1BJO).

Materials and Methods

SERC

PDB:3E77

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC004863
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:SERCA-k006
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host:E.coli BL21(DE3) R3 pRARE, where R3 denotes a derivative of BL21(DE3) resistant to a strain of T1 bacteriophage (SGC Oxford) and the pRARE plasmid originating from the Rosetta strain (Novagen) supplies tRNAs for rare codons.

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smLPHSVLLEIQKELLDYKGVGISVLEMSHRSSDFAKIINNTENLVRELLAVPDNYKVIFLQGGGCGQFSAVPLNLIGLKAGRCADYVVTGAWSAKAAEEAKKFGTINIVHPKLGSYTKIPDPSTWNLNPDASYVYYCANETVHGVEFDFIPDVKGAVLVCDMSSNFLSKPVDVSKFGVIFAGAQKNVGSAGVTVVIVRDDLLGFALRECPSVLEYKVQAGNSSLYNTPPCFSIYVMGLVLEWIKNNGGAAAMEKLSSIKSQTIYEIIDNSQGFYVCPVEPQNRSKMNIPFRIGNAKGDDALEKRFLDKALELNMLSLKGHRSVGGIRASLYNAVTIEDVQKLAAFMKKFLEMHQL
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 20 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 °C overnight. The overnight culture was used to inoculate 0.75 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 0.5 ml 204 Antifoam A6426 (Sigma). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD600 reached ~2. The bottle was down-tempered to 18 °C over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x g, 10 min, 4 °C). The resulting cell pellet (17.6 g wet cell weight) was resuspended in lysis buffer (1.5 ml/g cell pellet), supplemented with 1250 U Benzonase (Merck) and 0.5 tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 °C.

Purification

Procedure
Columns
IMAC: Ni-charged 1 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 200 Prep Grade (GE Healthcare)

Procedure
Purification of the protein was performed as a two step process on an ÄKTAxpress system (GE Healthcare). Prior to purification, columns were equilibrated with IMAC wash1 buffer and gel filtration buffer, respectively. The filtered lysate was loaded onto the HiTrap Chelating column and washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC column with IMAC elution buffer and automatically loaded onto the gel filtration column. Fractions containing the target protein were pooled and fresh TCEP was added to a final concentration of 2 mM. The protein was subsequently concentrated using an Amicon Ultra-15 centrifugal filter device with 10,000 NMWL (Millipore) to 41.0 mg/ml in a volume of 0.8 ml and stored at -80 ºC. The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,100 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.2 µl of the protein sample (41 mg/ml) was mixed with 0.1 µl of well solution consisting of 2.1 M DL-malic acid. The plate was incubated at 20 ºC and crystals appeared within 2 days. The crystals were quickly transferred to cryo solution consisting of well solution complemented with 20% glycerol, and flash-frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 2.5 Å resolution were collected at the ESRF beam line ID29.
Data Processing:The structure was solved by molecular replacement with PHASER, using the structure of phosphoserine aminotransferase from E.coli (PDB entry 1BJO) as search model. The crystal belonged to space group P3121 with cell dimensions a= b= 171.00 Å, c= 103.88 Å, α= 90°, β= 90°, γ= 120°. Three monomers were located in the asymmetric unit. REFMAC5 was used for refinement and Coot for model building. Data in the interval 19.8 - 2.5 Å was used and after refinement the R values were: R= 19.9% and Rfree= 24.4%. Coordinates were deposited in the Protein Data Bank, accession code 3E77.

References

Baek, J.Y., et al. (2003) Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of L-serine biosynthesis. Biochem J 373: 191-200.
Ojala, P., et al. (2002) mRNA differential display of gene expression in colonic carcinoma. Electrophoresis 23: 1667-1676.
Hart, C.E., et al. (2007) Phosphoserine aminotransferase deficiency: a novel disorder of the serine biosynthesis pathway. Am J Hum Genet 80: 931-937.
Friederichs, J., et al. (2005) Gene expression profiles of different clinical stages of colorectal carcinoma: toward a molecular genetic understanding of tumor progression. Int J Colorectal Dis 20: 391-402.
Vie, N., et al. (2008) Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells. Mol Cancer 7: 14.