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Originally published In Press as doi:10.1074/jbc.M703121200 on July 6, 2007

J. Biol. Chem., Vol. 282, Issue 38, 28117-28125, September 21, 2007
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Integration of Inositol Phosphate Signaling Pathways via Human ITPK1*

Philip P. Chamberlain{ddagger}1, Xun Qian§1, Amanda R. Stiles, Jaiesoon Cho§, David H. Jones{ddagger}, Scott A. Lesley{ddagger}, Elizabeth A. Grabau, Stephen B. Shears§2, and Glen Spraggon{ddagger}3

From the {ddagger}Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, §Inositol Signaling Section, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, and Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Received for publication, April 13, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Cellular inositol phosphate metabolism is an intricate web of kinase and phosphatase reactions that produces a number of important signaling molecules (for review see Ref. 1). A now classic example of these signaling activities is the release of Ca2+ into the cytoplasm through an intracellular channel that is gated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)4 (2). Additional roles are continually being discovered: inositol phosphates have recently been shown to be critical to the activity of RNA-editing enzymes (3), to participate in telomere maintenance (4), and to be the phosphate donors in certain protein phosphorylation events (5). It is of critical interest, therefore, to establish the regulatory mechanisms that govern the metabolism of inositol phosphates.

An interesting feature of inositol phosphate metabolism is the promiscuity with which several key kinases phosphorylate multiple substrates (6). For example, ITPK1 (also known as inositol 1,3,4-trisphosphate 5/6-kinase) adds either a 5- or 6-phosphate to Ins(1,3,4)P3 and also attaches a 1-phosphate to Ins(3,4,5,6)P4 (6, 7) (inositol phosphate structures shown in Fig. 2). These reactions have been demonstrated to be reversible: ITPK1 can also dephosphorylate Ins(1,3,4,5,6)P5 back to Ins(3,4,5,6)P4 (8). An especially puzzling aspect of this phenomenon is that dephosphorylation of Ins(1,3,4,5,6)P5 by human ITPK1 is stimulated, rather than competitively inhibited, by one of its alternate substrates, Ins(1,3,4)P3 (8).

The fact that mammalian ITPK1 reversibly phosphorylates both Ins(3,4,5,6)P4 and Ins(1,3,4)P3 takes on special significance because the two substrates occur in separate branches of the metabolic pathway (9). Ins(3,4,5,6)P4 is formed in a metabolic cycle with Ins(1,3,4,5,6)P5, whereas Ins(1,3,4)P3 is formed by a kinase/phosphatase pathway as a consequence of receptor-dependent phospholipase C activity. The metabolic coupling of these two distinct pathways has been demonstrated in cells (10).

The metabolic interaction between ITPK1 substrates is of particular biological significance because Ins(3,4,5,6)P4 decouples calcium-activated chloride channels from activation by cytoplasmic Ca2+ (11). In this way Ins(3,4,5,6)P4 regulates a range of biological processes, including salt and fluid secretion, neurotransmission, and insulin secretion from pancreatic beta-cells (8, 11-13). Recently it was shown that changes in ITPK1 expression influence Ins(3,4,5,6)P4 levels, possibly modulating the phenotypic severity of the cystic fibrosis condition (7). Changes in ITPK1 expression have also been reported to affect tumor necrosis factor-{alpha}-induced cell death (14).

Recently, Miller et al. (15) described the crystal structure of an ITPK1 homologue in Entamoeba histolytica (eITPK1). This structure revealed an unusually versatile catalytic cleft that possesses little or no stereospecific constraints. Ligand binding was further proposed to be unaffected by either the presence of hydroxyl groups or by their orientation (i.e. equatorial versus axial); up to 18 inositol phosphate analogues could be modeled into the active site (15). Most of these characteristics of eITPK1 were proposed to apply to Homo sapiens ITPK1 (hITPK1) (15).

However, other data indicated that there are limitations in the extent to which this information can be exploited to improve our molecular understanding of the human enzyme. For example, the ligand specificity of hITPK1 depends on the stereochemistry of the inositol ring and the orientation of its hydroxyl groups (16). Most important of all, the eITPK1 structure did not offer any rationalization for how Ins(1,3,4)P3 can stimulate Ins(1,3,4,5,6)P5 dephosphorylation by mammalian ITPK1. In fact, in the current study we demonstrate that this characteristic has evolved specifically in higher organisms; we show that Ins(1,3,4)P3 does not activate Ins(1,3,4,5,6)P5 dephosphorylation in ITPK1 homologues from soybean and in E. histolytica. Moreover, we describe the crystal structure of the human enzyme and identify structural features likely to contribute to this and other important differences in the catalytic activities of different homologues of ITPK1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
A rapid Escherichia coli expression, purification, and crystallization procedure for hITPK1 has been developed. Human ITPK1 was cloned into an MH4 vector (17) by PCR and expressed in the HK100 strain of E. coli. Briefly, 12 x 65-ml cultures were grown at 37 °C in a Genomics Institute of the Novartis Research Foundation fermenter (18), and hITPK1 expression was induced by the addition of 0.02% L-arabinose for 3 h. Proteins were extracted by sonication into 20 ml of 50 mM Tris, pH 8, 100 mM NaCl, 1 mM tri(2-carboxyethyl)phosphine hydrochloride (TCEP), 10 mM imidazole, with 1 Complete EDTA-free protease inhibitor tablet (Roche Applied Science). Cell debris was removed by centrifugation at 30,000 x g for 45 min. The recombinant enzyme was purified by nickel-nitrilotriacetic acid-agarose chromatography and eluted into a buffer composed of 20 mM Tris, pH 8, 100 mM NaCl, 10% glycerol, 0.5 mM TCEP, 100 mM imidazole. S-200 size exclusion chromatography followed. Selenomethionine-substituted protein was produced by inhibition of methionine biosynthesis (19) and subsequently purified in the same manner as the native protein.

Protein was concentrated in a Centriprep 15 10-kDa concentrator (Amicon) prior to crystal screening. Native protein was concentrated to 15-22 mg/ml for crystallization trials, whereas selenomethionine protein appeared insoluble beyond 12 mg/ml and was used at this concentration. 480 crystallization conditions were set up in Greiner low profile 96-well sitting drop vapor diffusion plates at two temperatures (4 and 20 °C) (conditions described in Ref. 17). Full-length ITPK1 failed to crystallize despite extensive coarse crystallization screening and showed some proteolytic degradation during purification and storage. Proteolytic products, presumably a consequence of the action of endogenous E. coli proteases, were analyzed by mass spectroscopy, and sequences corresponding to the truncated products were cloned back into the MH4 vector. The construct composed of residues 1 to 335 proved amenable to crystallization and was used in subsequent structural studies. Crystals grew in 2 weeks at 4 °C after mixing the protein solution 1:1 with, and subsequently equilibrating against, a reservoir solution of 0.2 M citrate, pH 5.6, 2.0 M (NH4)2SO4, 0.2 M potassium/sodium tartrate. Further crystals were obtained by screening around this condition. Nucleotide complexes were obtained by co-crystallization with either 1 mM ATP or AMPPNP (Sigma), along with 5 mM MnCl2. Crystals were cryo-protected in reservoir solution containing 20% glycerol.


Figure 1
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  		FIGURE 1.
Intersubstrate phosphate transfer by hITPK1. The panels show HPLC analyses of inositol phosphate metabolism by hITPK1 (Partisphere SAX or Q100 column as indicated). For details see "Experimental Procedures." Identities of reaction products were all confirmed using genuine standards. A and B, hITPK1 was incubated with 1 mM ADP and 5 µM [32P-1]Ins(1,3,4,5,6)P5 in either the presence (open circles) or absence (closed circles) of 5 µM Ins(1,3,4)P3. The elution position of Pi is shown by an arrow. B, the InsP3/InsP4 region of the chromatograph from a different experiment than that shown in panel A. C, elution of [3H]Ins(1,3,4,5)P4 and [14C]Ins(1,3,4,6)P4 standards. D, the InsP4 region of the chromatograph obtained from an assay in which hITPK1 was incubated with 1 mM ADP, 5 µM [3H]Ins(1,3,4)P3, and 5 µM Ins(1,3,4,5,6)P5. E, hITPK1 was incubated with 1 mM ADP and 5 µM Ins(1,3,4)P3 in either the absence (filled hexagons) or presence of 5 mM ATP (open hexagons).

 
Data were collected at the Advanced Light Source (ALS, Berkeley, CA) beamlines 5.0.2 and 5.0.3. Data were integrated, reduced, and scaled using HKL2000 (20) and the CCP4i suite (21). Crystallographic statistics are summarized in Table 1. ITPK1 was initially phased using a three-wavelength multiwavelength anomalous dispersion experiment on selenomethionine-derivative protein, and the structure was subsequently solved using Solve/Resolve (22). Subsequently, non-isomorphous structures were solved by molecular replacement using Phaser (23). Iterative rounds of manual model building in Coot (24) were followed by restrained refinement using Refmac5 (25). Ligand solvent accessibility was calculated using AREAIMOL (26) with a probe radius of 1.4 Å. The protein-protein interaction server was used to evaluate the hITPK1 crystal interactions (27). Figures were prepared using PyMOL.


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  		TABLE 1
Crystallographic statistics for hITPK1

 
All in vitro assays of inositol phosphate metabolism were performed as previously described (28). For these experiments, recombinant hITPK1 was prepared after expression in Sf9 cells as previously described (28) except that the gene was first shuttled into the pDEST605 kinase vector, which also contains a green fluorescent protein reporter gene that was then used to generate bacmid insect viral DNA (Invitrogen). Site-directed mutagenesis was performed as previously described (28).

An expressed sequence tag clone encoding gmITPK4 was obtained from the Public Soybean Data Base (Biogenetic Services, Brookings, SD). PCR amplifications were performed with a 5'-primer containing a SmaI restriction site and the 3'-primer containing XhoI. PCR products were digested and ligated into pGEX4T-1 (encoding an N-terminal glutathione S-transferase tag), which was used to transform competent TOP10 bacterial cells. Protein expression was induced with isopropyl-1-thio-beta-D-galactopyranoside, cells were lysed, and protein was purified using glutathione S-transferase-Sepharose beads. Recombinant ITPK1 from E. histolytica was generously provided by Dr. Greg Miller (Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada). [32P-1]Ins(1,3,4,5,6)P5 was made by phosphorylating Ins(3,4,5,6)P4 with [32P]ATP (PerkinElmer) using ITPK1, after which the product was HPLC-purified.


Figure 2
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  		FIGURE 2.
Sequential intersubstrate phosphate transfer hypotheses for hITPK1. This graphic shows hypothetical intersubstrate phosphate transfer mechanisms by which levels of Ins(1,3,4)P3 could regulate the synthesis of Ins(3,4,5,6)P4. Panel A shows the two candidate phosphate carriers with enzyme represented as "E" with the transferred phosphate highlighted in red. Panel B expands the nucleotide-mediated phosphate transfer hypothesis in which enzyme-bound nucleotide acts as the phosphate carrier. Unliganded ITPK1 is represented by E; phosphotransferase reactions are indicated by red graphics. The position of the [32P] group that is transferred between inositol phosphates is shown in red.An asterisk indicates a reaction for which Ins(1,3,4,5)P4 is also produced at a reduced rate. We do not rule out the possibility that a phosphoenzyme intermediate might also participate in these reactions.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Intersubstrate Phosphate Transfer Activity of hITPK1—We investigated the mechanism by which Ins(1,3,4)P3 stimulates Ins(1,3,4,5,6)P5 dephosphorylation (Figs. 1 and 2). Dephosphorylation assays used Ins(1,3,4,5,6)P5 in which the 1-phosphate was [32P]-labeled. When incubated with ADP, hITPK1 dephosphorylated [32P-1]Ins(1,3,4,5,6)P5 to yield unlabeled Ins(3,4,5,6)P4. As Ins(3,4,5,6)P4 was not detectable in the HPLC profiles shown in Fig. 1, the accumulation of the Ins(3,4,5,6)P4 product was separately verified using [3H]Ins(1,3,4,5,6)P5 as substrate (data not shown). Note that there was no release of [32P]Pi when [32P-1]Ins(1,3,4,5,6)P5 was dephosphorylated to Ins(3,4,5,6)P4 (Fig. 1A). Instead, [32P]-labeled ATP accumulated (Figs. 1, A and B), which was separately verified using a luciferase reporter assay (data not shown). This is the first demonstration that the 1-phosphate from Ins(1,3,4,5,6)P5 is transferred to ADP rather than being released to the medium as inorganic phosphate by ITPK1.

The addition of non-radiolabeled Ins(1,3,4)P3 to these reactions increased the rate of dephosphorylation of Ins(1,3,4,5,6)P5 despite the fact that both are substrates of ITPK1 (Fig. 1A). This is a biologically significant phenomenon that enables Ins(1,3,4)P3 to regulate Ins(3,4,5,6)P4 accumulation in vivo (8), but it has not previously been rationalized at a molecular level. We now show that Ins(1,3,4)P3 considerably reduced the quantity of [32P]ATP that accumulated when [32P-1]Ins(1,3,4,5,6)P5 was dephosphorylated (Fig. 1, A and B). Instead, the [32P]1-phosphate that was removed from Ins(1,3,4,5,6)P5 was now transferred to Ins(1,3,4)P3, thereby forming [32P]InsP4 (Fig. 1, A and B). It has been known for some time that ITPK1 shows both 5- and 6-kinase activities toward Ins(1,3,4)P3 (29).Using[3H]Ins(1,3,4)P3 we confirmed that two [3H]InsP4 isomers were formed, Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (Fig. 1, C and D). The above results imply that Ins(1,3,4)P3 can bind to an enzyme-ATP state faster than ATP can be exchanged for ADP. Thus, the enzyme may return to its ADP state more quickly (ready for another cycle of Ins(1,3,4,5,6)P5 dephosphorylation) if enzyme-bound ATP is used to phosphorylate Ins(1,3,4)P3. Note also that a small amount of newly formed [32P]InsP4 was itself dephosphorylated to a [32P]-labeled InsP3, which consistently eluted one fraction after ATP (Fig. 1B).

The assays described above contained a physiologically relevant concentration of ADP (1 mM) (30) but no ATP. In further experiments, non-radioactive ATP was added to 5 mM and it is striking that there was only a 30% decrease in both the extent of Ins(1,3,4,5,6)P5 dephosphorylation and the rate of [32P] transfer from Ins(1,3,4,5,6)P5 to Ins(1,3,4,6)P4 (Fig. 1E) compared with incubations containing 1 mM ADP and no ATP. These results support our hypothesis that there is a relatively slow rate of exchange of bulk phase ATP with either enzyme-bound ADP or enzyme-bound [32P]ATP. Clearly, the intersubstrate phosphate transfer activity of ITPK1 occurs even in the presence of physiologically relevant levels of adenine nucleotides (i.e. [ATP] > [ADP]) (30).

Simultaneous binding of both Ins(1,3,4)P3 and Ins(1,3,4,5,6)P5 to the same active site seems implausible because of the topological and electrostatic constraints imposed by the binding pocket (described below). In order for the same phosphate to be transferred between different substrates via the same binding site, a sequential reaction model has been proposed (Fig. 2A). The efficient operation of this proposed nucleotide-mediated intersubstrate phosphate transfer may be facilitated by the enzyme withholding the nucleotide from the bulk phase during this series of reactions (Fig. 2B).


Figure 3
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  		FIGURE 3.
Both eITPK1 and gmITPK4 dephosphorylate Ins(1,3,4,5,6)P5, but Ins(1,3,4)P3 does not stimulate the reaction. Either eITPK1, hITPK1, or gmITPK4 were separately incubated as described under "Experimental Procedures" with 1 mM ADP and 5 µM either [3H]Ins(1,3,4,5,6)P5 (A and B) or [32P-1]Ins(1,3,4,5,6)P5 (C) in either the presence (closed symbols) or absence (open symbols) of 5 µM Ins(1,3,4)P3. Assays were analyzed either by HPLC (A and C) or by gravity-fed columns (B) (means ± S.E., n = 3). The elution gradient was steeper in the chromatograph described in panel C, hence the arrow marks the elution position of InsP4, determined in an adjacent run.

 
Homologues of hITPK1 from Plant and Protozoa Dephosphorylate Ins(1,3,4,5,6)P5 but Ins(1,3,4)P3 Does Not Stimulate This Reaction—Although the inositol phosphate kinase activity of eITPK1 has been well documented in previous reports (15, 31), the data shown in Fig. 3A represent the first demonstration that eITPK1 can dephosphorylate Ins(1,3,4,5,6)P5 to Ins(3,4,5,6)P4. It is particularly notable that the rate of Ins(1,3,4,5,6)P5 dephosphorylation was not stimulated by Ins(1,3,4)P3 (Fig. 3A). This is an important functional difference between eITPK1 and hITPK1 and may represent a divergence in cellular function. Homologues of hITPK1 are also present in plants (32). Plant enzymes have higher homology with hITPK1 than does eITPK1 (Fig. 4C). The plant enzymes are also functionally more similar to hITPK1 in that neither show physiologically significant kinase activity toward Ins(1,4,5)P3 (Table 2 and Ref. 32), unlike eITPK1 which shows robust Ins(1,4,5)P3 3-kinase activity (31). We therefore felt useful information could be obtained by comparing some of the characteristics of hITPK1 with a plant homologue.


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  		TABLE 2
Catalytic comparison between WT and H162D mutant of hITPK1

Assays were performed as described under "Experimental Procedures." The units for the kinase reactions are 103 x k (the first-order rate constant)/ng protein. Ins(1,3,4,5,6)P5 dephosphorylation is measured as nmoles/mg protein/min. The effect of Ins(1,3,4)P3 upon Ins(1,3,4,5,6)P5 dephosphorylation is calculated as a ratio: plus Ins(1,3,4)P3/minus Ins(1,3,4)P3. The values for "phosphate transfer" are the ratios of [32P]-labeled InsP4 vs. ATP, formed after either enzyme dephosphorylated ~50% of 5 µM [32P-1]/Ins(1,3,4,5,6)P5 substrate, in the presence of 5 µM Ins(1,3,4)P3 (e.g. see Fig. 1B).

 
Candidate homologues of hITPK1 were identified in the Glycine max (soybean) Expressed Sequence Tag data base (gmITPK1 to gmITPK4), which we have numbered based on their similarity to known genes from Arabidopsis thaliana (32) and Zea mays (33). Recombinant gmITPK4 was expressed in E. coli as an N-terminal glutathione S-transferase fusion protein and was found to phosphorylate both Ins(1,3,4)P3 and Ins(3,4,5,6)P4 (data not shown), verifying its nature as a homologue of hITPK1. Additionally, both gmITPK4 and hITPK1 showed Ins(1,3,4,5,6)P5 dephosphorylation activity when assayed under identical conditions. The soybean enzyme was up to 40-fold more active (Fig. 3B). However, Ins(1,3,4)P3 did not stimulate Ins(1,3,4,5,6)P5 dephosphorylation by gmITPK4 (Fig. 3B). Instead, Ins(1,3,4)P3 was a slight inhibitor, which is what would be expected if the two substrates competed for the same active site in gmITPK4. Moreover, gmITPK4 did not show substantial intersubstrate phosphate transfer activity (Fig. 3C) compared with hITPK1 (Fig. 1).

Structural Overview of hITPK1—To rationalize the species differences in substrate regulation, the crystal structure of hITPK1 was determined. The structure of hITPK1 was initially solved by selenomethionine multiwavelength anomalous dispersion and has been subsequently refined in a variety of ligand-bound forms and crystal forms to a maximum resolution of 1.6 Å (Table 1). The entire catalytic domain is defined as far as residue 335. In both crystal forms the C-terminal residues form a small dimer interface. Although this interface corresponds to ~10% of the total surface area of the protein, other biophysical methods such as gel filtration and static light scattering indicate a monomeric state for ITPK1 (data not shown).

hITPK1 exhibits the same overall topology as the eITPK1 structure, which is related to the former by 24% amino acid sequence identity between the catalytic domains (Fig. 4C). Superposition of the hITPK1 structure with eITPK1 results in a root mean square deviation of 2.2 Å on 242 topologically equivalent C{alpha} atoms (Fig. 4A). As would be expected for enzymes with variations in enzymatic function, structural diversity is exhibited in regions proximal to the active site.


Figure 4
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  		FIGURE 4.
Sequence and structural comparisions between hITPK1 and eITPK1. Panels A and B show a structural alignment of hITPK1 in complex with AMPPNP (blue) with eITPK1 (orange). Panel B highlights topological differences proximal to the ATP binding site. Selected hITPK1 residues are shown in stick representation and selected hydrogen bonds as broken lines. AMPPNP is from the hITPK1 structure and is shown in stick form, with carbons shown in yellow. Panel C shows a sequence alignment of hITPK1, eITPK1, and gmITPK4. Secondary structural elements from hITPK1 are shown in blue above the alignment, and eITPK1 secondary structure is shown in orange below the alignment. aX, betaX, and 310X refer to {alpha} helices, beta-sheets, and 310 helices, respectively, numbered in order from the N terminus. The position of His-162 is indicated by an asterisk.

 
ATP Binding Site—The ATP binding sites of eITPK1 and hITPK1 are each sandwiched between two sets of four-stranded anti-parallel beta-sheets (Fig. 4A), a structure commonly described as an ATP-grasp fold (34, 35). In hITPK1, one beta-sheet is composed of beta11, beta10, beta13, and beta14; the opposed sheet is composed of beta12, beta8, beta7, and beta9 (Fig. 4, A and C). A further two extended strands provide contacts to the purine ring of the bound nucleotide. Two well defined Mn2+ ions are present in the active site of the hITPK1 structure. One Mn2+ ion is coordinated by Asp-281 and Asp-295 and interacts with oxygens from both the beta- and {gamma}-phosphates of the bound ATP, adjacent to the scissile bond. The second Mn2+ ion is coordinated by the side chains of Asp-295 and Asn-297. These Mn2+ ions are structurally equivalent to the Mg2+ ions in the eITPK1 structure (15).

It was previously noted that the nucleotide binding site of eITPK1 encloses much of the ligand; only 22% was accessible to solvent (15). There is only one entry and exit site for nucleotides in both eITPK1 and hITPK1 structures that would not require considerable rearrangement of the protein backbone, and this opening occurs next to active site residues Asp-295 and Asn-297. In eITPK1, one face of the entry/exit site is formed by a turn in the polypeptide chain just after the conserved structural element beta12 and is followed by two helices (eITPK1 residues 212 to 236) arranged anti-parallel to each other and oriented roughly parallel to beta12 (Fig. 4B). However, the structure of hITPK1 diverges after beta12, with a 14-residue deletion relative to the eITPK1 sequence. Instead, hITPK1 substitutes a 310 helix (3104), composed of residues 232 to 236, in a lateral orientation to the ATP phosphates (Fig. 4B). The two crystal forms (Table 1) each contain two unique hITPK1 molecules in the asymmetric unit, and in each crystal form these molecules vary in the degree to which helix 3104 encloses the nucleotide, indicating flexibility within this motif. In the conformation in which the nucleotide is most enveloped, the side chain of His-233 is able to reach hydrogen bonding distance to the AMPPNP {gamma}-phosphate, although the equivalent distance is 6 Å in the second molecule, beyond interacting range. Nevertheless, in both cases the entry/exit site for nucleotide is more restricted for hITPK1 than eITPK1, with solvent accessibilities of only ~5% for AMPPNP (Fig. 5, A and B).

The 3104 helix also offers several extra interactions with the bound nucleotide that are not conserved in eITPK1. These include a bond from Ser-236 to the ADP-ribose o3 hydroxyl (2.7 Å, Fig. 4B), and extra Van der Waals interactions from Val-235 to the ribose ring (4.2 Å, Fig. 4B). hITPK1 Ser-232 makes a tight polar contact with the beta-phosphate (2.5 Å) and also approaches the side chain of His-167 (3.4 Å). Note also that the helix 3104 in hITPK1 aligns with a sequence in gmITPK4 that contains a proline residue (Fig. 4C), which would be expected to disrupt the intra-helix hydrogen bonding and geometry.

Inositol Phosphate Binding Site of hITPK1—A particularly interesting feature of the eITPK1 structure is that there are no direct interactions between inositol hydroxyl groups and the protein, reflecting a limited capability for stereochemical substrate discrimination (15). In contrast, experiments with hITPK1 indicate a much greater ability to discriminate between substrates, including those that are stereoisomers (16). The inositol phosphate binding site of hITPK1 can be visualized as a strongly electropositive cleft that is stationed at the entrance to the ATP binding pocket (Fig. 5). The hITPK1 inositol phosphate binding site is composed of residues from helices {alpha}1, {alpha}3, and 3104, Arg-212 from sheet beta11, and loop regions between beta14-{alpha}9 and beta7-beta8 (Fig. 4, A and C). Despite repeated attempts, we have been unable to co-crystallize or soak inositol phosphates into the hITPK1 crystals, possibly due to the highly polar nature of the binding site in combination with the presence of ammonium sulfate in the crystallization buffer. Sulfate molecules are, in fact, bound in the inositol phosphate binding site (Fig. 5C).

In comparing the inositol phosphate binding sites of hITPK1 and eITPK1, structural rearrangements and amino acid substitutions contribute to a considerable reduction in binding pocket volume for hITPK1 (Fig. 5, A and B). Helix 3104 in hITPK1 is followed by a loop composed of residues Ser-236 to Ser-258 where the eITPK1 binding site is open to solvent. The consequences of this loop are the further encapsulation of the bound nucleotide, the constriction of the inositol phosphate binding site, and the introduction of residues capable of mediating novel interactions with the bound inositol phosphate. For example, Lys-237 reaches from this loop into the inositol binding pocket where it would be able form a bond with either a phosphate group or with a hydroxyl group (Fig. 5, B and C). From the opposite face of the inositol phosphate binding cleft, two additional lysine residues, Lys-17 and Lys-21, both project from the {alpha}1 helix into the inositol phosphate binding pocket (Fig. 5, B and C). Lys-21 is a substitution for Thr-20 in eITPK1 and in hITPK1 could form a bond with a phosphate or hydroxyl group on an inositol ring modeled into the binding site. Lys-17 is also capable of interacting with inositol phosphate groups (Fig. 5C). A kink in the loop formed from residues Ile-296 to Glu-307, and a substitution from a glycine in eITPK1 to an glutamate at position 303 in hITPK1, closes one further face of the inositol binding site (Fig. 5, B and C).


Figure 5
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  		FIGURE 5.
Detail of the hITPK1 inositol phosphate binding pocket. Identical projections for eITPK1 (A) and hITPK1 (B), showing the reduction in inositol phosphate binding site volume and ATP solvent accessibility for hITPK1. Ins(1,3,4)P3 is shown modeled into the hITPK1 based on structural alignment with eITPK1 and exhibits a steric clash with His-162. A solvent-accessible surface is shown for each molecule. The position of the ATP {gamma}-phosphate is indicated by "{gamma}". Metal ions are shown as green spheres. Panel C is a stereodiagram showing structural superposition of hITPK1 with eITPK1. hITPK1 is shown with blue carbons atoms, with a bound sulfate shown in gold and red. Residues capable of carrying a phosphate that are proximal to the ATP {gamma}-phosphate are shown in yellow. eITPK1 in complex with Ins(1,3,4)P3 is shown with orange carbon atoms for protein, magenta carbons for the inositol.

 
A further reduction of the inositol phosphate binding pocket occurs as a consequence of structural changes at hITPK1 {alpha}3 and the loop between beta7 and beta8 (Fig. 4, A and C) that contains His-162 (Fig. 5, B and C). Helix {alpha}3 is composed of residues 60-69 and corresponds to a loop region in eITPK1. In hITPK1, Asp-62 protrudes from {alpha}3 and forms a tight polar interaction with His-162 from the loop between beta6 and beta7 (2.6 Å, Fig. 5C). The side chain of His-162 is positioned over the side chain amine of residue Lys-59 at a distance of 3.4 Å, which lines the inositol phosphate binding site below the imidazole ring. Interestingly, when Ins(1,3,4)P3 is modeled into hITPK1 in the same binding mode as eITPK1, the imidazole ring of His-162 clashes with the 4-phosphate group of the ligand (Fig. 5, B and C). His-162 is equivalent to Gln-141 in eITPK1, which also hydrogen bonds with the 4-phosphate of Ins(1,3,4)P3 (15). The conformation of {alpha}3 in hITPK1 leads to a shift of 5.0 Å for Thr-61 (equivalent to Thr-59 in eITPK1) toward the inositol phosphate binding site and within 3.6 Å of the imidazole ring of His-162. Thus, a steric barrier exists preventing His-162 from accommodating Ins(1,3,4)P3 in the manner of eITPK1 and indicating that rearrangements of protein and/or substrate are necessary to facilitate binding.

His-162 in hITPK1 Contributes to Intersubstrate Phosphate Transfer—Ins(1,3,4)P3 stimulated Ins(1,3,4,5,6)P5 dephosphorylation by hITPK1, but this was not observed for either eITPK1 or gmITPK4 (Fig. 3). Based on comparative structural and sequence analysis, residues were identified that may contribute to this phenomenon. His-162 and His-233 emerged as especially interesting candidates as in our comparative analysis both are unique to hITPK1 (Fig. 4C). Our structural studies show that His-162, and its polar interaction with Asp-62, makes a significant contribution to the geometry of the substrate binding cleft (Fig. 5, B and C). His-233 is present on helix 3104 and protrudes into the substrate binding site (Fig. 5, B and C). However, the ability of Ins(1,3,4)P3 to enhance Ins(1,3,4,5,6)P5 dephosphorylation was preserved in a H233S mutant of hITPK1 (data not shown). In contrast, when His-162 in hITPK1 was mutated to its equivalent residue in the soybean enzyme, Asp, Ins(1,3,4)P3 did not stimulate Ins(1,3,4,5,6)P5 dephosphorylation (Table 2). This loss-of-function for Ins(1,3,4)P3 in the H162D mutant was matched by a 200-fold reduction in its Ins(1,3,4)P3 kinase activity (Table 2), yet the H162D mutant dephosphorylated Ins(1,3,4,5,6)P5 almost twice as fast as the wild-type enzyme (Table 2). In previous work we found that the ability to dephosphorylate Ins(1,3,4,5,6)P5 was compromised whenever kinase activity was reduced (28), making H162D unique (Table 2). The H162D mutant also displayed very limited ability to transfer phosphate from Ins(1,3,4,5,6)P5 to Ins(1,3,4)P3 in the presence of physiological concentrations of nucleotide (Table 2). Instead, most of the phosphate removed from Ins(1,3,4,5,6)P5 was trapped in ATP and released into the bulk phase (Table 2; data not shown). The position of His-162 on a loop forming part of both the inositol phosphate binding site and the nucleotide binding site indicates that a role in substrate binding or catalysis is plausible. Our data indicate that the structural motif featuring His-162 plays an important role in the intersubstrate phosphate transfer activity of hITPK1, in part by promoting the phosphorylation of Ins(1,3,4)P3 and also regulating ATP exchange with the bulk phase.

Concluding Comments—This study provides a molecular basis for understanding how hITPK1 couples steady-state levels of Ins(3,4,5,6)P4 to a metabolically separate pool of inositol phosphates, including Ins(1,4,5)P3 and Ins(1,3,4)P3, that arise from receptor-regulated phospholipase C activity (9). During metabolic coupling, a phosphate is transferred between inositol phosphates by hITPK1, but not by eITPK1 or gmITPK4. Although our studies reveal that ADP does act as a phosphate carrier, we do not exclude the possibility that a phosphoenzyme intermediate might also play a role in the catalytic cycle. hITPK1 residues capable of carrying a phosphate and that are proximal to the ATP {gamma}-phosphate are shown in yellow in Fig. 5C.

We propose that these differences between ITPK1 homologues represent the evolution in mammals of a mechanism for regulating the signaling activities of Ins(3,4,5,6)P4. This function may be superimposed upon a more general role of ITPK1 homologues in eukaryotes: contributing to the synthesis of higher inositol polyphosphates such as InsP6 (33, 37). However, the extent to which ITPK1 contributes to higher order inositol phosphate synthesis in mammals remains to be demonstrated in a range of conditions and cell types. For example, it has been shown that two other inositol kinases, IPK1 and IPK2, are sufficient to recapitulate InsP6 synthesis from Ins(1,4,5)P3 (38, 39). In contrast, the function of gmITPK4 in soybean is likely restricted to the synthesis of higher inositol polyphosphates. Furthermore, Ins(1,3,4)P3 has not been identified in any plant, nor has an enzyme that can synthesize it.

It seems likely the capacity for inositol phosphate signal integration has co-evolved as Ins(3,4,5,6)P4 developed into an intracellular signal that, through its control over Cl- channel conductance, regulates many biological processes, including epithelial salt and fluid secretion, insulin secretion, and likely smooth muscle contraction and neurotransmission. Together, the biochemical and structural studies on ITPK1 provide an example how evolution can transform a metabolic enzyme into one with a signaling function. The characterization of the structure of hITPK1 and its mechanism of substrate regulation together offer new avenues for intervention in the inositol phosphate metabolic pathway.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2Q7D and 2QB5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was based on experiments conducted at beamlines 5.0.3 and 5.0.2 of the Advanced Light Source (ALS). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Material Sciences Division of the U.S. Department of Energy under contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. This research was in part supported by the Intramural Research Program of the NIEHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence may be addressed. E-mail: shears{at}niehs.nih.gov.

3 To whom correspondence may be addressed. E-mail: gspraggo{at}gnf.org.

4 The abbreviations used are: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins(1,3,4)P3, inositol 1,3,4-trisphosphate; ITPK1, inositol 1,3,4-trisphosphate 5/6-kinase; Ins(1,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; Ins(3,4,5,6)P4, inositol 3,4,5,6-tetrakisphosphate; Ins(1,3,4,5,6)P5, inositol 1,3,4,5,6-pentakisphosphate; eITPK1, E. histolytica ITPK1; hITPK, H. sapiens ITPK1; HPLC, high pressure liquid chromatography; ALS, Advanced Light Source; gmITPK4, Glycine max ITPK1 homologue. Back


   ACKNOWLEDGMENTS
 
We thank colleagues at Novartis, Scott Brittain, Dan McMullan, Polat Abdubek, Michael DiDonato, Andreas Kreusch, Christian Lee, Joanna Hale, Donna Eckert, Eric Peters, Christopher Stroh, Garrett Hampton, and Linda Okach for technical assistance and Mike Cooke for useful discussions. Andrew Riley (University of Bath, UK) is also thanked for helpful discussions. Thanks to Peter Schultz for continued support. We thank all the staff at the ALS beamlines for continued support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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A. Saiardi and S. Cockcroft
Human ITPK1: A Reversible Inositol Phosphate Kinase/Phosphatase That Links Receptor-Dependent Phospholipase C to Ca2+-Activated Chloride Channels
Sci. Signal., January 29, 2008; 1(4): pe5 - pe5.
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