Structural elucidation of the binding site and mode of inhibition of Li+ and Mg2+ in inositol monophosphatase Anirudha Dutta*, Sudipta Bhattacharyya*, Debajyoti Dutta and Amit Kumar Das Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India

Keywords bipolar disorder; crystal structure; inositol monophosphatase; lithium ion inhibition; post-catalytic inhibition Correspondence A. K. Das, Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India Fax: +91 3222 255303/278707/283757 Tel: +91 3222 283756 E–mail: [email protected] *These authors contributed equally to this work (Received 25 July 2014, revised 16 September 2014, accepted 23 September 2014) doi:10.1111/febs.13070

Mg2+-dependent, Li+-sensitive phosphatases are a widely distributed family of enzymes with significant importance throughout the biological kingdom. Inositol monophosphatase (IMPase) is an important target of Li+-based therapeutic agents in manic depressive disorders. However, despite decades of intense research efforts, the precise mechanism of Li+-induced inhibition of IMPase remains obscured. Here we describe a structural investigation of the Li+ binding site in staphylococcal IMPase I (SaIMPase I) using X–ray crystallography. The biochemical study indicated common or overlapping binding sites for Mg2+ and Li+ in the active site of SaIMPase I. The crystal structure of the SaIMPase I ternary product complex shows the presence of a phosphate and three Mg2+ ions (namely Mg1, Mg2 and Mg3) in the active site. As Li+ is virtually invisible in X–ray crystallography, competitive displacement of Mg2+ ions from the SaIMPase I ternary product complex as a function of increasing LiCl concentration was used to identify the Li+ binding site. In this approach, the disappearing electron density of Mg2+ ions due to Li+ ion binding was traced, and the Mg2+ ion present at the Mg2 binding site was found to be replaced. Moreover, based on a detailed comparative investigation of the phosphate orientation and coordination states of Mg2+ binding sites in enzyme–substrate and enzyme–product complexes, inhibition mechanisms for Li+ and Mg2+ are proposed. Database The atomic coordinates for the SaIMPase I ternary complex, SaIMPase I in 50 mM LiCl, SaIMPase I in 100 mM LiCl and SaIMPase I in 0 mM MgCl2 have been submitted to the Protein Data Bank under accession numbers 4G61, 4I40, 4I3Y and 4PTK, respectively. Structured digital abstract ●

SaIMPase-I and SaIMPase-I bind by x-ray crystallography (View interaction)

Introduction Lithium ions are therapeutically important in the treatment of a form of manic depression termed bipolar disorder [1]. Therapeutically low concentrations of Li+ serve as a mood stabilizer for manic depressive disorder, and have been found to inhibit several Mg2+-dependent enzymes, including inositol monophosphatase (IMPase) [2,3]. The phosphoinositol signaling pathway is known

to be hyperactive in individuals suffering from manic depression. IMPase is involved in the phosphoinositol signaling pathway, generating free inositol [4,5]. The synthesis of inositol in brain tissue from glucose-6– phosphate also requires IMPase in the final dephosphorylation step [6]. The inhibition of IMPase by Li+ is thus an effective method for inhibiting the

Abbreviations Hal2p, yeast 30 -phosphoadinosine-50 -phosphate phosphatase; IMPase, inositol monophosphatase; SaIMPase I, staphylococcal inositol monophosphatase I.

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phosphoinositol signaling cycle. Recent studies have demonstrated the efficacy of Li+ therapy in several other acute neurological disorders and neurodegenerative diseases [7]. The pharmacological effects of Li+ as a psychotropic drug have been thoroughly described by Pasquali et al. [8]. Despite their therapeutic efficiency, Li+ treatments have toxic side-effects [9]. Because of this, researchers have explored alternative inhibitors with similar modes of action that have reduced toxicity. However, to determine an appropriate substitute, the precise molecular mechanism of Li+ inhibition must be understood. IMPase catalyzes hydrolysis of the phosphoester bond of inositol monophosphate using Mg2+ as a cofactor. The presence of three activating Mg2+ ions in the catalytically competent IMPase has been observed. Two metal binding sites were initially identified from the structures of human IMPase, and a third Mg2+ ion binding site was identified in an archaeal IMPase ortholog. In human IMPase, the Mg1 binding site is consisting of Glu70, Asp90 and the carbonyl oxygen of Ile92 [10], and the Mg2 binding site is consisting of Asp90, Asp93 and Asp220 residues [11]. The Mg3 binding site comprises a single amino acid (Glu65) in archaeal IMPase, corresponding to Glu70 in humans [12–14]. Mg1 and Mg3 activate the catalytic water nucleophile (W1) (Fig. 1A), whereas Mg2 activates another water molecule (W2) that protonates the inositol oxyanion (Fig. 1B). The proposed reaction scheme mediated by three Mg2+ ions was verified by X–ray crystallographic studies on IMPase [15] as well as in fructose-1,6–bisphosphatases and 30 –phosphoadinosine-50 –phosphate phosphatases [16–18]. Computational methods have also been used to examine the two/three Mg2+ ion-mediated phosphate hydrolysis mechanism of IMPase [19,20]. Biochemical studies have shown that Li+ displays concentration-dependent bimodal inhibition. At low concentrations, Li+ inhibition is uncompetitive, whereas at high concentrations, it is non-competitive

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with respect to the substrate [21,22]. Furthermore, despite its traditional role as a cofactor, a high concentration of Mg2+ (e.g. > 5 mM in human IMPase) inhibits the enzyme [23]. Kinetic studies suggest that the inhibition observed at high concentrations of Mg2+ is uncompetitive with respect to the substrate [21,24,25]. Thus, the mode of inhibition exerted by low concentration of Li+ and high concentration of Mg2+ is speculated to be the same. Additionally, the competitive binding of Li+ to the Mg2+ binding site and the mutually exclusive nature of Li+ and Mg2+ inhibition [21,25,26] indicate the existence of common or overlapping binding site(s) for Li+ and Mg2+ in IMPase. By virtue of their ‘diagonal relationship’ in the periodic table, the ionic radii of Mg2+ and Li+ are comparable  for Li+) [27]. Thus,  for Mg2+; r = 0.76 A (r = 0.72 A 2+ ion may easily be in proteins, the bound Mg replaced with Li+. However, unlike Mg2+, the Li+ ion cannot activate the water nucleophile involved in an in–line attack during phosphoester bond hydrolysis. Numerous approaches, such as enzyme kinetics [21,24,25], site-directed mutagenesis (e.g. H217Q and C218A) [23,28] and spectroscopic studies [29–31], have been performed to characterize the Li+ ion binding site in IMPase. It has been proposed that the Mg2 site is responsible for Li+ inhibition. However, these results were biased because of the assumed mechanism for two metal ion-mediated phosphate hydrolysis [11,32,33] and a lack of direct evidence. Another approach that may explain the Li+-induced inhibition suggests that the Mg3 site is the target of Li+ binding [12–14]; however, this hypothesis suffers from several shortcomings. First, the mechanism cannot explain the ‘burst phase’ release of inositol during Li+ inhibition [21]. Second, it does not explain the linear non-competitive mode of Li+ inhibition at higher Li+ concentrations [21,22]. The non-competitive mode indicates that Li+ is able to bind the free enzyme (low-affinity binding site) and the enzyme–substrate or enzyme– product complex (high-affinity binding site). However,

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Fig. 1. Schematic diagram of the three metal-assisted reaction mechanism of IMPase. (A) Pre-catalytic substrate-bound state in which the Mg1 and Mg3 activate the water nucleophile (W1) involved in in–line attack. (B) Protonation of the departing inositol oxyanion by a water molecule (W2), activated by Mg2.

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only the high-affinity binding site is occupied in uncompetitive inhibition at low concentrations of Li+. At high concentrations, Li+ is proposed to bind the free enzyme at the Mg2 site [22]. Sophisticated methods, such as 7Li NMR spectroscopy [34] and magic angle spinning solid-state NMR spectroscopy [35], have also been used to study the high-affinity Li+ ion binding site in IMPase. Using solution NMR, only a single high-affinity Li+ binding site was observed in IMPase. Solid-state NMR studies revealed that the Li+ ion binds to either the Mg1 site or the Mg2 site, but not to the Mg3 site. Therefore, the precise location of the high-affinity Li+ binding site and whether binding of Li+ is pre-catalytic or post-catalytic remain unclear. In an effort to explain the molecular basis of linear non-competitive Li+ inhibition at higher Li+ concentrations, we proposed the occurrence of competitive binding of Li+ at the Mg2 site (designated as Mg1 site in Bhattacharyya et al. [22]) of the free enzyme [22]. In the present study, in order to ascertain the mode of Li+ inhibition of IMPase family proteins and to discern the precise high-affinity binding site of the Li+ ions in uncompetitive inhibition, we obtained the crystal structures of the Mg2+-bound and Li+-soaked product complexes of staphylococcal inositol monophosphatase I (SaIMPase I), which has significant structural homology with human IMPase I. The structure presented herein is a ternary product complex (SaIMPase I–3 Mg2+–PO43) obtained from co-crystallization of SaIMPase I in the presence of an inhibitory high concentration of Mg2+ (0.2 M) and substrate NADP+. Additionally, two LiCl-soaked inhibitory product complexes are described, which were obtained by soaking the crystals of the aforementioned ternary product complex in 50 and 100 mM LiCl. The replacement of Mg2 by Li+ allows determination of the location of the uncompetitive (high-affinity) Li+ binding site. Moreover, molecular mechanisms of inhibition of IMPase family members by Li+ and Mg2+ are also proposed.

Results Biochemical studies of Mg2+ inhibition The essential activator of SaIMPase I, Mg2+, exerts concentration-dependent bimodal activation and inhibition with respect to the substrate. At low concentrations (< 10 mM), Mg2+ acts as an activator (Fig. 2A); however, at high concentrations (> 10 mM), Mg2+ is an uncompetitive inhibitor (Fig. 2B). The inhibition at a high concentration of Mg2+ is mediated by formaFEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

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tion of the inhibitory product complex [24,26]. To address the precise kinetic mechanism of inhibition by Mg2+ as a function of product retardation, the order of product release from SaIMPase I was determined. This was defined by the product inhibition patterns using 20 –AMP as the substrate. Because adenosine is sparingly soluble in the reaction buffer (i.e. 50 mM Tris/HCl, pH 8), a substitute product, inositol, was used with PO43. The non-competitive inhibition by inositol (Fig. 2C) and the competitive inhibition by PO43 (Fig. 2D) were compatible with the sequential ordered product release mechanism [36]. As a noncompetitive inhibitor, inositol was found to be the first product released by the enzyme, followed by release of PO43 to free the enzyme for the next round of catalysis. Finally, a multiple inhibition kinetics experiment was performed at inhibitory concentrations of the last product PO43, and the cofactor Mg2+. The data were plotted using the method described by Yonetani and Theorell (see Eqn 1 below) [37], in which KI and KJ are the inhibition constants of PO43 (KPO4 = 0.46 mM) and the cofactor Mg2+ (KMg = 88 mM) respectively, as determined from secondary slope plots. The increase in slope with an increasing concentration of PO43 in the plot of 1/Vo versus [MgCl2] (Fig. 3A) suggests simultaneous binding of Mg2+ and PO43. The interaction constant (b) of 0.6 in multiple inhibition studies between PO43 and inhibitory Mg2+ suggests the existence of an E–Mg2+–PO43 inhibitory complex. Biochemical studies of Li+ inhibition The activity of SaIMPase I, similar to the other members of the IMPase superfamily, is inhibited by Li+. Biochemical experiments suggested that Li+ acts as a competitive inhibitor of the cofactor Mg2+ [22]. Moreover, at low concentrations, Li+ acts as an uncompetitive inhibitor with respect to the substrate [22]. The Mg2+-induced inhibition pattern resembles that exerted by Li+ at low concentrations. Notably, the similar inhibition patterns indicate the presence of common or overlapping Li+ and Mg2+ binding sites in SaIMPase I. Hence, to ascertain the correlation between the low-concentration Li+ binding and highconcentration Mg2+ binding inhibitory sites, multiple inhibition kinetics was performed (Fig. 3B). Multiple inhibition kinetics data were analyzed using Eqn (1), where KI and KJ are the inhibition constants for Li+ (KLi = 81 mM) and Mg2+ (KMg = 88 mM), respectively, as determined from secondary slope plots. The results show a series of parallel lines with various fixed concentrations of Mg2+. The Li+ and inhibitory

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Fig. 2. Biochemical studies of Mg2+ inhibition in SaIMPase I. (A) At a low concentration (< 10 mM), Mg2+ acts as a non-competitive activator with respect to the substrate used. Double reciprocal plots of initial velocity versus [20 –AMP] at Mg2+ concentrations of 2 mM (black squares), 5 mM (open squares) and 9 mM (black triangles). (B) At a high concentration (> 10 mM), Mg2+ acts as an uncompetitive inhibitor with respect to the substrate used. Double reciprocal plots of initial velocity versus [20 –AMP] at fixed Mg2+ concentrations of 10 mM (black squares), 50 mM (open squares) and 100 mM (black triangles). (C) Non-competitive inhibition of 20 –AMPase activity by inositol. Double reciprocal plots of initial velocity versus [20 –AMP] at inositol concentrations of 100 mM (black squares), 200 mM (open squares) and 300 mM (black triangles). (D) Competitive inhibition of 20 –AMPase activity by PO43. Double reciprocal plots of initial velocity versus [20 – AMP] at PO43 concentrations of 100 lM (black squares), 200 lM (open squares) and 300 lM (black triangles). Experiments were performed at 310 K and pH 8, in the presence of 0.25 lM of enzyme. Each experiment was performed at least twice independently in triplicate, and the deviations were < 5%.

Mg2+ binding sites were found to be mutually exclusive, as reflected by the infinite value of the interaction constant b (b = ∞). Hence, these two cations bind to a common inhibitory site on the enzyme. Interestingly, the common inhibitory site may be occupied by either Li+ or Mg2+, and binding of either metal ion precludes binding of the other. The absence of slope effects (common for non-competitive inhibition) in the inhibition kinetics exerted by low Li+ concentrations and high Mg2+ concentrations indicates that these 5312

cations inhibit the enzyme by binding to either of the enzyme–substrate or enzyme–product complex. Structure of the Mg2+-bound inhibitory and dissociable product complexes The overall three-dimensional structure of the enzyme appeared to comprise an ababa penta-layered sandwichlike arrangement, which is a signature fold of the IMPase superfamily (Fig. 4A). Structural superimposition with FEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

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Fig. 3. Multiple inhibition kinetics. (A) Yonetani–Theorell plot for PO43- and high Mg2+-mediated multiple inhibition analysis. The inhibitory effect of Mg2+ and PO43- are synergistic. Inhibition of 20 –AMPase activity by a high concentrations of Mg2+ (> 10 mM) and 0 mM (black squares), 100 mM (open squares) and 300 mM (black triangles) of different fixed inhibitory concentrations of PO43 at 3 mM 20 –AMP. (B) Yonetani–Theorell plot for Li+- and high Mg2+-mediated multiple inhibition analysis. The Li+ and high-concentration Mg2+ binding sites are mutually exclusive. Inhibition of 20 –AMPase activity by Li+ and 50 mM (black squares), 75 mM (open squares) and 100 mM (black triangles) of different fixed inhibitory [Mg2+] at 3 mM 20 –AMP. Experiments were performed at 310 K and pH 8, in the presence of 0.25 lM of enzyme. Each experiment was performed at least twice independently in triplicate, and the deviations were < 5%.

human IMPase I [11] shows that both structures are  over a range of very similar, with an RMSD of 2.4 A 173 Ca atoms (Fig. 4B). The active site of SaIMPase I is situated at the interface of the N- and C–terminal domains between the two flanking sets of helices (N– terminal a1 and a2 helices and C–terminal a4 and a7 helices). SaIMPase I crystals were grown in the presence of NADP+ substrate and at a high MgCl2 concentration (0.2 M) to obtain an Mg2+-bound inhibitory product complex. The difference Fourier map shows several connecting blobs in the active-site cavity of the enzyme. The central globular part with the highest contour levels (8.8 r) was assigned as the phosphate ion. The source of the bound phosphate is certainly NADP+, which was added to the protein prior to crystallization, because the crystallization conditions did not include any phosphate ions and no bound phosphate was observed in the crystal structures determined in the absence of NADP+. However, no extended electron density indicative of NAD+ was observed. It is likely that NADP+ is hydrolyzed in the presence of Mg2+, and, after hydrolysis, the NAD+ is diffused, leaving the phosphate entrapped in the active site. Three Mg2+ ions and water molecules were fitted into the difference Fourier maps around the phosphate moiety based on the geometric parameters previously reported in the structure of bovine IMPase [15]. A refinement shows that the map is in good agreement with the placement of metals and phosphate (Fig. 5A). FEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

All three Mg2+ ions in the active site were found to be octahedrally coordinated (Fig. 5B). The Mg1 site includes Glu70 OE2, Asp88 OD1, Ile90 O, one oxygen atom from the phosphate moiety, and two water molecules. The octahedral Mg2 site consists of Asp88 OD2, Asp91 OD1, Asp209 OD1, two oxygen atoms from the phosphate moiety, and a water molecule. The Mg3 is coordinated to Glu70 OE1. Of the remaining five coordinating ligands, one is a phosphate oxygen and four are water molecules. The geometric parameters of all three metal-binding sites are summarized in Table 1. The structure of the three Mg2+- and phosphate-bound ternary product complex (SaIMPase I3 Mg2+–PO43) represents the inhibitory product based on the results of the biochemical study. It is well established that hydrolysis of phosphates in IMPase occurs through an in–line attack of nucleophilic water, and is activated by the Mg2 and Mg3 [12,18]. The in–line attack results in an inversion of the phosphate [38,39]. Surprisingly, the orientation of the bound phosphate in the ternary product complex was found to be retained and mimic that of the unhydrolyzed phosphate group of the substrate when superimposed on the substrate-bound structure of human IMPase (PDB ID 1AWB; Fig. 6A) [40]. At 100% occupancy, the phosphate showed a B–factor compatible with the interacting metals and active-site residues; it also showed a good fit to the electron density map. This indicates firmly ordered binding of the phosphate

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Fig. 4. Overall three-dimensional structure of SaIMPase I. (A) The structure is composed of five alternative layers of a–helices and b–sheets (ababa). Pink residues in the interface of the two domains indicate the active-site metal binding residues Glu70, Asp88, Asp91 and Asp209, together with Thr93, which is involved in activation of the water nucleophile W1. (B) Structural alignment of SaIMPase I (orange) and human IMPase (blue) (PDB ID 2HHM).

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to the active site. Moreover, an additional positive difference density, which appeared to be the nucleophilic water (W1), further supports the unusual substrate-like orientation of the phosphate (Fig. 5A). The electron density of the nucleophilic water (W1) and the phos apart) at a contour phate were distinctly visible (2.7 A level of 2.8 r. To obtain a Mg2+-bound dissociable product complex, the inhibitory excess Mg2+ ions were removed from the crystal by soaking it in a reservoir solution 5314

Fig. 5. Stereodiagrams representing the active site of the high Mg2+-bound ternary product complex of SaIMPase I (SaIMPase I–3 Mg2+–PO43). (A) Fobs– Fcalc density map calculated by omitting the ligands model and contoured at the 2.8 r level. The three Mg2+ ions are referred to as Mg1, Mg2 and Mg3 (blue spheres). A distinct electron density corresponding to the nucleophilic water molecule (W1) (red sphere) is observed 2.7  A from the phosphorus atom of the phosphate ion. (B) Octahedral coordination states of the three Mg2+ ions referred to as Mg1, Mg2 and Mg3 (green spheres). Of the water molecules (shown as red spheres), W1 and W2 indicate nucleophilic water molecules involved in in–line attack for phosphate hydrolysis and protonation of the inositol oxyanion, respectively.

containing 0 mM MgCl2 for 24 h (batch A). After removal of excess MgCl2, the orientation of the bound phosphate was altered from an unhydrolyzed substrate-like orientation to a product-like orientation. The resulting post-soaking complex fully resembled the Mg2+- and phosphate-bound product complex of human IMPase (PDB ID 4AS4; Fig. 6B) [41]. Therefore, it represents the dissociable product complex that appears in the normal course of the catalytic cycle. The refinement showed a good fit of the modeled FEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

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Fig. 6. Orientation of bound phosphate with changes in the concentration of Mg2+. (A) Structural superimposition of the active sites of three Ca2+- and inositol monophosphate-bound human IMPase (yellow) (PDB ID 1AWB) with the high Mg2+-bound ternary product complex of SaIMPase I (PDB ID 4G61) (pink). The orientation of the bound phosphate of the ternary complex resembles that of the unhydrolyzed phosphate group of the substrate. The catalytic nucleophilic water (W1) is shown as a red sphere. (B) Structural superimposition of the active sites of three Mg2+- and phosphate-bound human IMPase (yellow) (PDB ID 4AS4) with the structure of the SaIMPase I dissociable product complex (batch A; PDB ID 4PTK) (pink). The orientation of the phosphate is converted to a product-like state on removal of excess MgCl2.

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phosphate into the electron density map (Fig. 7A). Simultaneously, the coordination state of Mg2 was converted from octahedral to tetrahedral in the postsoaking complex (Fig. 7B). The tetrahedral Mg2 sites comprised Asp88 OD2, Asp91 OD1, Asp209 OD1 and one oxygen atom from the phosphate moiety (Table 1). The octahedral Mg1 site comprised Glu70 OE2, Asp88 OD1, Ile90 O, two oxygen atoms from the phosphate moiety, and one water molecule. The Mg3 is coordinated to Glu70 OE1, two oxygen atoms from the phosphate moiety, and three water molecules. Moreover, superimposition of the pre- and post-soaking complexes (PDB IDs 4G61 and 4PTK, respectively) did not reveal any significant structural  for 487 Ca). Theredifferences (RMSD = 0.215 A fore, the change in orientation of the phosphate is attributed to the change in concentration of Mg2+ ions. Structure of the Li+-bound inhibitory product complex To identify the specific uncompetitive binding site of Li+ in IMPase, the crystals of three magnesium and phosphate-bound ternary product complexes of SaIMPase I were soaked in increasing concentrations of FEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

LiCl prior to X–ray data collection. As Li+ and Mg2+ share common or overlapping binding sites, LiCl soaking leads to dissociation of Mg2+ from the active site of the enzyme. Therefore, the Li+ binding site was identified by investigating the difference Fourier maps of the active site of each soaked structure. Crystals were divided into two batches (B and C). Batch B crystals were subjected to soaking with a crystallization reservoir solution containing 0.2 M MgCl2 and increasing concentrations of LiCl (50, 75 and 100 mM). Batch C crystals were soaked in a reservoir solution containing 50 mM LiCl but lacking MgCl2. It was found that the binding efficiency of the Li+ ion strongly depends on the concentration of MgCl2 present in the soaking solution. In the presence of excess MgCl2, batch B crystals required at least 100 mM LiCl for replacement of the Mg2+. No replacement of Mg2+ was observed in batch B crystals soaked in 50 and 75 mM LiCl. However, in the absence of an inhibitory excess of MgCl2, Mg2+ replacement was observed in the 50 mM LiCl-soaked structure (batch C crystals). Therefore, the soaking experiments showed complete agreement with the results of the biochemical study, such as an IC50 value of Li+ in SaIMPase I (53 mM) [22] and the mutually exclusive nature of Mg2+ and Li+ binding. No dissociation of Mg2+ was observed

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when the crystals were soaked for 24 h in a reservoir solution lacking both Mg2+ and Li+ (batch A) (Table 2). The result unambiguously verifies the presence of Li+ in the soaking solution to be responsible for Mg2+ replacement. Analysis of the difference Fourier maps clearly revealed that LiCl soaking leads to the replacement of Mg2 in both subunits in 100 mM LiCl, and in subunit B in the 50 mM LiCl-soaked structure (Fig. 8A). The 50 mM LiCl-soaked structure contains Mg2 in subunit A, albeit with a tetrahedral coordination state and an occupancy of 0.5. The occupancy of Mg2 (B– 2) was manually adjusted according factor of 13.7 A 2) of the interacting to the mean B–factor (13.65 A 2, Or2 Asp88 = ligand atoms (Or1 Asp209 = 11.7 A 2 2  and O2P = 19.1 A 2).  , Or1 Asp91 = 10.5 A 13.3 A Interestingly, the amino acid residues of the vacant Mg2 binding site in both soaked structure showed a perfect tetrahedral orientation even after replacement of Mg2+. This may indicate the presence of bound Li+ in the Mg2 binding site. The Li+ was manually modeled in a vacant Mg2 site. The tetrahedral geometry of the modeled Li+ is attributed to Asp88 OD2, Asp91 OD1 and Asp209 OD1 residues, in addition to one oxygen atom from the phosphate moiety 5316

Fig. 7. Stereodiagram of the active site of the SaIMPase I dissociable product complex (batch A; PDB ID 4PTK). (A) Fobs–Fcalc density map calculated by omitting the ligands model and contoured at the 4.0 r level. The three Mg2+ ions are referred to as Mg1, Mg2 and Mg3 (blue spheres). (B) Coordination states of the three Mg2+ ions referred to as Mg1, Mg2 and Mg3 (green spheres). The water molecules are shown as red spheres. The coordination state of Mg2 is converted to tetrahedral from octahedral in the ternary product complex.

 and a bond angle of at a bond distance of 1.9 A approximately 109° (Fig. 8B). The values for the geometric parameters were comparable with existing data for stable Li+ complexes [42]. Moreover, no significant change in the electron density of the Mg1 and Mg3 was observed in soaked structures. The conformation of the active-site residues and the coordination state of Mg1 and Mg3 remained unaltered in both soaked structures (Tables 1 and 2). The results strongly suggest that Li+ ions bind explicitly at the Mg2 site. Superimposition of the LiCl post-soaking complexes with the ternary product complex showed no significant structural difference (the RMSD of  over PDB ID 4G61 versus PDB ID 4I3Y is 0.33 A 470 Ca; the RMSD of PDB ID 4G61 versus PDB  over 495 Ca). However, modeling ID 4I40 is 0.23 A and subsequent refinements showed a product-like orientation of the phosphate ion (Fig. 8B) in both the 50 and 100 mM LiCl-soaked structures. In case of batch B, the composition of the soaking solution was completely identical with that of the reservoir solution, except for addition of 100 mM LiCl, resulting in a change in the orientation of the bound phosphate. Therefore, the efficacy of Li+ in reorientation of bound phosphate is demonstrated. FEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

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A a Fig. 8. SaIMPase I ternary product complex soaked with LiCl. (A) Fobs–Fcalc electron density map (contoured at the 3.0 r level) for the active site of (a) the 100 mM LiCl-soaked structure of SaIMPase I (batch B), (b) the ternary product complex of SaIMPase I, and (c) the 50 mM LiCl-soaked structure of SaIMPase I (batch C). The three Mg2+ binding sites are referred to as Mg1, Mg2 and Mg3 (green spheres). Mg2 dissociates upon soaking in 100 and 50 mM LiCl. A change in the orientation of the phosphate was observed on Li+ ion binding. (B) Model of the Li+ ion (purple sphere) in the vacant Mg2 binding site of the 100 mM LiCl-soaked structure of SaIMPase I. Mg2+ and water molecules are shown as green and red spheres. The binding site of Li+ is perfectly tetrahedral, and the geometric parameters match existing data for a stable Li+ complex [42].

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Discussion Inhibition by a high Mg2+ concentration is facilitated by a change in the coordination state of Mg2 The inhibition of IMPase by Mg2+ is thought to be mediated by product trapping [24,26,43]. To investigate the mode of inhibition by a high concentration of Mg2+, SaIMPase I was crystallized in the presence of substrate (NADP+) and an inhibitory concentration (0.2 M) of MgCl2. The Mg2+-bound enzyme hydrolyzed the substrate, and a ternary product complex (SaIMPase I–3 Mg2+–PO43) was obtained, which represents the inhibitory product complex speculated from the biochemical study. The stereochemistry of phosphate hydrolysis by IMPase has been thoroughly studied. Previous reports suggested that the substrate hydrolysis is mediated by in–line nucleophilic attack, which results in inversion of the phosphate [38,39]. However, the phosphate was found to retain a substrate-like orientation in the ternary product complex of SaIMPase I (Fig. 6A). This unusual phosphate orientation is achieved by in situ substrate hydrolysis during the co-crystallization process and yielded with the product phosphate at the active site. Therefore, the substrate-like orientation of the phosphate is unlikely to be a crystallographic artifact. Moreover, the presence of nucleophilic water (W1) at the Mg2 and Mg3

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sites signifies the substrate-like orientation of phosphate. By removing the inhibitory excess of MgCl2 from the crystal through soaking, the effect of a high Mg2+ concentration on the orientation of the phosphate was validated. In the absence of an inhibitory excess of MgCl2, the orientation of the bound phosphate was altered, and resembled the orientation in the previously reported product complex (Fig. 6B) [41]. The result unequivocally indicates that the unique orientation of the phosphate is a consequence of Mg2+ inhibition. Magnesium ions form both tetrahedral and octahedral complexes; however, octahedral complexes are more stable and are abundant in protein structures [44]. A crystal structure of the IMPase simultaneously bound to the cofactor Mg2+ and the substrate is difficult to obtain and is thus unavailable. However, Ca2+ is a competitive inhibitor with respect to Mg2+, and, based on the structure of a three Ca2+-bound IMPase–substrate complex [40] and the ternary product complex of SaIMPase I, it may be assumed that the Mg2 is octahedral in the substrate-bound state (Fig. 5B). Two amongst the six ligands of the octahedral Mg2 binding site are the nucleophilic water (W2) and ester-linked oxygen of the unhydrolyzed substrate. Thus, by removal of these two after hydrolysis, the coordination state of Mg2 is converted to tetrahedral in the product complex (Fig. 7B). However, even in the product complex, the Mg2 can be found in a stable octahedral state in the

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presence of a high concentration of Mg2+. The octahedral coordination state of the Mg2 has previously been identified in the high Mg2+-soaked product-bound complex of yeast 30 -phosphoadinosine-50 -phosphate phosphatase (PDB ID 1K9Y) [18]. Additionally, the octahedral coordination state of the Mg2 has also been observed in the high Mg2+-bound crystal structure of bovine IMPase (PDB ID 2BJI) [15]. The change in Mg2 coordination state from tetrahedral to octahedral in the product complex requires restoration of the donor position of the ester-linked oxygen of the substrate by acquiring a ligand from the surrounding moieties (the 30 –OH of AMP in PDB ID 1K9Y, and H2O/A 2242 and H2O/B 3223 in PDB ID 2BJI). In the SaIMPase I ternary product complex, the octahedral geometry of the Mg2 site was achieved when the phosphate mimicked the altered substrate-like orientation. Thus, high concentrations of the Mg2+ ion promote a change in the coordination state to stabilize Mg2 binding. The octahedral state of Mg2 prevents its dissociation from the product complex, and, as a consequence, retards phosphate release. Therefore, the reaction ends with generation of an E–Mg2+–PO43 dead-end inhibitory product complex. +

Li binds to the Mg2 site in uncompetitive inhibition To detect the uncompetitive Li+ binding site in IMPase, crystals of the ternary product complex (SaIMPase I–3 Mg2+–PO43) were soaked with increasing concentrations of LiCl. An Li+ soaking experiment with crystals of the fructose-1,6–bisphosphatase– 3 Tl+–fructose-1,6–bisphosphate complex has been described previously [45]. However, to the best of our knowledge, our study is the first to report an Li+ ion binding study performed using a physiologically relevant three Mg2+-bound enzyme–product complex. The soaking experiments suggest that Li+ binds specifically to the Mg2 site of SaIMPase I. The preference of Li+ for the Mg2 binding site over the Mg3 site may be explained by analyzing their coordination states. When comparing the substrate-bound complex with the product-bound complex, it was found that the Mg2 switches from the octahedral coordination state to the tetrahedral coordination state, whereas the Mg3 maintained its octahedral geometry. Li+ prefers a tetrahedral coordination geometry over an octahedral coordination geometry [42,46]. Given the coordination plasticity of the Mg2 site, Li+ prefers this site. Additionally, the hydration free energy of Li+ and Mg2+ ions are significantly different (123.5 kcalmol1 for Li+ and 455.5 kcalmol1 for Mg2+) due to a differ5318

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ence in their charge density [47]. The hydration energy released upon replacement of Mg2+ results in Li+ binding being energetically favorable. Among the six ligands of the Mg3 site, three are water molecules (Fig. 7B). The replacement of Mg3, which coordinates three water molecules in its inner sphere, is energetically less favorable than that of the Mg2 coordinating three Asp residues and a phosphate oxygen, in terms of hydration energy. Similarly, binding of Li+ to the active site of the apo-enzyme (low-affinity site) is also energetically less favorable. This explains the requirement for a higher concentration of Li+ ions in the non-competitive mode of inhibition. The discrepancy in Li+ ion sensitivity between IMPases from various organisms and the effect of activesite mobile loop mutations on Li+ inhibition [14] are difficult to explain if Li+ binding occurs at the Mg2 site. However, recent theoretical studies on IMPase showed a correlation between the Mg2+–Li+ exchange free energy and the solvent exposure of the binding site [48]. Based on this information, we propose that the solvent accessibility of the active-site groove may affect the Li+ ion sensitivity of different IMPases. Binding of Li+ in uncompetitive inhibition is a post-catalytic event The LiCl soaking study performed on the ternary product complex showed a change in the orientation of bound phosphate from an unhydrolyzed substratelike orientation to an inverted product-like orientation. The change in phosphate orientation provided an ideal tetrahedral coordination state for binding of Li+ (Fig. 8B). Therefore, it indicates a post-catalytic mode of uncompetitive Li+ binding. Moreover, by virtue of phosphate orientation, the Mg2 is octahedral in the substrate-bound complex. The octahedral coordinated state of Mg2+ is more stable than the tetrahedral state, and it is difficult to replace by Li+. Because Li+ prefers the tetrahedral coordination state over the octahedral state, the tetrahedral Mg2, which is attributed to the post-catalytic complex, would be replaced by Li+. The identification of a tetrahedral Mg2 as the intermediate state in subunit B of the 50 mM LiCl-soaked structure (batch C) further supports this hypothesis. Therefore, Li+ binds a post-catalytic complex rather than a substrate-bound pre-catalytic complex, and does not interfere with substrate hydrolysis. However, Mg2 activates W2 involved in protonation of the inositol oxyanion (Fig. 1B). On replacement of Mg2 by Li+ even after phosphoester bond cleavage, release of inositol from the active site may also be retarded. However, Leech et al. [21] found no retardation of FEBS Journal 281 (2014) 5309–5324 ª 2014 FEBS

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inositol release upon Li+ inhibition. This may be possible if the phosphoester bond cleavage and protonation of oxyanion are concomitant. We have demonstrated that Li+ binds dissociable post-catalytic product complexes, replacing the tetrahedral Mg2. However, binding of Li+ did not alter the conformation of the active-site amino acid residues. The question arises as to how Li+ inhibits the enzyme or prevents dissociation of the product complex. We propose that the stability of the post-catalytic complex is dependent on the physicochemical properties of the bound metal ion and not on the conformation of the active-site residues. The detailed biochemical and structure-based investigation suggest that the product phosphate dissociates, leaving the active site only after release of tetrahedral Mg2. The tetrahedral coordination state of Mg2+ is unstable and is less frequently observed in protein structures [44]. Thus, a tetrahedral Mg2+ can easily be dissociated from the post-catalytic complex. However, the most common and stable coordination state of Li+ is tetrahedral [42,46]. Binding of Li+ to the Mg2 site stabilizes the post-catalytic complex and prevents dissociation of the phosphate. This proposition is further supported by the fact that various structurally and functionally unrelated Mg2+-dependent phosphatases are inhibited by Li+ regardless of their amino acid sequences. In summary, we have described the mode of inhibition of Mg2+ and Li+ in SaIMPase I based on biochemical and structural investigations. The Mg2 binding site has been identified as the common binding site for both inhibitory Li+ and for Mg2+. Both Li+ and high concentrations of Mg2+ modify the Mg2 site to stabilize the product complex. The Mg2+ inhibition appears to be mediated by transformation of the less stable tetrahedral Mg2 to a stable octahedral state, whereas Li+ replaces less stable tetrahedral Mg2 and generates a stable enzyme–product complex. Moreover, changes in the relative orientation of bound phosphates during LiCl soaking suggest a post-catalytic mode of Li+ binding in uncompetitive inhibition. Based on our structural findings, it may be proposed that Li+ at a low concentration and Mg2+ at a high concentration do not interfere with the substrate hydrolysis reaction; however, they stabilize the post-catalytic complex to prevent the next round of the catalytic cycle.

Experimental procedures Protein purification and crystallization Recombinant staphylococcal inositol monophosphatase I (SaIMPase I, SAS2203 from Staphylococcus aureus

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MSSA476) was purified as described previously [49]. In brief, N–terminally hexahistidine-tagged recombinant SaIMPase I was purified by Ni–NTA affinity chromatography followed by gel filtration chromatography. The final gel filtration buffer contains 10 mM Tris/HCl pH 8.0, 50 mM NaCl, 10 mM CaCl2, 5 mM dithiothreitol. The gel filtration buffer used in protein purification for the biochemical assay contains 50 mM Tris/HCl pH 8.0, 5 mM dithiothreitol and 1 mM EDTA. The purity of the protein was verified by 12% SDS/PAGE, and the protein concentration was determined using Bradford reagent (Bio–Rad, Hercules, CA, USA) [50]. Finally, the highly purified SaIMPase I in gel filtration buffer was concentrated to 30 mgmL1 using a 10 kDa cut-off Vivaspin 20 concentrator (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and used for crystallization trials. The concentrated protein was incubated with 25 mM NADP+ (substrate) dissolved in gel filtration buffer for 1 h prior to the crystallization drop set-up experiment. The crystallization was performed with 2 lL of concentrated protein solution mixed with an equal volume of crystallization reagent, and equilibrated with 1 mL reservoir solution in a hangingdrop vapor diffusion method. After 48 h of incubation at room temperature (approximately 298K), large plateshaped crystals (0.5 mm 9 0.07 mm 9 0.1 mm) were obtained under crystallization conditions comprising 0.2 M MgCl2, 0.1 M Na/HEPES pH 8, 18% w/v poly(ethylene glycol) 3350.

Data collection and structure solution X–ray diffraction data were collected at our in–house X–ray facility, which is equipped with a Raxis IV++ image plate detector and a Micromax HF007 CuKa rotating anode generator (both from Rigaku, Woodlands, TX, USA). Prior to data collection, the crystals were quick-soaked with reservoir solution containing 10% glycerol, and then flash-cooled in a liquid N2 stream at 100 K. A full set of diffraction data with 1.0° oscillation and an exposure time of 2 min per frame were collected from a single crystal. For soaking experiments, the crystals were divided into three batches (A, B and C). Batch A crystals were soaked in reservoir solution lacking of MgCl2. The batch A data is for the dissociable product complex, which was collected after 24 h of soaking. Batch B crystals were soaked in reservoir solution containing increased concentrations of LiCl (50, 75 and 100 mM). Batch C crystals were soaked with reservoir solution containing 50 mM LiCl but lacking MgCl2. Two sets of data, after 10 min and 24 h soaking periods, were collected from 50 to 75 mM LiCl-soaked batch B crystals. A 10 min soaking period was used for 100 mM LiCl-soaked batch B crystals and 50 mM LiCl-soaked batch C crystals. All soaking experiments were performed by incubating the crystals in 2 lL soaking solution for the various time periods prior to data collection.

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angle data reported in the Cambridge Structural Database [37]. The data collection and refinement parameters are summarized in Table 3. Structures have been submitted to the Protein Data Bank (PDB IDs 4G61, 4I40, 4I3Y and 4PTK). All structural representations were prepared using PyMOL (www.pymol.org).

Diffraction data were processed using XDS [51] and scaled using SCALA [52]. The presence of screw axes was determined by observing the systematic absences, and confirmed by Pointless [53]. The phase information was obtained by the molecular replacement method using MOLREP [54], with the SaIMPase I structure (PDB ID 3QMF) as the template. The solution was obtained in the P21212 space group with a dimeric assembly of SaIMPase I in asymmetric unit. The initial model was refined by rigid body refinement using Refmac5 [55]. Iterative cycles of manual model building in Coot [56] followed by restrained refinement using Refmac5 yielded the final structural models. Identification of the ligands was achieved using difference Fourier electron density maps. In LiCl-soaked structures, Li+ was modeled in the vacant second metal binding site manually, obeying the bond distance and bond

Phosphatase assay Biochemical assay were performed by colorimetric determination of released free phosphate using an EvolutionTM 300 UV/Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) as described previously [22]. Briefly, the assay was performed in 100 lL of assay mixture containing 10 mM MgCl2 and 3 mM of substrate dissolved in 50 mM Tris/HCl buffer, pH 8. For all kinetic experiments, 20 –AMP

Table 1. Metal–ligand interactions and distances in subunit A/subunit B. Distances ( A)

Interactions Mg1–Or1 Asp88 Mg1–O Ilu90 Mg1–Oe2 Glu70 Mg1–O4P Mg1–O water401 Mg1–O water401/427 Mg1–O water401/403 Mg1–O3P/O2P Mg1–O water403/406 Mg1–O water402/406 Mg2–Or1 Asp91 Mg2–Or2 Asp88 Mg2–Or1 Asp209 Mg2–O3P Mg2–O2P Mg2–O4P Mg2–O water405/473 Mg3–Oe1 Glu70 Mg3–O1P Mg3–O2P Mg3–O4P Mg3–O water406/403 Mg3–O water407/404 Mg3–O water404/403 Mg3–O water405/402 Mg3–O water428/440 Mg3–O water430/418 Mg3–O water429 Mg3–O water401/402 Mg3–O water403 Mg3–O water402 Mg3–O water404/405 Mg3–O water406/403 Mg3–O water407/403

5320

SaIMPase I ternary complex

SaIMPase I in 0 mM MgCl2

SaIMPase I in 100 mM LiCl

SaIMPase I in 50 mM LiCl

2.0/2.0 2.0/2.0 1.9/2.0 1.8/1.9 2.1/2.3

2.0/2.0 2.1/2.3 1.9/2.2 2.2/2.3

2.0/2.0 2.1/2.0 2.05/1.9 2.3/2.2

2.1/1.9 2.15/2.1 2.0/2.1 2.2/2.4

2.2/2.4

2.2/2.2 2.2/2.3

2.4/2.2 2.35/2.6 2.3/2.3 2.3/2.2 2.1/2.0 2.2/2.3 2.0/2.1 1.9/1.9

2.4/2.0 2.3/2.0 2.1/2.0 1.6/1.7

2.3 2.0 2.0 1.7

2.5/2.1 3.1/3.0 2.1/2.2 1.8/1.9

2.05/2.25 2.35 2.2 2.6/2.5

2.2/2.2 2.0/2.1

2.1/2.3 2.4/2.2

2.2/2.4

2.85/2.4

2.2/2.4 2.1/2.1 2.0/2.0 2.2/2.1 2.3/2.5 2.1/2.0 1.8 2.3/2.4 1.7 2.1 2.2/1.9 2.2/2.0 2.2/2.05

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Table 2. Occupancy and B–factors of metal ions for subunit A/subunit B. Values for ligands are the mean B–factor of interacting amino acid ligand atoms. SaIMPase I ternary complex SIMPase I in 0 mM MgCl2 (4PTK)

(4G61) B–factor ( A2)

SaIMPase I in 50 mM LiCl (4I40)

B–factor ( A2)

SaIMPase I in 100 mM LiCl (4I3Y)

B–factor ( A2)

B–factor ( A2)

Metal

Occupancy

Metal

Ligand

Occupancy

Metal

Ligand

Mg1

1/1

15.1/19.4

11.2/12.9

1/1

18.7/25.9

17.4/19.2

1/1

13.0/15.7

1/1

20.0/12.3

12.0/15.6

Mg2

1/1

22.8/24.4

15.6/15.8

1/1

19.9/32.9

17.8/21.6

0.5/0

13.7/–

13.6/15.9

0/0

–/–

14.3/15.1

Mg3

1/1

16.6/22.2

10.8/17.2

1/1

25.6/27.3

14.9/22.2

1/1

20.4/25.4

14.0/14.8

1/1

20.9/18.0

16.4/19.4

Occupancy

Metal 8.5/16.1

Ligand

Occupancy

Metal

Ligand

Table 3. Data collection and refinement statistics.

PDB ID Space group Cell parameters a, b, c Resolution range ( A) Observed reflections Unique reflections Completeness (%) Rmerge (%)a CC1/2 Multiplicity Mean I/r(I) Solvent content Rworkb Rfreec Wilson B factor ( A2)  Mean B factor (A2) Number of atoms Amino acids Water Ligands/ions RMSD length ( A) RMSD angle (°) Ramachandran plot Favorable/allowed

SaIMPase I ternary complex

SaIMPase I in 0 mM MgCl2 (batch A)

SaIMPase I in 100 mM LiCl (batch B)

SaIMPase I in 50 mM LiCl (batch C)

4G61 P21212 60.71, 63.05, 141.62 2.3 174 142 (25 489) 24 511 (3589) 98.3 (100) 9.3 (40.8) 99.9 (93.7) 7.1 (7.1) 20.9 (5.4) 43.34 0.17 0.24 28.1 21.5

4PTK P21212 60.4, 62.4, 140.5 2.5 139 296 (19 864) 18 411 (2588) 97.7 (97.3) 14.9 (69.5) 99.6 (81.1) 7.7 (7.6) 14.8 (3.3) 43.8 0.17 0.25 25.0 27.6

4I3Y P21212 60.14, 62.82, 141.58 2.0 248 955 (35 402) 34 974 (5037) 99.9 (99.9) 10.9 (58.3) 99.8 (83.1) 7.1 (7.0) 17.5 (3.5) 44.84 0.18 0.23 20.5 22.12

4I40 P21212 59.98, 62.4, 140.64 2.5 125 653 (17 642) 18 941 (2684) 99.7 (99.3) 16.0 (58.2) 99.3 (85.8) 6.6 (6.6) 11.9 (3.5) 43.95 0.17 0.25 19.5 22.65

4184 251 76/9 0.016 1.95 90.3/9.5

4193 71 27/6 0.013 1.8 87.5/11.0

4215 218 61/4 0.017 2.1 90.1/8.8

4172 131 46/5 0.012 1.7 89.7/9.3

Rmerge = ΣhklΣi|Ii(hkl)  〈I (hkl)〉|/ΣhklΣi Ii (hkl), where Ii (hkl) is the observed intensity of the ith reflection, and is the mean value for P P all equivalent measurements of reflection hkl. b Rwork ¼ hkl jjFobs j  jFcalc jj= jFobs j; where Fobs and Fcalc are the observed and calculated c structure factors, respectively. Rfree is calculated in the same way as Rwork, based on 5% of total reflections excluded from refinement. Values in parenthesis are for the highest resolution shell.

a

was used as the substrate. The reaction mixture was incubated for 2 min at 310 K before immediately being chilled on ice, followed by addition of 100 lL of 10% trichloroacetic acid to quench the enzymatic reaction. The concentration of the released phosphate was then measured colorimetrically using ammonium molybdate/ascorbic acid reagent as described previously [57]. The absorbance of the assay mixture at 280 nm was compared to a standard curve prepared using various concentrations of Na(H2PO4) to determine the concentration of the released phosphate. A control assay lacking either enzyme or substrate was performed for each set of experiments.

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Data analysis of multiple inhibition kinetics experiments Multiple inhibition experiments with two inhibitors, a high concentration of Mg2+ versus a low concentration of Li+ and a high concentration of Mg2+ versus PO43, were performed as described previously [37]. The effect of the two inhibitors on the velocity of the enzymatic reaction was analyzed using the following equation: 1=VIJ ¼ 1=V0 ½1 þ Km =½S þ ½I=KI þ ð1 þ ½I=bKI Þ½J=KJ  ð1Þ

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where VIJ is the velocity of the reaction in the presence of two inhibitors (I, J), V0 is the velocity of the reaction in the absence of inhibitors, [I] and [J] are the concentrations of the two inhibitors I and J, respectively, KI and KJ are the inhibition constants for inhibitors I and J, respectively, and b is a constant that accounts for any interaction between two inhibitors. The Km is the Michaelis–Menten constant of the substrate, and [S] is the substrate concentration.

7

8

9

Acknowledgements This work was performed with financial assistance (sanction order number SR/SO/BB-065/2008) from the Department of Science and Technology, Government of India. We thank H.G. Wiker (Department of Clinical Science, University of Bergen and Haukeland University Hospital, Bergen, Norway) for providing the genomic DNA of Staphylococcus aureus MSSA476. We gratefully acknowledge the Department of Biotechnology, Government of India, for establishing the macromolecular crystallography facility at the Indian Institute of Technology, Kharagpur, India. S.B and A.D thank the Indian Institute of Technology for providing individual fellowships.

10

11

12

13

Author contributions AD performed the experiments, analyzed data and contributed to writing the manuscript. SB planned and performed experiments and analyzed data. DD helped with crystal structure determination. AKD initiated the project and wrote the final manuscript.

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