Human Inosine Triphosphatase

Target ID:

ITPAA

Aliases:

C20orf37dJ794I6.3HLC14-06-PIP3KIP3K-BIP3KBITPaseITPKBPIG37

Deposition Date:

Thursday 22nd December 2005

Families:

Phosphatases

Related Diseases:

CancerMetabolism

Structure type:

apo

Download Coordinates:

2CAR

Authors:

P.STENMARK,P.KURSULA,C.ARROWSMITH,H.BERGLUND,A.EDWARDS,M.EHN,S.FLODIN,A.FLORES,S.GRASLUND,M.HAMMARSTROM,B.M.HALLBERG,M.HOGBOM,L.HOLMBERG SCHIAVONE,T.KOTENYOVA,P.NILSSON-EHLE,T.NYMAN,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

2CAR

tabs

Structure Details

Inosine monophosphate (IMP), the precursor of adenosine and guanosine monophosphates (AMP and GMP),
is an essential metabolite in the biosynthesis of purines.
IMP is generated predominantly by de novo biosynthesis and by interconversion of nucleotide monophosphates.
ITP is present at low levels in the cell as byproducts generated either by deamination of purine bases
or by phosphorylation of IMP.

IMP is also generated by enzymatic action of the enzyme inosine triphosphatase (ITPase; ITPA; EC 3.6.1.19)
that catalyzes the conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP) and pyrophosphate (PPi).
The enzyme also catalyses the hydrolysis of dITP and XTP.

ITP --> IMP + PPi

ITPase activity prevents the cytosolic accumulation of ITP, dITP and XTP,
which may be incorporated into RNA and DNA by the respective polymerases, posing the risk of direct mutagenesis.
ITPase exists as a dimer under physiological conditions. Several naturally occurring mutations
in the ITPA gene have been found that cause ITPase deficiency.
The most common mutation is proline 32 to threonine (P32T).
Interestingly, ITPase activity is completely lost in patients homozygous for the mutation,
but reduced to 25% in patients heterozygous for the mutation.
This indicates that ITPase activity depends on the integrity of both protomers of the dimer,
and suggests that dimer activity might be regulated by crosstalk between the two protomers.

Several studies conclude that ITPase deficiency is responsible for adverse drug reactions
in patients treated with the purine analog azathioprine,
although other studies did not support such a correlation.
Azathioprine is an immunosuppressive drug used in the treatment of inflammatory bowel diseases,
leukemia, and autoimmune complications in connection with organ transplants.

We have solved the structure of human ITPA without any ligand bound to 1.1Å resolution.
We have also solved the substrate complex to 2.8 Å.
We here identify the metal binding site and substrate binding site of this protein family.
In the ITP complex structure (PDB entry 2J4E), one molecule of ITP is bound in the cleft
between the dimerization lobe and N-terminal lobe of the protein.
The purine base is wedged between the Phe149 and Trp151 side chains of the dimerization lobe.
The substrate binds by an induced fit mechanism,
the active site cleft is closed by a large movement of an N-terminal helix. This positions several conserved residues for coordination of the substrate. The ITPases are dependent on Mg2+ or Mn2+ for activity, the location of this binding site has previously not been known. In our ITP complex we see this metal bound between Glu44 and the triphosphate tail of ITP.

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Apo structure";
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Binary structure with ITP";
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Superposition of the apo (grey) and ITP-bound (pink) structures.
The upper dimerization domain and the lower N-terminal domain form a cleft that closes upon substrate binding.
ITP is shown as sticks

Arg 178, His177 and Lys 172 are within hydrogen bonding distance from the 6-keto oxygen of the inosine ring. These residues constitute a positively charged environment that interacts with the 6-keto oxygen. In ATP this position of the inosine ring is occupied by an amino group, this explains the high specificity for ITP over ATP. Because of the high levels of ATP in the cell it is essential that the enzyme does not break down this nucleotide.

In one of the eight protein chains in the asymmetric unit (D) the density showed that
the hydrolysis reaction had proceeded, and IMP and pyrophosphate occupied the active site.
The alpha- and beta-phosphates have moved apart, but otherwise no major structural rearrangements can be seen.

The P32T mutation causing ITPA deficiency is located in a loop region after the helix
that closes the active site upon substrate binding.
This loop constitutes a hinge region, and we believe that the P32T mutation
leads to a partial miss folding that prevent the dynamics necessary for substrate binding or substrate release.
Glu44 is located very close to the dimer interface; Glu44 and neighboring residues make hydrogen bonds
over the interface (Glu44, Tyr45, Gln46).
This interaction could transmit the structural problems in one monomer carrying the P32T mutation
to the other (wild type) monomer of the dimer.
This would explain the observation that individuals heterozygous for the mutations only retain 25% of ITPA activity;
which would indicate that the heterodimer between mutated and wild type ITPA is inactive.

Materials and Methods

ITPA

PDB:2CAR

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC010138
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated thrombin protease cleavage site: mgsshhhhhhssglvprg*s(m).
Host:E. coli BL21(DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMAASLVGKKIVFVTGNAKKLEEVVQILGDKFPCTLVAQKIDLPEYQGEPDEISIQKCQEAVRQVQGPVLVEDTCLCFNALGGLPGPYIKWFLEKLKPEGLHQLLAGFEDKSAYALCTFALSTGDPSQPVRLFRGRTSGRIVAPRGCQDFGWDPCFQPDGYEQTYAEMPKAEKNAVSHRFRALLELQEYFGSLAA
Vector:p28A-LIC

Growth

Medium:
Antibiotics:
Procedure:20 microL competent BL-21 cells were transformed with 1 microL plasmid. 10 min on ice, 45 sec at 42 degC. Added 100 microL SOC then 30min at 37 degC. Cells were plated on Kanamycin. Colonies were grown in 50mL of TB + 4% glycerol at 37degC, until OD600 of 0.5 then diluted into 400 mL TB + 4% glycerol. Growth at 37degC until OD600 of 1.2 - 1.5. Then at 18 degC for 1h. Induction with 0.5 mM IPTG at OD600 of 1.4 - 1.8. Left culture at 18degC over night.

Purification

Procedure
Columns: HisTrap HP 1 mL (IMAC); MonoS 5/50 GL (Cation Exchange)

Procedure: All chromatography equipment was obtained from GE Healthcare. Purification was conducted on an ÄKTA purifier 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 IEX buffer A (MonoS). The protein sample was loaded on the HisTrap HP column, and the column was washed with IMAC Bind/Wash Buffer 1 followed by IMAC Wash Buffer 2. Bound protein was eluted from the IMAC columns with 7.5 mL of IMAC Elution Buffer. Total protein (ca. 70 mg) was concentrated to a volume of 1 mL in a Vivaspin cartridge and cleaved with 30 units of thrombin over night at room temperature. The sample was diluted 1:20 with IEX buffer A and applied to a MonoS column. The protein passing the MonoS column was essentially pure ITPA liberated from the His6-tag as judged by SDS-PAGE and TOF-MS analysis. The material eluting with IEX buffer B contained traces of uncleaved fusion protein, thrombin, as well as other contaminants. Pure ITPA protein was dialyzed into storage buffer, concentrated to 50 mg/mL and stored as aliquots at -80 ºC. Analytical gel filtration on a Superdex 75 column in storage buffer showed that the protein migrated as a dimer.

Extraction

Procedure

Concentration:
Ligand
MassSpec:
Crystallization:Crystals of the apo protein were obtained using haning drop method at 4degC. Drops were prepared using 1 µL of protein (concentration 25 mg/mL) and 1 microL of the well solution (0.2 M potassium chloride, 27 % (w/v) PEG 3350, pH 6.9). The crystals diffracted to 1.1 Å.

Crystals of the complex were obtained using sitting drops at 4degC with 900 nanoL of protein and 900 nl of well solution. The well solution contained 0.2 M potassium chloride, 31% (w/v) polyethylene glycol 3350, and the protein was added from a 50 mg/ml stock solution containing 10 mM ITP and 20 mM HEPES, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing:The crystal was pseudo merohedrally twinned and the structure was refined using ShelX.

References

Stenmark P, Kursula P, Flodin S, Graslund S, Landry R, Nordlund P, Schuler H.
Crystal structure of human inosine triphosphatase. Substrate binding and implication of the inosine triphosphatase deficiency mutation P32T.
J Biol Chem. 2007 Feb 2;282(5):3182-7. Epub 2006 Nov 29.
Lin S, McLennan AG, Ying K, Wang Z, Gu S, Jin H, Wu C, Liu W, Yuan Y, Tang R, Xie Y, Mao Y. Cloning, expression, and characterization of a human inosine triphosphate pyrophosphatase encoded by the itpa gene. J Biol Chem. 2001 Jun 1;276(22):18695-701.
Hwang KY, Chung JH, Kim SH, Han YS, Cho Y. Structure-based identification of a novel NTPase from Methanococcus jannaschii. Nat Struct Biol. 1999 Jul;6(7):691-6.
Derijks LJ, Gilissen LP, Hooymans PM, Hommes DW. Review article: thiopurines in inflammatory bowel disease. Aliment Pharmacol Ther. 2006 Sep 1;24(5):715-29.
Stenmark P, Kursula P, Flodin S, Graslund S, Landry R, Nordlund P, Schuler H, Crystal structure of human inosine triphosphatase. Substrate binding and implication of the inosine triphosphatase deficiency mutation P32T.
J Biol Chem. 2007 Feb 2;282(5):3182-7.