Human CTP Synthetase - Synthetase Domain

Target ID:

CTPSA

Aliases:

CTPS

Deposition Date:

Friday 28th October 2005

Families:

Nucleotide metabolism

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

Superceded by 2VO1

Authors:

P.STENMARK,P.KURSULA,C.ARROWSMITH,H.BERGLUND,A.EDWARDS,M.EHN,S.FLODIN,S.GRASLUND,M.HAMMARSTROM,B.M.HALLBERG,L.HOLMBERG SCHIAVONE,T.KOTENYOVA,P.NILSSON-EHLE,D.OGG,C.PERSSON,J.SAGEMARK,H.SCHULER,M.SUNDSTROM,A.G.THORSELL,S.VAN DEN BERG,J.WEIGELT,P.NORDLUND

Site:

Stockholm

ICM Datapack:

ICM Datapack

2C5M

tabs

Structure Details

CTP synthetase is an essential protein for all organisms since it catalyses the final step in the biosynthesis of cytidine triphosphate (CTP). We have solved the structure of the N-terminal synthetase domain of CTP synthetase where UTP first is phosphorylated utilizing ATP, and then ammonia is incorporated into the ATP-phosphorylated UTP. Ammonia is generated at the glutaminase domain and shuttled to the synthetase domain through a molecular tunnel. The activity of the protein is increased 2-fold when it is phosphorylated by protein kinase A.

ATP + UTP + NH3 => ADP + phosphate + CTP

High levels of CTP synthetases activity are characteristic of several human cancers types and necessary for the viability of these cells. Cyclopentenyl cytosine, acivicin and 3-deazauridine are anti-CTP synthetase inhibitors that slow down or arrest proliferation of tumour cells. Resistance to some of these compounds is caused by mutations in CTP synthetase.

The asymmetric unit contains the synthetase domain tetramer, in the full-length structure the glutaminase domains would form knobs pointing out from this tetramer core. UTP and ATP promote tetramerization of CTP synthetase. The active site of the protein is located at the tetramer interface; this is the cause of the positive cooperative kinetics toward UTP and ATP. A disulfide bond is present between Cys218 and Cys243 in the structure, this bond is only present in two of the four molecules in the asymmetric unit. Several sulphate ions are found in the active sites of the protein, occupying the positions where the phosphates of the substrates likely should be positioned.

The asymmetric unit contains the synthetase domain tetramer, in the full-length structure the glutaminase domains would form knobs pointing out from this tetramer core. UTP and ATP promote tetramerization of CTP synthetase. The active site of the protein is located at the tetramer interface; this is the cause of the positive cooperative kinetics toward UTP and ATP. A disulfide bond is present between Cys218 and Cys243 in the structure, this bond is only present in two of the four molecules in the asymmetric unit. Several sulphate ions are found in the active sites of the protein, occupying the positions where the phosphates of the substrates likely should be positioned.

CTP synthetase is an interesting drug target and the structure of the human protein will facilitate structure based drug design where existing compounds can be developed further.

Re-refinement 2008: The CTPSA structure (2C5M) scored badly in MolProbity and was therefore selected for internal SGC Stockholm re-refinement. The first CTPSA (2C5M) structure was built in space group P 41 with some additional symmetry erroneously interpreted as perfect twinning. Recent re-processing of the data suggests the space group to be P 41 21 2 with no signs of twinning judged from the output of XTRIAGE from the PHENIX suite. A seven amino-acid register error in 2C5M between Phe233 and Val241 was corrected and the â€œnewâ€ CTPS structure was re-deposited (PDB code 2VO1).

Materials and Methods

CTPS

PDB:2C5M

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC009408
Entry Clone Source:MGC (RZPD)
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m).
Host:BL21(DE3)-Gold-tf2

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMKYILVTGGVISGIGKGIIASSVGTILKSCGLHVTSIKIDPYINIDAGTFSPYEHGEVFVLDDGGEVDLDLGNYERFLDIRLTKDNNLTTGKIYQYVINKERKGDYLGKTVQVVPHITDAIQEWVMRQALIPVDEDGLEPQVCVIELGGTVGDIESMPFIEAFRQFQFKVKRENFCNIHVSLVPQPSSTGEQKTKPTQNSVRELRGLGLSPDLVVCRCSNPLDTSVKEKISMFCHVEPEQVICVHDVSSIYRVPLLLEEQGVVDYFLRRLDLPIERQPRKMLMKWKEMADRYDR
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:20 mL TB media was inoculated with BL21gold-DE3 cells, and all cultures were grown overnight at 30 ºC in 100 mL shake flasks at 175 rpm. 750 mL TB media supplemented with 8 g/L glycerol and 50 µg/mL kanamycin was inoculated with 10 mL of the over night culture. The large scale cultivations were grown in TunAir shake flasks at 37 °C, 225 RPM. The cells were harvested by centrifugation in the SLC-6000 rotor (6x1000mL) at 5000 rpm for 10 minutes.

Purification

Procedure
Purification was conducted automatically on an ÄKTA xpress system operated by UNICORN software at a flow of 0.8 mL/min. Prior to purification columns were equilibrated with IMAC Bind/Wash1 Buffer (HisTrap HP) and Gel filtration buffer (Superdex 200). The protein sample was loaded on the HisTrap HP column that was washed with IMAC Bind/Wash1 Buffer followed by IMAC Wash2 Buffer. Bound protein was eluted from the IMAC columns with IMAC Elution Buffer and loaded onto the Gelfiltration column. The chromatogram from Gelfiltration showed one major protein peak that consisted of highly pure CTPSA-c007 as shown by SDS-PAGE analysis. TCEP was added to the sample to a final concentration of 2 mM. The protein was concentrated to 5.34 mg/mL and stored at -80ºC.

Extraction

Procedure
A stock solution of Complete EDTA-free (protease inhibitor) was prepared by dissolving 2x12 tablets in 2x30 mL of IMAC binding/washing buffer 1. Cells were harvested by centrifugation for 10 min at 4500 g. The cell pellet were resuspened in 90mL of IMAC lysis buffer complemented with 1mL of Complete EDTA-free protease inhibitor stock and then frozen at -80˚C. The frozen cell pellets were briefly thawed by warm water. 4 µL (1000 U) benzonase was added to the sample priorto high-pressure homogenization with the HPH. Cells were disrupted by high-pressure homogenization with the HPH before centrifugation for 30 min at 30 000 g in the Sorvall SS-34 rotor. The soluble fraction was decanted and filtered through 0.45mm prior to loading onto the ÄKTAxpress for furher purification.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained by the hanging drop vapour diffusion method using by mixing 1uL of the protein solution (5.3 mg/mL) with 1µL of the well solution consisting of 0.1 M Tris pH 8.8, 1.2 M AmSO4 and 0.1 M Malonic acid at room temperature. Crystals appeared after 3 days and were briefly transferred to cryosolution containing 0,1 M Tris pH 8.8, 1.4 M AmSO4, 50 mM Malonic acid, 25% glycerol, 0.2 M NaCl and 2 mM TCEP and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:ESRF ID29, 20051001
Data Processing:The structure was initially solved by MR using MOLREP and then refined using COOT, REFMAC and CNS. Initially the structure was built and deposited (PDB code 2C5M) in space group P 41 with some additional symmetry erroneously interpreted as perfect twinning. Recent re-processing of the data suggests the space group to be P 41 21 2 with no signs of twinning judged from the output of XTRIAGE from the PHENIX suite. A ten amino-acid register error was corrected in 2C5M and the CTPS structure was re-deposited (PDB code 2VO1).

References

Politi PM, Xie F, Dahut W, Ford H Jr, Kelley JA, Bastian A, Setser A, Allegra CJ, Chen AP, Hamilton JM, et al. Phase I clinical trial of continuous infusion cyclopentenyl cytosine. Cancer Chemother Pharmacol. 1995;36(6):513-23.
Whelan J, Phear G, Yamauchi M, Meuth M. Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells. Nat Genet. 1993 Apr;3(4):317-22.
Han GS, Sreenivas A, Choi MG, Chang YF, Martin SS, Baldwin EP, Carman GM. Expression of human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A. J Biol Chem. 2005 Sep 22
Goto M, Omi R, Nakagawa N, Miyahara I, Hirotsu K. Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites. Structure (Camb). 2004 Aug;12(8):1413-23.
Endrizzi JA, Kim H, Anderson PM, Baldwin EP. Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets. Biochemistry. 2004 Jun 1;43(21):6447-63.
Simon C. Lovell, Ian W. Davis, W. Bryan Arendall III, Paul I. W. de Bakker, J. Michael Word, Michael G. Prisant, Jane S. Richardson, David C. Richardson Structure validation by C-alpha geometry: phi, psi, and C-beta deviation. Proteins: Structure, Function, and Genetics. 2003, 50: 437-450.
Kursula P, Flodin S, Ehn M, HammarstrÃ¶m M, SchÃ¼ler H, Nordlund P, Stenmark P. Structure of the synthetase domain of human CTP synthetase, a target foranticancer therapy. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006, 613-7.