Crystal Structure of Human Inosine Triphosphatase

Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1Å, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer. Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1Å, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer. Inosine monophosphate (IMP) is an essential metabolite in purine biosynthesis; it is the precursor of adenosine and guanosine monophosphates (AMP and GMP) (1Berg J.M. Tymoczko J.L. Stryer L. Biochemistry.5th Ed. W. H. Freeman, New York2002: 698-701Google Scholar). IMP is generated predominantly by de novo biosynthesis and by interconversion of nucleotide monophosphates. Inosine nucleotide triphosphates are present at low levels as byproducts generated either by deamination of purine bases or by phosphorylation of IMP. They are removed by the housecleaning enzyme inosine triphosphatase (ITP pyrophosphohydrolase; ITPase; ITPA 4The abbreviations used are: ITPA, inosine triphosphatase; PDB, Protein Data Bank. ;EC 3.6.1.19) that catalyzes the conversion of inosine triphosphate (ITP) to IMP and pyrophosphate (PPi). ITPase activity prevents accumulation of ITP and dITP, which may be incorporated into RNA and DNA, posing a risk for mutagenesis. ITPA orthologs are found in organisms from all kingdoms. In human and mouse, the protein is ubiquitously expressed (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 3Behmanesh M. Sakumi K. Tsuchimoto D. Torisu K. Ohnishi-Honda Y. Rancourt D.E. Nakabeppu Y. DNA Res. 2005; 12: 39-51Crossref PubMed Scopus (20) Google Scholar). Human ITPA is a 194-amino acid homodimer that is reliant upon Mg2+ or Mn2+ ions for catalytic activity. ITP, dITP, and xanthine triphosphate are converted with similar kinetics, whereas the enzyme has very low affinity for other nucleotides. The pH optimum of ITPA is ∼10 (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 4Holmes S.L. Turner B.M. Hirschhorn K. Clin. Chim. Acta. 1979; 97: 143-153Crossref PubMed Scopus (41) Google Scholar, 5Vanderheiden B.S. J. Cell. Physiol. 1979; 98: 41-47Crossref PubMed Scopus (21) Google Scholar). Genetic studies of individuals with detectable ITP levels in erythrocytes have linked ITPA deficiency to several single nucleotide polymorphisms in the ITPA gene. Of these, the mutation P32T abolished ITPase activity and caused accumulation of ITP in erythrocytes (6Sumi S. Marinaki A.M. Arenas M. Fairbanks L. Shobowale-Bakre M. Rees D.C. Thein S.L. Ansari A. Sanderson J. De Abreu R.A. Simmonds H.A. Duley J.A. Hum. Genet. 2002; 111: 360-367Crossref PubMed Scopus (224) Google Scholar, 7Cao H. Hegele R.A. J. Hum. Genet. 2002; 47: 620-622Crossref PubMed Scopus (89) Google Scholar). ITPase activity was lost in individuals homozygous for the P32T mutation, and it was reduced to ∼ 25% in heterozygous subjects (8Maeda T. Sumi S. Ueta A. Ohkubo Y. Ito T. Marinaki A.M. Kurono Y. Hasegawa S. Togari H. Mol. Genet. Metab. 2005; 85: 271-279Crossref PubMed Scopus (73) Google Scholar). This indicates that ITPase activity depends on the integrity of both protomers of the ITPA dimer. The ITPA P32T allele is present in all ethnic groups, being highest (11–19%) in Asian and lowest (1–2%) in Central and South American populations (7Cao H. Hegele R.A. J. Hum. Genet. 2002; 47: 620-622Crossref PubMed Scopus (89) Google Scholar, 9Marinaki A.M. Sumi S. Arenas M. Fairbanks L. Harihara S. Shimizu K. Ueta A. Duley J.A. Nucleosides Nucleotides Nucleic Acids. 2004; 23: 1399-1401Crossref PubMed Scopus (19) Google Scholar, 10Marsh S. King C.R. Ahluwalia R. McLeod H.L. J. Hum. Genet. 2004; 49: 579-581Crossref PubMed Scopus (84) Google Scholar, 11Shipkova M. Lorenz K. Oellerich M. Wieland E. von Ahsen N. Clin. Chem. 2006; 52: 240-247Crossref PubMed Scopus (89) Google Scholar). ITPA deficiency is not linked to pathology in afflicted individuals, but perturbed ITP levels may be harmful under circumstances such as cellular stress. For instance, ITPA deficiency may be responsible for adverse drug reactions in patients treated with the purine analog azathioprine (12Derijks L.J. Gilissen L.P. Hooymans P.M. Hommes D.W. Aliment. Pharmacol. Ther. 2006; 24: 715-729Crossref PubMed Scopus (139) Google Scholar), an immunosuppressive drug used in the treatment of inflammatory bowel diseases, leukemia, and autoimmune complications in connection with organ transplants. Crystal structures of several bacterial ITPA homologs have been solved in the absence of physiological ligands (13Hwang K.Y. Chung J.H. Kim S.H. Han Y.S. Cho Y. Nat. Struct. Biol. 1999; 6: 691-696Crossref PubMed Scopus (124) Google Scholar, 14Zheng J. Singh V.K. Jia Z. Structure. 2005; 13: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 15Galperin M.Y. Moroz O.V. Wilson K.S. Murzin A.G. Mol. Microbiol. 2006; 59: 5-19Crossref PubMed Scopus (170) Google Scholar). The location of the substrate binding site has been postulated on the basis of weak binding of the non-physiological ATP analog, AMPPNP, to the Methanococcus enzyme (13Hwang K.Y. Chung J.H. Kim S.H. Han Y.S. Cho Y. Nat. Struct. Biol. 1999; 6: 691-696Crossref PubMed Scopus (124) Google Scholar). However, the mode of substrate binding proposed could not account for the strong binding preference for ITP, dITP, and xanthine triphosphate over other nucleotides (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 14Zheng J. Singh V.K. Jia Z. Structure. 2005; 13: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We have determined the crystal structure of human ITPA alone and in complex with one of its physiological substrates, ITP, and the products of hydrolysis, IMP + PPi. Our results identify the substrate and Mg2+ binding site of ITPA and show that it is different from the site previously proposed for this class of enzymes. Our structures provide an explanation for the high specificity of ITPA toward inosine and xanthine nucleotides. Comparison of the ITP-bound and unbound structures show that substrate binding induces closure of the nucleotide binding cleft and positions key side chains for catalysis. The structural implications of the P32T mutation causing ITPA deficiency are discussed. Cloning and Protein Purification—Human ITPA cDNA (Mammalian Gene Collection clone 19624) was subcloned into pET28 (Novagen). Protein expression and purification were as described before (16Kursula P. Flodin S. Ehn M. Hammarstrom M. Schuler H. Nordlund P. Stenmark P. Acta Crystallogr. Sect. F Struct. Biol. Crystalliz. Commun. 2006; 62: 613-617Crossref PubMed Scopus (30) Google Scholar) involving expression in Escherichia coli BL-21(DE-3) cells (Stratagene), cell lysis using a cell disruptor (Stansted Fluid Power), and purification on HisTrap HP columns (GE Healthcare). Eluted proteins were concentrated using Vivaspin cartridges (Millipore) to a volume of 1 ml and treated with 30 units of thrombin (GE Healthcare) overnight at room temperature. The protein sample was diluted 20-fold with 20 mm sodium citrate, pH 5.0, 20 mm NaCl, 10% glycerol, 1 mm Tris(2-carboxyethyl)phosphine and applied to a MonoS column (GE Healthcare). The protein passing the MonoS column was essentially pure ITPA liberated from the His6 tag as judged by SDS-PAGE and time-of-flight mass spectrometry analysis. ITPA was diluted 1:5 with 50 mm HEPES, pH 7.5, 300 mm NaCl, 10% glycerol, concentrated to 50 mg/ml, and stored as aliquots at -80 °C. Protein Crystallization—Crystal screens were performed using a JCSG screen (17Page R. Stevens R.C. Methods. 2004; 34: 373-389Crossref PubMed Scopus (55) Google Scholar) with minor modifications. Monoclinic and trigonal crystals were obtained using the sitting drop method at 4 °C using 900 nl of protein (concentration 22 mg/ml) and 900 nl of well solution (0.2 m potassium chloride, 20% (w/v) polyethylene glycol 3350, pH 6.9). For the ITP complex crystals, 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. Data Collection and Processing—Crystals were briefly soaked in a cryoprotectant (well solution containing 15–20% glycerol). Synchrotron diffraction data were collected at BESSY beamline BL14.1. The data were processed and scaled using XDS (apo structure) (18Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3242) Google Scholar,19Kursula P. J. Appl. Crystallogr. 2004; 37: 347-348Crossref Scopus (48) Google Scholar) or MOSFLM (ITP complex) (20Leslie A.G. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography. 1992; 26Google Scholar). Structure Determination and Refinement—Using data from trigonal crystals (space group P32), the structure was solved at 2.25 Å resolution by molecular replacement in Phaser/Phenix (21McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1600) Google Scholar, 22Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3664) Google Scholar) with structure 1B78 (“A hypothetical protein from Methanococcus jannaschii: Mj0226”) as model. After initial refinement the atomic resolution structure was solved using data from monoclinic crystals by molecular replacement in Molrep (23Vagin A. Teplyakov A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1622-1624Crossref PubMed Scopus (690) Google Scholar) using the partially refined structure as a model. After refinement in Refmac5 (24Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13911) Google Scholar) and manual rebuilding, automated model building was carried out using ARP/wARP (25Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar). Refinement was continued using SHELX (26Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1891) Google Scholar), applying the twinning operator (-h,-k, l) and using intensities instead of amplitudes. The twinning fraction refined to 50%. In the final refinement rounds, anisotropic atomic displacement parameters were refined and hydrogen atoms were added. A total of 35 residues were built in two conformations, and the occupancies of the conformers were also refined in SHELX. The ITP complex structure was solved by molecular replacement using the apo structure as a model and refined using Refmac5 with TLS parameters (27Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1654) Google Scholar). Both structures were built using Coot (28Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23605) Google Scholar). The complete protein chains were traceable; in the ITP complex the 122–128 loop was not built in all chains. Figures were made using PyMOL (www.pymol.org) and Ligplot (29Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4428) Google Scholar). Superpositioning of structural models was performed using SSM (30Krissinel E. Henrick K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3188) Google Scholar). Domain motion analysis was carried out using DynDom (31Hayward S. Berendsen H.J. Proteins. 1998; 30: 144-154Crossref PubMed Scopus (712) Google Scholar). We have determined the crystal structure of human apoITPA at 1.1 Å resolution, as well as the structure of its complex with the physiological substrate (2.8 Å). ITPA with or without bound substrate crystallized as a homodimer, which is the physiological form of the protein (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). There were two and eight molecules/asymmetric unit in the apo and ITP complex crystals, respectively. Data collection, processing, and structure refinement statistics are summarized in Table 1.TABLE 1Summary of crystallographic data The dataset obtained from trigonal crystals was not fully refined; see “Experimental Procedures” for a detailed explanation. R.m.s.d., root mean square deviation.Initial datasetApoITP complexBeamlineBESSY beamline 14.1Wavelength (Å)0.9781Space groupP32P21P1Unit cell dimensionsa = 50.0, b = 50.0, c = 309.2;a = 31.2, b = 105.0, c = 50.1;a = 68.0, b = 75.3, c = 110.8;(Å; degrees)α = 90.0, β = 90.0, γ = 120α = 90.0, β = 90.0, γ = 90.0α = 85.1, β = 77.7, γ = 69.2Unique resolution resolution resolution resolution ITP and in a structure of human ITPA of a a for two of structural pyrophosphate from have a similar such as M. 1B78 and (13Hwang K.Y. Chung J.H. Kim S.H. Han Y.S. Cho Y. Nat. Struct. Biol. 1999; 6: 691-696Crossref PubMed Scopus (124) Google Scholar) and E. coli J. Singh V.K. Jia Z. Structure. 2005; 13: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). A the human apo ITPA structure and the Methanococcus enzyme an root mean square for positions of Å residues with The human ITPA structure for the first physiological substrate to an enzyme of this structural The two from a single form a substrate binding cleft The dimer is made up by interactions the of The the monomers of the apo structure is with hydrogen and the dimer and the ITP complex structure one of ITP is bound in the cleft the and of the protein 1 and The purine base is the and side chains of the that are on of has in toward the purine base with the apo residues are hydrogen from the of the inosine and side chains into the purine site from it is that the base is by this specificity may be by the hydrogen by side chains and the group in the with the hydrogen the of the enzyme nucleotides nucleotides are also ITPA (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). specificity in xanthine binding may be obtained by a hydrogen the of the xanthine base and the The binding is not to an the bases The is in a and both into the solution without strong with the This is with the that are also ITPA (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We also with a Mg2+ as judged by its and in the active site. The Mg2+ ion and triphosphate with side chains from and and the all side chains are in the of The is the which is a in bacterial this is a replacement in the that the side also with the nucleotide Comparison of ITPA structures a closure of the cleft by upon ITP by displacement of structural from the 1 and are of residues and the two structures are the positions of residues show a Å root mean square the apo and ITP-bound and of by residues and This also results in a shift of These changes upon substrate binding be summarized as a of the toward the These may explain crystals of bacterial upon of nucleotides (13Hwang K.Y. Chung J.H. Kim S.H. Han Y.S. Cho Y. Nat. Struct. Biol. 1999; 6: 691-696Crossref PubMed Scopus (124) Google Scholar, 14Zheng J. Singh V.K. Jia Z. Structure. 2005; 13: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In the crystals the ATP analog was bound to one of the monomers at a site that from the ITP binding site in human ITPA crystals (13Hwang K.Y. Chung J.H. Kim S.H. Han Y.S. Cho Y. Nat. Struct. Biol. 1999; 6: 691-696Crossref PubMed Scopus (124) Google Scholar). The are by of the two structures with the nucleotides bound In the structure the base of from the protein without interactions that explain specificity for the physiological In the enzyme in the cleft similar to apo with which it also the of the high binding of the ATP analog at a or site in the Methanococcus We that the substrate binding site in human ITPA which has been by Murzin and M.Y. Moroz O.V. Wilson K.S. Murzin A.G. Mol. Microbiol. 2006; 59: 5-19Crossref PubMed Scopus (170) Google Scholar), is this enzyme and ITPA crystals resulting from with the substrate ITP, of the eight the unit ITP bound The Mg2+ ion is in to the ITP and and is by the ITPA Mg2+ or Mn2+ for activity and has a high Mg2+ optimum of 10 mm or (2Lin S. McLennan A.G. Ying K. Wang Z. Gu S. Jin H. Wu C. Liu W. Yuan Y. Tang R. Xie Y. Mao Y. J. Biol. Chem. 2001; 276: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), of an metal binding site. ITPA protein substrate complex contained metal ions high affinity binding in the active metal ions were not added to the were also for with to pH and temperature. These may explain we were to the substrate We under physiological Mg2+ at the site found in In one of the protein chains the that the and IMP and the active site The and and to model in ITP in on the However, changes in nucleotide coordination from The group from the of ITP in the before and the of interactions catalytic In the ITP-bound the triphosphate of ITP is in the of side chains and whereas the is by the of and the A by the be for on the the could a for on the A of the be by the of the or side chains. In the D IMP + the has by Å toward the group of a for as a of the of the ITPA that individuals heterozygous for the P32T mutation 25% ITPase activity A.M. Sumi S. Arenas M. Fairbanks L. Harihara S. Shimizu K. Ueta A. Duley J.A. Nucleosides Nucleotides Nucleic Acids. 2004; 23: 1399-1401Crossref PubMed Scopus (19) Google Scholar) that both protomers of the physiological dimer to be for catalytic activity. The ITPA dimer structure an by the The loop structure and the is to structural changes upon binding and to the The of a hydrogen with the of the the of a hydrogen with the of the We that and/or nucleotide in the two monomers are the dimer could explain the the a minor of the dimer we that the and may as triphosphate structural changes upon to the active site of the its activity. The structures a for catalytic deficiency in P32T Comparison of two structures and of as a for nucleotide as detailed in the of the is a key for of side chains involved in substrate binding and catalysis. The loop structure and residues of catalytic and is by Its be disturbed by the P32T a disturbed nucleotide or a of the dimer the dimer populations the highest of the P32T ITPA that are by in populations S. K. N. N. H. H. H. Y. Ito M. K. J. Mol. 12: Google Scholar). the that is to a non-physiological in the P32T

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