Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities

Abstract

Human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) 3-kinase activities. This catalytic profile is not conserved; orthologs from Arabidopsis thaliana and Saccharomyces cerevisiae are predominantly Ins(1,4,5)P₃ 6-kinases, and the plant enzyme cannot phosphorylate PtdIns(4,5)P₂. Therefore, crystallographic analysis of the yeast and plant enzymes, without bound inositol phosphates, do not structurally rationalize HsIPMK activities. Here, we present 1.6-Å resolution crystal structures of HsIPMK in complex with either Ins(1,4,5)P₃ or PtdIns(4,5)P₂. The Ins(1,4,5)P₃ headgroup of PtdIns(4,5)P₂ binds in precisely the same orientation as free Ins(1,4,5)P₃ itself, indicative of evolutionary optimization of 3-kinase activities against both substrates. We report on nucleotide binding between the separate N- and C-lobes of HsIPMK. The N-lobe exhibits a remarkable degree of conservation with protein kinase A (root mean square deviation = 1.8 Å), indicating common ancestry. We also describe structural features unique to HsIPMK. First, we observed a constrained, horseshoe-shaped substrate pocket, formed from an α-helix, a 3₁₀ helix, and a recently evolved tri-proline loop. We further found HsIPMK activities rely on a preponderance of Gln residues, in contrast to the larger Lys and Arg residues in yeast and plant orthologs. These conclusions are supported by analyzing 14 single-site HsIPMK mutants, some of which differentially affect 3-kinase and 6-kinase activities. Overall, we structurally rationalize phosphorylation of Ins(1,4,5)P₃ and PtdIns(4,5)P₂ by HsIPMK.

Introduction

Considerable attention is focused on the enzymes that regulate the metabolism and hence the myriad cell signaling activities of the inositol phosphates and the inositol lipids. These are two physicochemically and functionally distinct groups of intracellular signals (1), which typically each rely on separate families of kinases for their synthesis. The sole exception is the inositol phosphate multikinase, initially named for its ability to phosphorylate inositol phosphates (2, 3), but later found to also phosphorylate PtdIns(4,5)P₂ (4). This “dual-specificity” has endowed the inositol phosphate multikinase (IPMK)² family with multiple biological activities. For example, IPMK is indispensable for connecting PLC-mediated Ins(1,4,5)P₃ release to the generation of InsP₅ (5–7); the latter is a precursor for InsP₆ and the inositol pyrophosphates, which each have many cellular functions (1, 8). Activation of the inositol phosphate kinase activities of IMPK appears to be a key response in the Wnt/β-catenin signaling pathway (9). IPMK is mainly localized in the nucleus (4, 10, 11), where its kinase activities have been shown to mediate cellular differentiation programs (12), and transcript-selective mRNA export from the nucleus (13). Also in the nucleus, the PtdIns(4,5)P₂ 3-kinase activity of IPMK stimulates the transcriptional activity of the nuclear receptor steroidogenic factor 1 (14). In addition, mammalian IPMK has moonlighting functions, unrelated to its catalytic activities, which are mediated through interactions with a number of protein-binding partners, such as mTOR (mechanistic target of rapamycin) (15), p53 (16), and AMP-activated protein kinase (17).

The wide-ranging importance of the IPMKs is underscored by the observation that knock-out of the IPMK gene in mice is embryonic lethal (5). There are also some pathological consequences for genetic defects in human IPMK that have been associated with a reduction in its kinase activities. For example, a heterozygous, frameshift mutation in the human IPMK gene has been identified in six members of the same family who all developed small intestinal neuroendocrine tumors; these individuals also exhibited a reduction in InsP₅ synthesis (18). Additionally, impaired IPMK transcription and a decrease in IPMK stability has been linked to the pathology of Huntington's disease, by virtue of an attenuation of the PtdIns(3,4,5)P₃/AKT signaling cascade (19). Genome-wide association studies have described that decreased IPMK expression in brain tissue is associated with the pathogenesis of inflammation-associated neurodegeneration (20).

IPMK is a member of the so-called IP-kinase family that includes IP3Ks and IP6Ks (21). Characterization of the structures of each of these enzymes can rationalize their alternate catalytic specificities, assist in deciphering evolutionary relationships, and permits rational design of enzyme-specific inhibitors. Previous work has described crystal structures of an IP3K (21, 22) and an IP6K (23), each with substrates captured in the active site. Two previous studies have described the crystal structures of IPMK orthologues from Saccaromyces cerevisiae (24) and Arabidopsis thaliana (25), at resolutions of 2 and 2.9 Å, respectively. However, neither study captured inositol phosphate within the active site.

Molecular modeling has generated a consensus view that IPMKs host a relatively spacious and conformationally flexible substrate-binding pocket, in which mobile side chains of Lys and Arg residues play major roles (24, 25). Here, we demonstrate that this model does not apply to HsIPMK; we describe a catalytic pocket that is more constrained than those of the plant and yeast orthologs. Also unique to mammalian IPMK is a catalytically important proline-loop, and a preponderance of Gln residues in the active site. These conclusions are drawn from our description, for the first time, of the crystal structure of HsIPMK. Moreover, we present the first structures of any IPMK in complex with inositol phosphate substrate: we describe two versions of Ins(1,4,5)P₃ within the active site, first as a free inositol phosphate, and second as the headgroup of a soluble analogue of PtdIns(4,5)P₂. This allows a structural rationalization of 3-kinase activity toward Ins(1,4,5)P₃ and PtdIns(4,5)P₂.

Results and discussion

General structure of HsIPMK

Previous work (3, 4, 11, 14) has shown that HsIPMK is both an inositol phosphate kinase and a PtdIns(4,5)P₂ kinase. Here, we confirmed that recombinant HsIMPK uses 3-kinase activity to phosphorylate Ins(1,4,5)P₃ to Ins(1,3,4,5)P₄, and then phosphorylates the latter with a 6-kinase activity that yields Ins(1,3,4,5,6)P₅ (Fig. 1, A–E). In addition, we used a deacylated, soluble version of Ptd[³H]InsP(4,5)P₂ (i.e. GroPtd[³H]Ins(4,5)P₂) to confirm the PtdIns(4,5)P₂ 3-kinase activity of recombinant HsIPMK (Fig. 1, F–I). Our next goal was to rationalize these various kinase reactions structurally, at an atomic level.

Figure 1.

The inositol phosphate and inositol lipid kinase activities of HsIPMK. A, graphic depicting the successive Ins(1,4,5)P₃ 3-kinase and Ins(1,3,4,5)P₄ 6-kinase activities of HsIPMK; filled circles represent phosphate groups. B, HPLC analysis of [³H]Ins(1,4,5)P₃ at zero time (solid line). C, HPLC analysis of the phosphorylation of [³H]Ins(1,4,5)P₃ for 15 min by 0.9 ng HsIPMK (solid line). Also shown is the elution of a [¹⁴C]Ins(1,4,5,6)P₄ standard (open circles; dotted line) obtained in parallel to a run by collecting fractions. D, HPLC analysis of the phosphorylation of [³H]Ins(1,4,5)P₃ for 15 min by 90 ng of HsIPMK (solid line). E, the elution of a [¹⁴C]Ins(1,3,4,5,6)P₅ standard (dotted line) obtained in parallel to an assay in which [³H]Ins(1,4,5)P₃ was phosphorylated to [³H]Ins(1,3,4,5,6)P₅ by HsIPMK (solid line). F, graphic depicting GroPtd[³H]Ins(4,5)P₂ 3-kinase activity of HsIPMK. G, HPLC analysis of GroPtd[³H]Ins(4,5)P₂ at zero time. H, HPLC analysis of phosphorylation of GroPtd[³H]Ins(4,5)P₂ to GroPtd[³H]InsP₃ (elution peak = 38 min) by 4.5 ng of HsIPMK for 15 min. I, HsIPMK (9.5 ng) was incubated for 16 h with [³²P]ATP and diC₈-PtdIns(4,5)P₂; the diC₈-Ptd[³²P]Ins(3,4,5)P₃ product was deacylated to GroPtd[³²P]InsP₃, which was analyzed by HPLC (solid line). In a parallel HPLC run, we analyzed a GroPtd[³²P]InsP₃ standard (dotted line), derived from PtdIns(4,5)P₂ 3-kinase 110-kDa catalytic subunit α (PIK3CA). The elution peak = 37.7 min. Full details are provided under “Experimental procedures.”

We produced crystals of the core catalytic domain of the enzyme that contains residues 50 to 416, from which we deleted an internal domain comprising residues 263 to 377 (Fig. 2A); that deletion, which was necessary to obtain crystals, was replaced with a simple Gly-Gly-Ser-Gly-Gly linker (Fig. 2A). Previous work has shown that this deletion does not compromise catalytic activity (26); this is a non-catalytic region of the protein. It contains a nuclear localization sequence, flanked by residues that host protein kinase phosphorylation sites that regulate nuclear localization sequence functionality (26).

Figure 2.

Overall structure of the ADP-Ins(1,4,5)P₃-HsIPMK crystal complex. A, domain graphic of the HsIPMK construct used for structural characterization, based on previously published terminology (21). Note that amino acid residues in a domain responsible for nuclear localization (263 to 377) were replaced with a Gly-Gly-Ser-Gly-Gly linker. B, ribbon plot of the HsIPMK structure. NLS = nuclear localization sequence. ADP and InsP₃ are shown as stick models within an 2F_o − F_c electron density, which is contoured at 1.5 σ. Two magnesium atoms are depicted as magenta balls. C, electrostatic surface plot with blue and red coloration to denote positive and negative electrostatic potentials, respectively, at physiological pH. D, a manual alignment of amino acid sequences (HsIPMK, NP_689416.1; HsIP6K2, NP_057375.2; and HsIP3KA, NP_002211.1), guided by the structural elements that have been observed in crystal structures, and in the case of HsIP6K2, secondary structural predictions. The secondary structural elements from HsIPMK are depicted above its sequence and are color-coded orange for the N-lobe, yellow for the C-lobe, and blue for the IP-helices. Residues are involved in ATP binding are highlighted as magenta for polar contacts and green for Van der Waals interactions. Residues that are involved in Ins(1,4,5)P₃ binding are highlighted in red. The loop that contains three prolines (“3-P”), and the hinge between the N- and C-domains, are also highlighted. PDB codes for HsIPMK are 5W2G, 5W2H, and 5W2I.

The structure of the IPMK apoenzyme was determined by a molecular replacement approach using a model constructed from the template of ScIPMK (PDB accession code 2IF8). That information was then used for further elucidation of the structures of crystal complexes with ADP plus either Ins(1,4,5)P₃ (Fig. 2, A–D) or diC₄-PtdIns(4,5)P₂ (see below).

For each asymmetric unit, there is one molecule of IPMK in space group P4₂2₁2 (Table 1). Analysis of the overall fold of HsIPMK (Fig. 2B) reveals domains that are similar to the so-called N- and C-lobes that comprise the ATP-binding sites in the orthologs ScIPMK (24) and AtIPMK (25), as well as EhIP6KA (23) and HsIP3KA (21). Superimposition structures of each of these proteins with HsIPMK reveals root mean square deviations (r.m.s. deviations) of 0.93 Å for ScIPMK (139 comparable Cα atoms), 1.37 Å for AtIPMK (149 comparable Cα atoms), 1.65 Å for EhIP6KA (145 comparable Cα atoms), and 1.14 Å for HsIP3KA (144 comparable Cα atoms).

Table 1.

Data collection and structure refinement statistics

PDB accession IDs	5W2G	5W2H	5W2I
Ligand	Apo	ADP-Ins (1,4,5) P3-Mg	ADP-DiC4-PtdInsP2-Mg
Data collection
Space group	P42212	P42212	P42212
Cell dimensions a, c (Å)	78.47, 85.65	78.02, 85.92	78.09, 86.32
Resolution (Å)a	50–1.80 (1.83)	50–1.9 (1.93)	50–1.6 (1.63)
Rmeasa	0.121 (0.857)	0.116 (0.953)	0.104 (0.782)
CC1/2 in the highest shell	0.754	0.763	0.853
I/σIa	15.5 (3.6)	18.0 (3.3)	22.1 (2.9)
Completeness (%)a	99.4 (99.9)	99.9 (100.0)	99.9 (100.0)
Redundancya	8.7 (8.1)	9.0 (8.9)	7.7 (7.3)
Refinement
Resolution(Å)a	1.80 (1.85)	1.9 (1.95)	1.6 (1.64)
No. reflections	23558	20189	34007
Rworka	17.1 (24.6)	16.3 (19.1)	16.9 (18.7)
Rfreea	21.5 (25.7)	23.0 (30.2)	21.5 (23.7)
No. atoms
Protein	2034	2046	2035
Mg		2	2
Inositol phosphates		24	24
ADP		27	27
Solvent	154	177	182
B-factors (Å2)
Protein	25.1	24.7	17.8
Mg		17.4	15.6
Inositol phosphates		34.5	25.2
ADP		17.6	13.7
Solvent	35.2	36.4	33.7
R.m.s. deviations
Bond length(Å)	0.008	0.010	0.009
Bond angle (°)	1.285	1.54	1.39

^a The numbers in parentheses are for the highest resolution shell.

The N-terminal lobe in HsIPMK is designated by residues 65–135, which consist of four antiparallel β-strands and two short helices α1 and α2 and the β1 and β3 strands. Residues 66–69 lie within a β-strand that has not been observed in either ScIPMK or AtIPMK; we designated this the β0 strand. We designated the C-terminal lobe as comprising residues 136–149 and 175–416, which is an α+β-fold with five, central antiparallel β-strands including β4–6, β8, and β9, a pair of small antiparallel β-strands (β7 and β10), and three α–helices (α5-α7). Also in the C-lobe of HsIPMK, a 3₁₀ helix was observed between the β6 strand and α5 helix. An equivalent, albeit longer 3₁₀ helix is present in EhIP6KA, where it is important for substrate binding (23).

Description of the nucleotide-binding region of HsIPMK

We soaked Ins(1,4,5)P₃, ATP, and magnesium into the apoenzyme crystals. Nucleotide binding did not alter protein conformation. The ADP moiety was observed (Fig. 3A), but not the γ-phosphate of ATP. A similar result was obtained in an earlier study with ScIPMK (24). Perhaps in our study, the γ-phosphate was hydrolyzed, or alternately, disordered in the crystal. The latter explanation is feasible, because the terminal phosphate of adenylyl-imidodiphosphate (AMP-PNP) was also the only portion of that non-hydrolyzable ATP analogue that we could not visualize, after it was soaked into the crystal structure with Ins(1,4,5)P₃.³

Figure 3.

Nucleotide binding by HsIMPK; conservation of a microenvironment in HsIPMK and protein kinase A. A, ADP is depicted as a stick and ball model. The HsIPMK-bound ADP are show in green for carbon, red for oxygen, blue for nitrogen, and orange for phosphorus atoms. Two magnesium atoms are depicted as magenta spheres. Polar contacts are shown with dashed lines. Amino acids side chains are shown as sticks. The backbones of the hinge residues are also shown as stick and ball models. B, superimposition of key residues for nucleotide binding between HsIPMK (orange or yellow stick, residues are numbered as in panel A) and ScIPMK (light green stick, with green-colored residues numbers). C, shows conservation of both the nature and the relative positions of the nucleotide-binding residues in HsIPMK (orange, blue, and yellow sticks) and PKA from the rat (white sticks; PDB code 1L3R). PKA-bound nucleotide is depicted in white. The magnesium atoms in HsIPMK are shown in magenta; those from PKA are colored pink. D–F, superimpositions of HsIPMK and PKA. D and E are ribbon plots of the N- and C-lobe, respectively, of HsIPMK (orange, blue, and yellow) and PKA (cyan). F, a surface representation of PKA (white) in complex with the peptide substrate; Ins(1,4,5)P₃ (green carbon stick and ball), from the HsIPMK structure (yellow and blue schematics), is located in the PKA catalytic center.

HsIPMK clasps the nucleotide between the N- and C-lobes, which are linked by a hinge that comprises residues Asp¹³² to Pro¹⁴⁰ (Figs. 2, A and C, and 3A). The N¹ and N⁶ atoms of adenine both make hydrogen bonds with the polypeptide backbone: N¹ contacts the amide nitrogen of Val¹³³ from the hinge, and N⁶ interacts with the carbonyl oxygen of Glu¹³¹ from the N-lobe (Fig. 3A). The ATP-ribose group is loosely confined by several van der Waals contacts with Leu²⁵⁴ and Ile³⁸⁴, plus one hydrogen bond with Asp¹⁴⁴. The α-phosphate of the nucleotide forms a salt bridge with Lys⁷⁵ (Fig. 3A). Asp³⁸⁵ interacts with two magnesium ions to make contact with the α- and β-phosphates of ADP. The particular importance of Asp³⁸⁵ is reflected in it being part of an Ile-Asp-Phe tripeptide that is conserved throughout the IP-kinase family (Figs. 2D and 3A, and see Ref. 21).

Key residues in the nucleotide-binding pocket of HsIPMK were superimposed upon those of ScIPMK, revealing a high degree of conservation (Fig. 3B; the same comparison could not be made with AtIPMK, because no nucleotide-bound crystal structures are available). Moreover, data in Fig. 3C show that five of the residues in HsIPMK that make contacts with ADP, namely, Lys⁷⁵, Glu¹³¹, Val¹³³, Asp¹⁴⁴, and Asp³⁸⁵, are also represented in the nucleotide-binding domain of protein kinase A (PKA) (27). Furthermore, a high degree of conservation of the entire N-lobes was revealed by superimposition of the secondary structure elements of the HsIPMK structure upon those in PKA (Fig. 3D): a core r.m.s. deviation of 1.80 Å (51/69 comparable residues). The C-lobes (Fig. 3E) are less conserved: a core r.m.s. deviation of 3.35 Å (78/168 comparable residues). The major structural differences between the C-lobes of the two proteins reflects specialization of the alternative substrate-binding pockets. The C-lobe of PKA, and indeed protein kinases in general, contains a greater degree of helical structure, and a wider binding site to accommodate a polypeptide (27, 28) (Fig. 3F). These data confirm and extend the idea (21, 29) that protein kinases and the so-called IP-kinase family share an evolutionary ancestry.

Description of inositol phosphate binding

The successful soaking of Ins(1,4,5)P₃ into HsIPMK crystals (Fig. 2, B and C) has yielded the first description of any inositol phosphate substrate captured in the active site of an IPMK. Simulated annealing omit maps and 2F_o − F_c maps clearly identify the inositol ring and individual phosphate groups for Ins(1,4,5)P₃ (Fig. 2B). The 2–5 axis of the Ins(1,4,5)P₃ substrate inserts vertically into a positively charged (at physiological pH) horseshoe-shaped pocket (Fig. 4A) constructed from (in anticlockwise rotation), a short 3₁₀ helix, the α3 helix, and a unique loop that is fabricated from three proline residues (Fig. 4B). Rigidity in the α3 helix is enhanced by virtue of a 2.8-Å hydrogen bond between the carboxyl and amine groups in the side chains of Gln¹⁶³ and Gln¹⁶⁴, respectively (Fig. 5A). This constrains the conformations of the two side chains and introduces planarity, resulting in a stacking effect between the inositol ring and the α3 helix.

Figure 4.

The proline loop of HsIPMK. A, electrostatic surface plot of the catalytic site of HsIPMK. Ins(1,4,5)P₃ and ADP are depicted as cyan sticks. Magnesium atoms are shown in magenta. B, the proline loop that is unique to HsIPMK (orange schematic) is shown in a structural superimposition of that protein with HsIP3K (pink, PDB code 1W2C), Entamoeba histolytica IP6K (cyan, PDB code 4O4F), and ScIPMK (green, PDB code 2IF8). ADP and Ins(1,4,5)P₃ are shown in stick and ball models (green stick for carbon, red for oxygen, and orange for phosphorus atoms). C, electrostatic surface plot of the catalytic site of HsIP3K; Ins(1,4,5)P₃ and ADP are shown in cyan sticks. D and E, electrostatic surface plots of the catalytic sites of AtIPMK and ScIPMK, respectively. These crystal complexes do not contain an inositol phosphate in the active site. ADP is shown in cyan sticks.

Figure 5.

Ins(1,4,5)P₃ binding by IPMKs. A, binding of Ins(1,4,5)P₃ within the catalytic site of HsIMPK (stick and ball model; green stick for carbon, red for oxygen, and orange for phosphorus atoms. The phosphate groups are numbered.). B, multiple sequence alignment of inositol phosphate-binding regions of IPMKs from the indicated organisms. The tri-Pro sequence is given in bold. Residues highlighted in red are those involved in binding Ins(1,4,5)P₃ by HsIMPK. C, significance of His³⁸⁸ in the catalytic center of HsIMPK (stick and ball model; green stick for carbon, red for oxygen, and orange for phosphorus atoms. D, superimposition of Ins(1,4,5)P₃-binding residues in HsIPMK (blue stick) upon the aligned residues (see B) in ScIPMK (green stick); E, superimposition of Ins(1,4,5)P₃-binding residues in HsIPMK (blue stick) upon the aligned residues (see B) in AtIPMK (pink stick), plus two additional AtIPMK residues (Lys¹⁵³ and Arg¹⁵⁶, also pink stick), which have been implicated in Ins(1,4,5)P₃ binding. The latter two residues align with His¹⁹⁷ and Arg²⁰⁰ in HsIPMK, which do not participate in Ins(1,4,5)P₃ binding; those two HsIPMK residues are shown as a transparent blue stick. Note that Arg⁸² in HsIPMK does not have a corresponding residue in either ScIPMK or AtIPMK.

The proline loop of HsIPMK (Fig. 4B) is a structural element that is absent from HsIP3K (Figs. 2D and 4C) and both the plant and yeast IPMKs (Fig. 4, D and E, and 5B). In fact, in our IPMK sequence alignment (Fig. 5B), there are gaps in the yeast and plant sequences in the region corresponding to the HsIPMK proline loop. Interestingly, the equivalent Drosophila IPMK sequence is Lys-Pro-Glu, suggesting that a nascent version of this loop is present in this, and perhaps, other invertebrates (Fig. 5B). Arg⁸² at the N terminus of the proline-loop makes 3 polar contacts with the 4- and 5-phosphates of Ins(1,4,5)P₃, indicating its particular importance in substrate binding. Gln⁷⁸, at the N terminus of the proline loop, interacts with two water molecules that coordinate with the β-phosphate of ADP and a magnesium atom (Fig. 4B).

We were unable to visualize the γ-phosphate of ATP within the catalytic center, apparently because it is disordered in the crystal (see above), but the ADP moiety is only 6.3 Å from the 3-OH of Ins(1,4,5)P₃ that is phosphorylated (Fig. 5, A and C). Thus, ATP may phosphorylate the Ins(1,4,5)P₃ substrate by direct, in-line transfer (Fig. 5C). There are also two magnesium ions that are in a position to stabilize the negative charge that would develop on the leaving γ-phosphoryl group. Additionally, His³⁸⁸ is also only 7.7 Å from the β-phosphate of the ADP moiety, and so it is possible that His³⁸⁸ may contribute to charge balance during catalysis. His³⁸⁸ can also hydrogen bond with the 4-phosphate of Ins(1,4,5)P₃ (Fig. 5A).

Other residues that form polar contacts with Ins(1,4,5)P₃ include Lys¹⁶⁰, Gln¹⁶³, Gln¹⁶⁴, and Lys¹⁶⁷ from the α3 helix, and Gln¹⁹⁶ from the 3₁₀ helix (Fig. 5A). A contact between Gln¹⁶⁴ and the axial 2-hydroxyl group appears to help locate the inositol ring near-parallel to the α3 helix (Figs. 4A and 5A). The interactions of Gln¹⁶⁴, Lys¹⁶⁷, and Gln¹⁹⁶ with Ins(1,4,5)P₃ may be particularly important for enforcing its phosphorylation at the 3-position. Thus, we have described a relatively constrained catalytic pocket in which a preponderance of Gln residues make contact with substrate. This contrasts with the description of the active site that emerged from the modeling of substrates into the active sites of AtIPMK (25) and ScIPMK (24). The latter two studies described a less-enclosed and conformationally-flexible substrate pocket in which mobile side chains of Lys and Arg residues play major roles in ligand binding (Fig. 5, D and E). Two such residues in particular, Lys¹⁵³ and Arg¹⁵⁶ in AtIPMK, were proposed to form key contacts with substrate (25). In a structural alignment (Fig. 5E), the latter two residues correspond to His¹⁹⁷ and Arg²⁰⁰ in HsIPMK (Fig. 5E), but these do not play any role in substrate-binding in our crystal structures (Fig. 5, A and E).

As noted above, HsIPMK phosphorylates Ins(1,4,5)P₃ to Ins(1,3,4,5)P₄ and then to Ins(1,3,4,5,6)P₅. We were unsuccessful in our efforts to soak Ins(1,3,4,5)P₄ into the crystals, so we modeled this particular substrate into the active site (Fig. 6, A and B). We found we could accomplish this, such that the 6-OH of Ins(1,3,4,5)P₄ is the group closest to the active site, by a 180° flip of the 3–6 axis of the inositol ring of Ins(1,3,4,5)P₄, relative to that of Ins(1,4,5)P₃ (Fig. 6, A and B). We therefore conclude that HsIMPK is unlikely to processively phosphorylate Ins(1,4,5)P₃ to Ins(1,3,4,5,6)P₅; instead, it appears that the Ins(1,3,4,5)P₄ intermediate must dissociate and rebind in a different orientation.

Figure 6.

Modeling of Ins(1,3,4,5)P₄ into the substrate-binding pocket of HsIPMK. A, the positions of Ins(1,4,5)P₃ (stick and ball model; green stick for carbon, red for oxygen, and orange for phosphorus atoms), magnesium, and ADP within the substrate binding pocket of HsIPMK. B, a model for Ins(1,3,4,5)P₄ (stick and ball model; green stick for carbon, red for oxygen, and blue for phosphorus atoms) in the active site, derived by flipping the inositol ring by 180 degrees relative to Ins(1,4,5)P₃. C, dashed lines depict the proposed interactions between substrate-binding residues and Ins(1,3,4,5)P₄. Carbon groups on the inositol phosphates are numbered.

In our model (Fig. 6B), the 4- and 5-phosphates of Ins(1,3,4,5)P₄ spatially mimic the orientation of the 5- and 4-phosphates of Ins(1,4,5)P₃, respectively. This model (Fig. 6) further predicts that the 3-phosphate of Ins(1,3,4,5)P₄ could gain up to two polar contacts with Gln¹⁶³ and Lys¹⁶⁷. Nevertheless, the overall conclusion is that, compared with Ins(1,4,5)P₃, the Ins(1,3,4,5)P₄ makes fewer total interactions with the protein. For example, the 1-phosphate of Ins(1,3,4,5)P₄ only makes 2 contacts with Lys¹⁶⁷ and Gln¹⁹⁶, whereas the 1-phosphate of Ins(1,4,5)P₃ makes 4 contacts with the same two residues. Second, Gln¹⁶⁴ interacts with Ins(1,4,5)P₃ at the axial 2-OH, but this group in Ins(1,3,4,5)P₄ does not directly interact with the protein (Fig. 6C). These comparisons suggest that, compared with Ins(1,4,5)P₃, IMPK may have a lower binding affinity for Ins(1,3,4,5)P₄; this may be the reason that we were unable to soak Ins(1,3,4,5)P₄ into the active site (see above). Differences in binding affinity may also explain why the rate of Ins(1,3,4,5)P₄ phosphorylation is 90-fold slower than that for Ins(1,4,5)P₃ (Table 2).

Table 2.

Effects upon the catalytic activities of HsIPMK following mutation of key residues to Ala

Rate equations (see under “Experimental procedures”) were used to derive the individual first-order rate constants k₁, k₂, and k₃. Data are mean ± S.E. from 3 to 6 determinations.

Mutant	Location	k1 (s−1) InsP3 metabolism (3-kinase)	Δ	k2 (s−1) InsP5 synthesis (6-kinase)	Δ	k3 (s−1) GroPIns(4,5)P2metabolism (3-kinase)	Δ
			-fold		-fold		-fold
None (WT)		56.1 ± 2.0		0.64 ± 0.02		11.3 ± 0.2
Q78A	Proline-loop	5.4 ± 0.6	−10	0.17 ± 0.03	−4	NDa
R82A	Proline-loop	1.6 ± 1.0	−35	0.01 ± 0.0005	−64	ND
K160A	IP-helix	0.8 ± 0.08	−67	0.01 ± 0.001	−64	ND
Q163A	IP-helix	20.6 ± 1.65	−3	0.36 ± 0.011	−2	ND
Q164A	IP-helix	0.5 ± 0.04	−119	0.006 ± 0.0003	−107	0.08 ± 0.002	−141
K167A	IP-helix	7.0 ± 0.56	−8	0.091 ± 0.004	−7	6.7 ± 0.4	−2
Q196A	310 helix	14.0 ± 1.4	−4	0.22 ± 0.012	−3	6.5 ± 0.4	−2
H388A	Catalytic center	0.1 ± 0.004	−584	0.00031 ± 0.00006	−206	ND

^a ND, not determined.

We do not exclude the possibility that the orientations of the amino acid side chains might be affected by the nature of the bound substrate. Nevertheless, no such movements were necessary for us to model Ins(1,3,4,5)P₄ into the active site, compared with their positions in the Ins(1,4,5)P₃-bound crystal complex. This situation contrasts with the conclusion that emerged after substrates were modeled into the plant and yeast IPMKs (24, 25). In the latter studies, it was proposed that conformational flexibility was likely an important aspect to accommodating the different substrates within a relatively spacious binding pocket. In those particular IPMK orthologs, such flexibility could be provided by the relatively long and mobile side chains of Lys and Arg (24, 25). In contrast, in the case of HsIPMK, the smaller side chains of Gln have a larger role in substrate-binding.

Mutagenesis of HsIPMK

Elements of the nucleotide-binding domain of HsIMPK are well-conserved within the IP-kinase family (Fig. 2D) (21). In contrast, our structural analysis has revealed unique features of the inositol phosphate-binding site, which presumably enforce its own particular set of catalytic activities. We interrogated these new findings using site-directed mutagenesis. We mutated to Ala seven residues that are revealed to interact with Ins(1,4,5)P₃ substrate, together with His³⁸⁸ at the catalytic center (Table 2). To varying degrees, each of these mutants exhibited reduced activity compared with wild-type enzyme, for both Ins(1,4,5)P₃ phosphorylation, as well as InsP₅ accumulation (Table 2). These mutagenic data validate the conclusions based on structural data (see above) that these particular residues are catalytically important. The H388A mutation had the largest effect, reflecting the critical nature of its role in the catalytic center (Fig. 5C).

We also performed a more subtle mutagenic approach to pursue further the particular significance of the three catalytically-important Gln residues at positions 163, 164, and 196: we mutated each to Arg and Lys, both of which have side chains that are larger and also positively charged at physiological pH. The results are quite dramatic (Table 3): in each case, the rate of Ins(1,4,5)P₃ 3-kinase activity declined, but in contrast, the rate of Ins(1,3,4,5)P₄ 6-kinase activity was not impaired; in fact, three of these mutants showed increased 6-kinase activity (Table 3). From our data on Ins(1,4,5)P₃ binding within the crystal complex (Fig. 5A), and our model of Ins(1,3,4,5)P₄ binding (Fig. 6C), we can propose 3 possible explanations for why these mutants exhibit a switch in 3-kinase/6-kinase preferences. First, the 3-kinase activity could be impeded by the mutation of Gln¹⁶⁴ to the larger Lys or Arg residue, because that would provoke a steric conflict with the 2-OH of Ins(1,4,5)P₃, with which Gln¹⁶⁴ has a favorable interaction in the wild-type enzyme (Fig. 5A). For Ins(1,3,4,5)P₄ binding, the 2-OH is rotated out-of-reach from any residue at position 164, because of the ring flip (Fig. 6C). Moreover, a gain of function of Ins(1,3,4,5)P₄ 6-kinase activity could result from this substrate's 6-OH and 3-phosphate groups making contact with the Lys or Arg replacement (Fig. 6C). Second, the relative spatial position of Gln¹⁶⁴ would be perturbed by mutation of Gln¹⁶³, because of elimination of the stabilizing electrostatic connection between the two Gln residues (Fig. 5A). Third, the substitution of Arg or Lys for Gln¹⁹⁶ would sterically disturb the latter's favorable interaction with the proximal 1-phosphate of Ins(1,4,5)P₃ (Fig. 5A); in the Ins(1,3,4,5)P₄ model, the 1-phosphate is rotated further away from Gln¹⁹⁶, but this gap could be bridged by multiple contacts with the larger Lys or Arg, thereby potentially contributing to a 6-kinase gain of function (Fig. 6C). These data provide a foundation for the generation and utilization of substrate-selective HsIPMK mutations for a synthetic biology approach to understanding each of the individual biological activities of this multifunctional enzyme.

Table 3.

Differential effects of mutagenesis of catalytically-important Gln residues upon 3-kinase and 6-kinase activities of IPMK

Data are mean ± S.E. from 3 to 6 determinations. There was no significant accumulation of Ins(1,4,5,6)P₄ in these experiments.

Mutant	k1 (s−1) InsP3 metabolism	Δ	k2 (s−1) InsP5 synthesis	Δ	k1/k2
		-fold		-fold
None (WT)	56.1		0.64		88
Q163K	6.3 ± 0.4	−9	0.41 ± 0.04	−2	16
Q163R	9.3 ± 0.9	−6	0.68 ± 0.02	0	14
Q164K	3.1 ± 0.2	−18	0.31 ± 0.05	−2	10
Q164R	8.3 ± 0.8	−7	8.5 ± 1.06	13	1
Q196K	19.4 ± 1.1	−3	2.4 ± 0.1	4	8
Q196R	12.3 ± 1.4	−4.5	3.1 ± 0.4	5	4

Structural rationalization of PtdIns(4,5)P₂ 3-kinase activity

The determination of the position of the 1-phosphate of Ins(1,4,5)P₃ in the substrate pocket is of particular interest for understanding why PtdIns(4,5)P₂ is also a substrate for HsIMPK. This 1-phosphate, which is doubly ionized (21), makes contacts with both Lys¹⁶⁷ and Gln¹⁹⁶ (Fig. 5A). In this configuration, a single uncharged oxygen is exposed to the bulk phase; the esterification of this particular oxygen to a diacyclyglycerol backbone would not be expected to impose any steric hindrance to substrate binding. To pursue that idea, we next soaked a soluble diC₄-analogue of PtdIns(4,5)P₂ (along with nucleotide and magnesium) into the HsIMPK apoenzyme crystal; the structure of the enzyme co-complex revealed that the Ins(1,4,5)P₃ headgroup of the inositol lipid was oriented in a near-identical configuration to that of free Ins(1,4,5)P₃ (Fig. 7). Furthermore, three mutations that compromised Ins(1,4,5)P₃ 3-kinase activity, Q164A, K167A, and Q196A, had quantitatively similar effects upon PtdIns(4,5)P₂ 3-kinase activity (Table 2). These data indicate that there has been co-evolution of Ins(1,4,5)P₃ and PtdIns(4,5)P₂ 3-kinase activities.

Figure 7.

Surface representation of HsIPMK crystal complexes with the Ins(1,4,5)P₃ headgroup of diC₄-PtdIns(4,5)P₂ superimposed upon free Ins(1,4,5)P₃. The figure describes the near-perfectly superimposed (r.m.s. deviation = 0.111 Å) positions of the Ins(1,4,5)P₃ headgroup of diC₄-PtdIns(4,5)P₂ (yellow carbon stick) and Ins(1,4,5)P₃ itself (green carbon stick). Phosphate groups are numbered. The likely position of the diester attachment is described with a black arrow.

No electron density was observed for the C₄-diacylglycerol moiety of the PtdIns(4,5)P₂ analogue, indicating that its mobility is not constrained upon binding to HsIPMK. This leaves the natural diacyglycerol backbone free to embed itself either into membranes, or the hydrophobic pockets of certain proteins (14). The position in the crystal complex of the Ins(1,4,5)P₃ headgroup of diC₄-PtdIns(4,5)P₂ clarifies that it can make the same contacts with the protein as Ins(1,4,5)P₃ itself, with just the one exception that the diester phosphate of the lipid at position 1 is held less tightly, because it only carries one negative charge, in contrast to the two in Ins(1,4,5)P₃ (30). Indeed, GroPIns(4,5)P₂ is a 5-fold weaker substrate than Ins(1,4,5)P₃ (Table 2).

Concluding comments

We have described several novel structural features of HsIMPK that clearly distinguish it from the orthologs in Arabidopsis and S. cerevisiae that predominantly phosphorylate the 6-hydroxyl of Ins(1,4,5)P₃ (24, 25). First, the horseshoe-shaped catalytic site in the human enzyme is more physically constraining. Second, HsIMPK hosts a smaller substrate-binding pocket in which Gln residues play major roles, unlike the plant and yeast orthologs that are more reliant on the longer and more flexible side chains of Arg and Lys. Third, the proline loop is a unique structural feature of the human enzyme that orients Arg⁸² and Gln⁷⁸ into functionally-important positions within the catalytic pocket. Our results indicate that these are all adaptations that optimize Ins(1,4,5)P₃ phosphorylation predominantly at the 3-position. Our crystal complex data also demonstrate that the Ins(1,4,5)P₃ headgroup of PtdIns(3,4,5)P₃ is near-perfectly superimposed upon free Ins(1,4,5)P₃ itself (Fig. 7). Thus, we propose that 3-kinase positional specificity toward Ins(1,4,5)P₃ has co-evolved along with the functional significance of PtdIns(4,5)P₂ 3-kinase activity of HsIPMK. Indeed, AtIPMK does not express PtdIns(4,5)P₂ 3-kinase activity (14, 31), whereas any PtdIns(3,4,5)P₃ product that might be formed in S. cerevisiae is not considered to be functional (32). In contrast, the biological importance of the PtdIns(4,5)P₂ 3-kinase activity of IMPK in mammals has been well demonstrated (14, 31, 33).

Experimental procedures

Protein expression and purification

The cDNA of HsIPMK was purchased from Addgene (plasmid number 23666). The Gateway expression system (Invitrogen) was used to subclone HsIPMK into the pDest-566 vector. This vector encodes a His₆ tag, a maltose-binding protein tag, and a tobacco etch virus protease cleavage site at the N terminus. Mutants were prepared using a site-directed mutagenesis kit (Stratagene) or a Q5 site-directed mutagenesis kit (Biolabs); all mutants were verified by sequencing. Each pDest-566 vector was used to transform DE3 competent Escherichia coli cells (Stratagene) that were pre-transformed with chaperone plasmid pGro7 (Takara, Clontech). An overnight culture of the transformed E. coli cells was inoculated into nutrient-rich 2× YT medium supplemented with 0.07% (w/v) l-arabionose at pH 7.5 and grown at 37 °C to A595 = 0.7. Isopropyl β-d-thiogalactopyranoside (0.1 mm) was then added and cultures were continued at 15 °C for 20 h. The cells were disrupted using a constant cell disruption system (Constant Systems LTD) under 20 KPsi. Recombinant wild-type and mutant proteins were purified by several chromatographic procedures performed at 4 °C. First, the protein was applied to a nickel-nitrilotriacetic acid-agarose column (Qiagen), washed with buffer containing 300 mm NaCl, 20 mm Tris-HCl, pH 7.5, 20 mm imidazole, then eluted by increasing the imidazole concentration to 400 mm. Second, the eluate was applied to a HiTrap^TM Heparin HP column (GE Healthcare) and eluted with 10 column volumes of a 50–2000 mm NaCl gradient in 20 mm Tris-HCl (pH 7.5). Next, after tobacco etch virus protease cleavage, the protein was further purified using another HiTrap^TM Heparin HP column, and finally, a Superdex^TM 200 gel filtration column (GE Healthcare) that was eluted with 150 mm NaCl, 20 mm Tris-HCl, pH 7.5. Purified proteins were concentrated to either 0.5–2 mg/ml (for assaying catalytic activities) or 30 mg/ml (for crystallization); storage was at −80 °C.

Crystallization

The core catalytic domain of HsIPMK (residues: ^50–262Gly-Gly-Ser-Gly-Gly^378–416; Fig. 2A) was initially screened for optimum crystallization conditions using the mosquito-LCP (TTP Labtech). Multiple conditions that each contained a high concentration of PEG 400 were identified. Final conditions were optimized by hanging drop vapor diffusion, against a well buffer of 35% (w/v) PEG 400, 0.1 m Li₂SO₄, 100 mm MES-imidazole buffer, pH 6.0, 50 mm β-mercaptoethanol at 25 °C (2 μl of 38 mg/ml of protein plus 2 μl of well buffer in the crystallization drop). To obtain complex structures, apoenzyme crystals were further soaked for 1 day in 35% (w/v) PEG 400, 100 mm Li₂SO₄, 100 mm HEPES, pH 7.5, at 25 °C, in the presence of 20 mm Ins(1,4,5)P₃ or a soluble diC₄-analogue of PtdIns(4,5)P₂, 10 mm MgCl₂, and 5 mm of either Na₂ATP or Li₂AMP-PNP; mother liquid was used for cryoprotection. The same conditions were used in attempts to soak Ins(1,3,4,5)P₄ into the crystals, in the presence of either ATP or ADP, but we were unable to define any clear electron density for the inositol phosphate.

Data collection, structure determination, and refinement

Diffraction data were collected using APS beamlines 22-ID and 22-BM. All data were processed with the program HKL2000 (34). Initial phases for the structure were determined by molecular replacement with the autoMR program in the CCP4 package (35, 36), using ScIPMK structure (PDB code 2IF8; sequence identity 31%) as a search model. This initial structure was manually rebuilt with COOT and refined with REFMAC from the CCP4 package. The other crystal structures were determined by using rigid body and direct Fourier synthesis, and refined with the equivalent and expanded test sets. The molecular graphics representations were prepared with the program PyMol (Schrödinger, LLC). Atomic coordinates and structure factors have been deposited with the Protein Data Bank with accession codes 5W2G, 5W2H, and 5W2I.

Enzyme assays and HPLC analysis

We used HPLC (37) to analyze the catalytic activities of HsIPMK toward [³H]Ins(1,4,5)P₃ (American Radiolabeled Chemicals). In these reactions, HsIPMK sequentially phosphorylates Ins(1,4,5)P₃ to Ins(1,3,4,5)P₄ to Ins(1,3,4,5,6)P₅ (see “Results and Discussions” and Ref. 11). We circumvented the problem that [³H]Ins(1,3,4,5)P₄ is not commercially available, by generating first-order rate constants for the two sequential reactions (38), from time-dependent changes in levels of Ins(1,4,5)P₃, Ins(1,3,4,5)P₄, and Ins(1,3,4,5,6)P₅. We assayed the PtdIns(4,5)P₂ 3-kinase activity of HsIPMK by using soluble GroPtd[³H]Ins(4,5)P₂ (prepared by deacylation (39) of Ptd[³H]Ins(4,5)P₂ (American Radiolabeled Chemicals)).

Kinase assays were performed by incubating either wild-type HsIPMK or mutants with trace quantities of either [³H]Ins(1,4,5)P₃ or GroPtd[³H]Ins(4,5)P₂ at 37 °C, in 100 μl of buffer containing 1 mm EDTA, 100 mm KCl, 20 mm HEPES, pH 7.2, 8 mm MgSO₄, 5 mm Na₂ATP. All reactions were acid quenched (0.2 volumes of 2 m perchloric acid + 1 mg/ml of InsP₆), and neutralized (40). For some experiments, assays contained 1 mm EDTA, 100 mm KCl, 20 mm HEPES, pH 7.2, 3 mm MgSO₄, 100 μm diC₈-PtdIns(4,5)P₂ (Cellsignals), 50 μm Na₂ATP plus 50,000 dpm of [³²P]ATP (MP Biomedicals); prior to HPLC analysis, the diC₈-Ptd[³²P]Ins(3,4,5)P₃ product was deacylated (39).

All assays were analyzed by ion-exchange HPLC, using an Adsorbosphere Q100 column. The elution gradient was generated by mixing Buffer A (1 mm Na₂EDTA) with Buffer B (Buffer A plus 2 m NH₄H₂PO₄, pH 3.9, with H₃PO₄) as follows: 0–5 min, 0% B; 5–10 min, 0–16% B, 10–60 min, 16–36% B, 60–61 min, 36–70% B. Radioactivity was measured either with an in-line counter using UltimaFlo AP (PerkinElmer Life Sciences), or by collecting 1-ml fractions, which were mixed with MonoFlow-4 (National Diagnostics); in all experiments, data are plotted as % of total radioactivity shown (in cpm or dpm).

Standards of both [¹⁴C]Ins(1,4,5,6)P₄ and [¹⁴C]Ins(1,3,4,5,6)P₅ were prepared as described previously (41). A standard of GroPtd[³²P]Ins(3,4,5)P₃ was prepared by deacylation (39) of Ptd[³²P]Ins(3,4,5)P₃ produced as follows: 50 ng of PIK3CA and PtdIns(4,5)P₂ (both supplied in a PI3K assay kit, product number 17–493, Millipore) were incubated at 37 °C for 16 h in 100 μl of buffer containing 1 mm EDTA, 100 mm KCl, 20 mm HEPES, pH 7.2, 3 mm MgSO₄, 50 μm Na₂ATP plus 50,000 dpm of [³²P]ATP (MP Biomedicals).

Author contributions

H. W. performed all of the experiments and analyzed the results. H. W. conceived and designed the experiments. H. W. and S. B. S. wrote the paper.