Báo cáo khóa học: How calcium inhibits the magnesium-dependent enzyme human phosphoserine phosphatase potx

How calcium inhibits the magnesium-dependent enzyme human phosphoserine phosphatase Yves Peeraer 1 , Anja Rabijns 1 , Jean-Franc¸ois Collet 2 , Emile Van Schaftingen 2 and Camiel De Ranter 1 1 Laboratory for Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, K.U. Leuven, Leuven, Belgium; 2 Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, Universite ´ Catholique de Louvain, Brussels, Belgium The structure of the Mg 2+ -dependent enzyme human phosphoserine phosphatase (HPSP) was exploited to examine the structural and functional role of the divalent cation in the a ctive site of phosphatases. Most interesting is the biochemical observation that a Ca 2+ ion inhibits the activity of HPSP, even in the presence of added Mg 2+ .The sixfold coordinated Mg 2+ ion present in the a ctive site of HPSP under normal physiological conditions, was replaced by a Ca 2+ ion by using a crystallization condition with high concentration of CaCl 2 (0.7 M ). The r esulting HPSP struc- ture now shows a sevenfold coordinated Ca 2+ ioninthe active site that might e xplain the inhibitory effect of Ca 2+ on the enzyme. Indeed, the Ca 2+ ion in the active site captures both side-chain oxygen atoms of the catalytic Asp20 as a ligand, while a Mg 2+ ion ligates only one oxygen atom of this Asp residue. The bidentate character of Asp20 towards Ca 2+ hampers the nucleophilic attack of one of the Asp20 side chain oxygen atoms on the phosphorus atom of the substra te phosph oserine . Keywords: calcium; HAD superfamily; magnesium-depend- ent enzymes; phosphoserine phosphatase; L -serine. Human phosphoserine phosphatase (HPSP) catalyses the last and irreversible step of the de novo biosynthesis of L -serine, i.e. the hydrolysis of phosphoserine l eading to the formation of L -serine and inorganic phosphate (Pi). HPSP is a member of the haloacid dehalogenase (HAD) superfamily of which the members are characterized by three short conserved sequence motifs (Fig. 1). The residues of these motifs cluster t ogether t o f orm t he active site. All enzymes o f the HAD superfamily use the aspartate r esidue of the first conserved DXXX(T/V) motif as a nucleophilic residue for catalysis [1]. T he second motif contains a conserved serine o r threonine residue, and the third motif contains a strictly conserved lysine residue followed, at some distance, by less conserved residues and a strictly conserved aspartate. Mutagenesis studies on these conserved residues show that all t hree motifs play an important role in the catalytic process [2–4]. Despite the low overall sequence homology among the enzymes of the HAD superfamily, all known structures o f enzymes of this superfamily display a conserved fold [5]. Indeed, 2-haloacid dehalogenase f rom Pseudomonas sp. YL and Xanthobacter autotrophicus [6,7], phosphonoacetalde- hyde hydrolase from Bacillus cereus [8], soluble epoxide hydrolase [9], the Ca 2+ -P-type ATPase [10], b-phospho- glucomutase from Lactococ cus lacti s [11], phosphoserine phosphatase (PSP) from Methanococcus j annaschii (MJ PSP) [12,13] and HPSP [14,15] all have a core a/b domain resembling the NAD(P)-binding Rossmann fold [5]. This fold is characterized by a central six-stranded b-sh eet flanked on both sides by two or three a-helices. The similar topology and common fold of the central domain, strongly suggest that the members of t he HAD superfamily evolved from a primordial, generic domain. Metals are found in a broad variety of proteins where they display important functional or structural roles. A bound Mg 2+ ion is an essential active site component of numerous metalloproteins including nucleases, kinases and phosphatases. Such proteins use Mg 2+ for phospho- substrate binding, catalysis, or both. The HAD superfamily members, except for 2-haloacid dehalogenases [6,7], utilize Mg 2+ as a cofactor during catalysis. The effects of various metal cations on the activity of PSP were described [16], but key features of their metal binding characteristics remained undetermined. Maximum activity of the enzyme, measured by the rate of Pi release from phosphoserine, is obtained with Mg 2+ . In the absence of added divalent cations, the activity of PSP is only 9–15% of the maximal a ctivity observed in the pr esence of Mg 2+ . Of p articular i nterest was our observation that the replacement of Mg 2+ by Ca 2+ in an activity test caused complete loss of activity of PSP. Furthermore Ca 2+ inhibited the activity measured in the presence of Mg 2+ . Two interesting questions arise from these observations: is there structural evidence for the fact that Mg 2+ in the active site cannot be replaced by another divalent cation without loss of activity, and how does an enzyme manage to select a specific cation from the surrounding fluids that contain a broad variety of cations? AdetailedstudyofthestructureoftheactivesiteofHPSP with Ca 2+ bound may provide an insight into the biological Correspondence to A. Rabijns, Laboratory for Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, K.U. Leuven, E. Van Evenstraat 4, B-3000 Leuven, Belgium. Fax:+3216323469,Tel.:+3216323421, E-mail: anja.rabijns@pharm.kuleuven.ac.be Abbreviations: HAD, haloacid dehalogenase; HPSP, human phos- phoserine phosphatase; Pi, inorganic phosphate; PSP, phosphoserine phosphatase. (Received 19 May 2004, revised 1 July 2004, accepted 7 July 2004) Eur. J. Biochem. 271, 3421–3427 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04277.x role of metal ions, especially divalent metal ions, in biological processes. Materials and methods Structure determination The expression, purification and crystallization of HPSP was carried out using methods described previously [17]. The C a 2+ containing crystal structure elucidated to a resolution of 1.53 A ˚ has been described elsewhere. The structure was deposited in the Protein Data Bank (code 1NNL; http://www.rcsb.org) [15]. The final HPSP model consists of 3203 protein atoms, 390 water molecules, six Cl – and three Ca 2+ ions and a summary of the crystallographic quality indicators for this final model is given in Table 1. HPSP activity assay After purification, HPSP was assayed at 3 0 °Cbythe release of Pi from unlabeled L -phosphoserine in an assay mixture (250 lL) containing 25 m M Mes (pH 6.5), 5 m M MgCl 2 ,1m M dithiothreitol, 5 m ML -phosphoserine, 0.1 m gÆmL )1 bovine serum albumin and 1–10 mU HPSP. Reactions were stopped by the addition of 250 lLof10% (v/v) trichloroacetic acid and the amount of Pi was measured using a spectrophotometer [18]. One unit o f enzyme is the amount that catalyses the conversion of 1 lmol of substrate per minute under these conditions. To address the effects o f Ca 2+ on the H PSP activity, HPSP was incubated with different concentrations of Mg 2+ (0.2, 1, 2, 5m M ) and all these set-ups were assayed in the presence of increasing concentrations of Ca 2+ (0.00, 0.025, 0.05, 0.10, 0.25 and 1.0 m M ). Results and Discussion Presence of Ca 2+ in the HPSP active site During the refinement of the HPSP model it b ecame clear that the electron density peak in the active site could best be explained by a Ca 2+ ion. The plausible reason for the presence of the Ca 2+ in the active site, instead of a Mg 2+ ion as in previously reported structures of the PSP family, is that we used CaCl 2 in the HPSP crystallization condition; at all times the presence of Mg 2+ was avoided. The two HPSP molecules in the asymmetric unit (Molecule A and B) were refined independently of each other. In both molecules, the atoms surrounding the divalent cation and the metal ion itself, were in the same range of B factor values (around 15 A ˚ 2 ); replacing the Ca 2+ ion by another ion (e.g. Mg 2+ ) causes the R factor to increase substantially (0.7%) during the refinement procedure, as commented by Peeraer et al. [15]. The presence of a Ca 2+ ion is further confirmed by the geometry and the metal-donor atom target distances (Table 2). Role of the divalent cation in the reaction mechanism of HPSP To understand the bio logical role of Mg 2+ in the catalytic mechanism of PSP, one should k eep in mind that the hydrolysis of phosphoserine by PSP proceeds through a stepwise phosphotransfer mechanism, as demonstrated by Table 1. Data collection, refinement and model statistics for the HPSP structure a t 1.53 A ˚ resolution. V alues in parentheses i nd icate data in the highest resolution shell, i.e. 1.56–1.53 A ˚ . Data collection statistics Resolution limit (A ˚ ) 1.53 (1.56–1.53) Completeness of all data (%) 99.8 (98.7) Completeness of the data I > 2r (%) 95.2 (80.7) Mean I/r 15.0 (3.1) R merge (%) 3.5 (18.7) R measure (%) 3.8 (21.9) Refinement statistics Final R work (%) 21.6 Final R free (%) 23.4 Model statistics Average atomic B factors (A ˚ 2 ) Main chain 24.6 Side chain 27.3 Water molecules 34.4 Ca 2+ atoms 17.6 Cl – atoms 23.8 rmsd of the model Bond lengths (A ˚ ) 0.005 Bond angles (°) 1.144 B, bonded main chain (A ˚ 2 ) 1.194 B, bonded side chain (A ˚ 2 ) 2.077 Fig. 1. Multiple sequence alignment of the members of the HAD superfamily. The first column indicates the protein and the species it comes from. PSP, phosphoserine phosphatase; PMM, phosphomannomutase; H AD, haloacid dehalogenase; ATP, ATPase (Human, Homo sapiens ;Meth, Methanococcus jannaschii;Sacc,Saccharomyces cerevisiae; Coli, Escherichia coli; Pssp, Pseudomonas sp.). Numbers indicate the d istances to the ends of each prot ein and numbers in parentheses indicate the sizes of the gaps between the aligned segments. The h ighlighted amino acids are conserved in the HAD superfamily. 3422 Y. Peeraer et al.(Eur. J. Biochem. 271) Ó FEBS 2004 mechanistic studies on MJ PSP [19]. Structural comparison between HPSP (PDB code 1NNL) and MJ PSP (PDB code 1L7O) structures ( rmsd 1.64 A ˚ for 176 residues super- imposedandrmsd0.64A ˚ for 16 active site residues superimposed), reveals that the reaction mechanism o f HPSP involves subsequent nucleophilic attacks and acid/ base catalysis. The conserved residues Arg65 and Glu29 play an essential role in o rientating the substrate in an appropriate manner for hydrolysis. The side-chain of Glu29 interacts with the amino group of phosphoserine, while the side-chain of Arg65 forms a hydrogen bond with the carboxyl group of the s ubstrate. When the substrate is positioned correctly, the enzyme closes and Asp20 performs a nucleophilic attack on the scissile phosphate. The substrate phosphoserine is then cleaved, resulting in the departure of the leaving group serine and the formation of a covalent phosphoaspartyl (Asp20) intermediate (Fig. 2 ). Asp22 serves as a general acid (Fig. 2, Enz-H) donating a proton to serine and thereby facilitating the expulsion of the leaving group. A water molecule takes the position in the just-vacated leaving group site. The Asp22 carboxylate anion (Fig. 2, Enz-B) that was formed during t he protona- tion of the leaving serine group, can now serve as a base catalyst in the dephosphorylation of the phosphoenzyme intermediate. Asp22 extracts a p roton from the water molecule in the active site, thereby activating the water molecule to perform a nucleophilic attack on the phospho- aspartyl intermediate. Opening of the enzyme and dissoci- ation of the inorganic phosphate completes the catalytic cycle. The Mg 2+ ion in the active site is essential for HPSP to perform the hydrolysis of phosphoserine. First of all Mg 2+ plays a catalytic role in the reaction mechanism. The Mg 2+ ion coordinates both an oxygen atom of the phosphate moiety of the s ubstrate a nd an oxygen a tom o f the attackin g Asp20 residue. In this way the Asp20 residue is stabilized in an optimal position t o p erform an attack on the phosphorus atom of phosphoserine. In addition, the positive charge of the divalent cation is essential to facilitate the nucleophilic attack of Asp20 by extracting negative charge from the phosphate group. The fact that haloacid dehalogenases do not need a divalent cation for activity, while the phospho- transferases of the same HAD superfamily do, supports the idea that a divalent c ation in HPSP i s needed to shield the negative charges of the phosphate group while the attacking nucleophile Asp20 is approaching. Of interest is that in haloacid dehalogenase, the corresponding attacking Asp residue approaches an electropositive carbon centre of the substrate and thus a cation is not required to promote the nucleophilic attack. Besides its catalytic role, the divalent cation in the HPSP active site also plays a purely structural role. In the H PSP active site, three Asp residues (20, 2 2 and 179) are in close proximity to each other and form a carboxylate cluster, thereby generating an excess of negative charge in the binding pocket. The positive charge of the divalent cation is therefore necessary to stabilize the overall architecture of this carboxylate cluster by diminishing the electrostatic repulsion between the negative charges of the Asp side- chains. The stabilizing, structural role of Mg 2+ is further illustrated b y the fact that Asp179, which belongs to sequence motif III and which coordinates the divalent cation in the active site, is conserved in all the HAD superfamily members w ith t he exception of the enzymes that are Mg 2+ -independent for their activity. Indeed, in the haloacid dehalogenases, the corresponding residue is a Ser which is not essential for catalytic activity [7]. This observation suggests that Asp179 is essential for binding of the divalent cation in the active site. Furthermore, mutagenesis studies on HPSP showed that mutation of Asp179 to an Asn or Glu results in a 10-fold decrease in the affinity for Mg 2+ [4]. The same functions for the Mg 2+ ion are observed in other Mg 2+ -dependent members of the HAD superfamily like phosphonoacetaldehyde hydrolase, b-pho sphoglucomutase and P-type ATPases [8,11,20]. Mg 2+ substituted by a Ca 2+ : implications for the reaction mechanism Neuhaus & Byrne [16] reported that HPSP activity depends on the presence of Mg 2+ . We confirmed this requirement and we d etermined t hat t he K a for Mg 2+ in the p resence o f a saturating concentration of substrate was 0.2 m M (not shown). From Fig. 3 it can be seen that Ca 2+ inhibited the enzyme activity, and the lower the Mg 2+ concentration the more apparent this effect was. Indeed, a 50% inhibition was observed a t 0.01, 0.025, 0.05 and 0.2 m M Ca 2+ in the presence of 0.2, 1, 2 and 5 m M Mg 2+ . Several experiments to also obtain a Mg 2+ -containing HPSP structure, i.e. soaking and cocrystallization experi- ments, failed. Therefore, to elucidate the inhibitory effect exerted by Ca 2+ on the activity of PSP, w e compared the active site of HPSP, which contains a Ca 2+ ion, with the M J PSP active site, con taining Mg 2+ (PDB codes 1F5S, 1L7P Table 2. Distances between the Ca 2+ ion and the neighbouring atoms in the active site for molecules A and B of the asymmetric unit of the HPSP structure. Th e typical metal-donor atom target distances for Ca 2+ and Mg 2+ are also given [24]. This s hows that the observed distances in the HPSP structure correspond to typical Ca 2+ distances. Ligating atom Ca 2+ (Mol A) (A ˚ ) Ca 2+ (Mol B) (A ˚ ) Target distance (A ˚ ) for Ca 2+ ion Target distance (A ˚ ) for Mg 2+ ion Asp20 OD1 2.37 2.38 2.36 2.26 Asp22 O 2.31 2.29 2.36 2.26 Asp179 OD2 2.30 2.33 2.36 2.26 H 2 O 2.33 2.30 2.39 2.07 H 2 O 2.44 2.49 2.39 2.07 H 2 O 2.37 2.41 2.39 2.07 Ó FEBS 2004 Inhibition of human phosphoserine phosphatase (Eur. J. Biochem. 271) 3423 and 1J97). The Mg 2+ ion in the MJ PSP active site displays almost perfect octahedral coordination geometry with six ligands. Four ligands are in a plane with O–Mg 2+ –O angles of nearly 90°, while the two other ligands are above and below this plane, r espectively. The c oordination of the Ca 2+ ion in the active site of HPSP is distorted from octahedral geometry as shown in Fig. 4. In open co nformation, three water molecules and three O atoms (OD1 of Asp20, the main-chain carbonyl group of Asp22 and OD2 of Asp179) occupy six of the coordination sites of the Ca 2+ , similar to the Mg 2+ ion in the MJ PSP active site. Nevertheless, it can be seen that one water molecule (Fig. 4B, Wat1) i s forced out of the plane. This distortion of the octahedral geometry is due to the fact that the Ca 2+ ion prefers seven ligands instead of six as Mg 2+ does. Because the coordination of spherical metal ions is optimized by maximum packing of ligand atoms, the preferred coordination number is p rimar- ily a function of the size of the ion [21]. The effective ionic radius of a Mg 2+ ion (0.72 A ˚ ) is considerably smaller than that of a Ca 2+ ion (1.06 A ˚ ) [22]. The smaller size of Mg 2+ determines its preference for a coordination number of six. In contrast, the effective ionic radius of Ca 2+ is s uch that seven or e ight coordinating ligands can be comfortably accommodated [23]. As a result the Ca 2+ ionintheHPSP active site accepts both side-chain oxygen atoms of Asp20 as a ligand, while a Mg 2+ ion ligates only one oxygen atom of this Asp residue. Besides the differences in geometry between Ca 2+ and Mg 2+ in the active site, the metal–ligand distances are also quite different. Comparison of the active sites of HPSP and MJ PSP shows t hat replacement of a Mg 2+ by aCa 2+ ion results in an increase in all metal–ligand Fig. 2. General scheme of the reaction cycle of PSP [22]. Open conformation of PSP (A). L -Phosphoserine binds to the active site presenting the phosphate group to Asp20 (B). Transition state with nucleophylic attack of Asp20 (C). Covalent phospho aspartyl enzyme i ntermediat e (D). Transition state with a nucleophylic attack of a water molecule cau sing the de phosphorylation of Asp20 (E). Phosphate noncovalently bo und in the active site (F). Enz-H indicates the general acid Asp22, which after the protonation of the leaving serine group serves as a base catalyst Enz-B. Fig. 3. Effect of C a 2+ on HPSP activity. Th e eff ect o f Ca 2+ on HPSP activity was assayed i n the pres ence of 0.2 (j), 1 ( m), 2 ( .)or5(r)m M Mg 2+ . HPSP activity was assayed as in Materials and methods. 3424 Y. Peeraer et al.(Eur. J. Biochem. 271) Ó FEBS 2004 distances, with average distances of 2.1 A ˚ for Mg 2+ and 2.4 A ˚ for Ca 2+ . The observed distances match to a large extent the ideal distances for Ca 2+ -donor atom combina- tions and similar distances are observed i n various metalloproteins [24]. Combination of the dissimilar geo- metry and changed metal–ligand distances drastically affects the reaction mechanism of HPSP when the Mg 2+ ion is substituted by a Ca 2+ ion (Fig. 5). Upon substrate binding, on e of the three water m olecules coordinating the divalent ion is replaced by an oxygen of the phosphate moiety of phosphoserine. The fact that Asp20 in HPSP acts as a bidentate ligand in the sevenfold coordination of Ca 2+ (OD1 and OD2 to Ca 2+ distances of 2.38 and 2.77 A ˚ , respectively), while the corresponding Asp11 in MJ PSP is a monodentate ligand in the sixfold Mg 2+ coordination, hampers th e nucleophilic attack of OD1 on the substrate. The corresponding OD1 of Asp11 in the Mg 2+ bound MJ PSP is 3.33 A ˚ away from the cation and therefore it is free to perf orm an attack on the phosphorus atom of the substrate. The distance between Fig. 4. Detailed o verview of the Ca 2+ + ion i n the active site of HPSP. (A) The residues are represented in ball and stick form with oxyge n, carbon a nd nitrogen atoms coloured red, light-blue and dark-blue, respectively. The Ca 2+ ion is shown in green. Thre e of t he Ca 2+ ligands ar e water molecules, shown as red balls. The dashed lines represent hydrogen bonds and metal–ligand interactions. Asp20, Asp22 and Asp179 directly coordinate the Ca 2+ ion. Asp179 and Gly180 interact with Asp183, thereby stabilizing the loop on which they are located. (B) The coordination of the Ca 2+ is distorted from ideal oct ahedral geometry with six l igands because it forms an extra interaction with one of the oxygen atoms of Asp20. This extra interaction between Ca 2+ and Asp20, shown in green, does not occur with a Mg 2+ ion in the active site. Fig. 5. Active s i te of M J PSP w ith a M g 2+ + and pho sphoserine in the active site (PDB codes 1F5S and 1L7P) (A) and HPSP (PDB c ode 1N NL) with a Ca 2+ ion bound and the modelled substrate in the active site ( B). For clarity only four ligands are shown, i.e . a ligating water mol ecule and Asp13/22 (HPSP/MJ PSP) are omitted i n this fi gure. In c ontrast to a Mg 2+ ion, the Ca 2+ ion i n HPSP liga tes both oxy gen atoms o f Asp20 there by preventing it to perform a nucleophilic attack on the phosphorus atom of the substrate. In addition, a C a 2+ ion displays longer metal–ligand d istances than a Mg 2+ ion. As a c onsequ ence the partial p o sitive charge o n t he ph osphorus ato m o f p hosphoserine i s sm aller if a Ca 2+ takes position i n the active site. In this manner, a Ca 2+ will further hamper the nucleophilic attack of the catalytic Asp residue on the substrate. Ó FEBS 2004 Inhibition of human phosphoserine phosphatase (Eur. J. Biochem. 271) 3425 the OD1 of the attacking Asp r esidue and the phos- phorus atom of the substrate is increased from 2.96 A ˚ with a Mg 2+ ionto3.36A ˚ with a Ca 2+ ,further hampering the nucleophilic attack on the substrate. In MJ PSP the distance between oxygen O2 of the phosphate part of L -phosphoserine and the Mg 2+ ion is 2.41 A ˚ [19]. Replacing the Mg 2+ ion by a Ca 2+ ion results in an increase of this distance to 3.20 A ˚ . This will undoubtedly result in a smaller attraction of negative charge from the phosphate moiety of the substrate, thereby suppressing the nucleophilic attack of Asp20 on the phosphorus atom. HPSP selectivity for Mg 2+ For the Mg 2+ binding site of the related CheY enzyme [25,26], it was proposed that the c arboxylate c luster in the active site provides charge specificity to the Mg 2+ binding site by excluding monovalent cations like Na + and K + , because they do not possess sufficient positive charge to stabilize the highly negative carboxylate cluster [27]. Ana- logously, HPSP can exploit the negative charge of the carboxylate cluster, composed of Asp20, Asp22 a nd Asp179, to provide the necessary charge specificity by excluding monovalent cations. On the other hand, it seems that t he Mg 2+ binding site in HPSP is weakly protected against the binding of other divalent cations like Ca 2+ ,asCa 2+ displays inhibiting properties even in the presence of Mg 2+ ([16] and this paper). The weak size-selectivity of HPSP can originate from the f act that in HPSP in open conformation three of the ligands to the divalent cation are water m olecules [15]. An interesting feature of this coordination structure is that one hemisphere of the bound ion is coordinated by three protein oxygens, while the other hemisphere is coordina- ted by three solvent molecules (Fig. 4). These water molecules can easily accommodate changing metal–ligand distances if Mg 2+ is replaced by a larger divalent cation such as Ca 2+ . In addition, the larger Ca 2+ ion can employ Asp20 as a bidendate ligand in o rder to complete its preferred sevenfold coordination geometry. Thus, the difference in ionic radii of Ca 2+ and Mg 2+ is not a sufficient criterion for HPSP to select Mg 2+ ,asthe binding cavity of the enzyme is flexible and able to adjust easily to different ionic radii and changing coordination geometry. In view of the facts outlined above i t becomes clear that the metal-binding pocket of HPSP is charge-selective in order to discriminate between mono- and divalent c ations, but not size-selective enough to single out particular divalent cations as Mg 2+ and Ca 2+ . Nevertheless, in living cells HPSP uses Mg 2+ as a cofactor and not the larger Ca 2+ . Thelatterseemslogical,asMg 2+ is the most abundant divalent cation in eukaryotic cells, with concentrations of free Mg 2+ ranging from 0.1 to 1.0 m M , while the Ca 2+ concentration is 10 4 -fold lower in resting eukaryotic cells [28]. Thus, HPSP has chosen Mg 2+ as a cofactor during evolution based mainly on its n atural abundance in living cells. In this scenario it is not the protein metal-binding pocket architecture itself but the cell homeostasis that controls the process of metal binding by regulating the appropriate concentrations of Mg 2+ and other cations in various biological compartments. Conclusions The HPSP reaction mechanism involves nucleophilic attack of Asp20 on the substrate with a cid/base catalysis mediated by Asp22. The Mg 2+ ion in the active site is essential for normal enzymatic activity, i.e. the Mg 2+ ion promotes the nucleophilic attack of Asp20 by withdrawing negative charge from the phosphorus atom of the substrate. In addition, the divalent cation is essential f or the correct orientation of the attacking Asp20 residue towards the substrate. A Ca 2+ ion h owever, employs Asp20 as a bidentate ligand, thereby inhibiting the nucleophilic attack of this catalytic residue. Furthermore, it seems that the Mg 2+ binding site in HPSP is weakly protected against the binding of other divalent cations, as Ca 2+ displays inhib- iting properties even in t he presence of M g 2+ . Therefore it is probable that HPSP h as chosen Mg 2+ as a cofactor during evolution based mainly on the natural abundance of Mg 2+ in living cells. Acknowledgements A.R. is a Postdoctoral Research Fellow of the Fund for Scientific Research-Flanders (Belgium) and J F.C. w as Charge ´ de R echerches of the Belgian FNRS. Work in the lab of E.V.S. is supported by the Interuniversity A ttraction Poles Program-Belgian Science Polic y and b y the FRSM. We thank the beam line scientists at DESY for technical support and the European Union for support of the work at EMBL Hamburg through the Access to Research Infra structure Action of the improving human potential programme, contact no. HPRI-CT-1999- 00017. References 1. Collet, J.F., Stroobant, V., Pirard, M., Delpierre, G. & Van Schaftingen, E. (1998) A new class of phosphotransferases phos- phorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. J. Biol. Chem. 273, 14107–14112. 2.MacLennan,D.H.,Clarke,D.M.,Loo,T.W.&Skerjanc,I.S. (1992) Site-directed m utagenesis of the Ca 2+ ATPase of sarco- plasmic reticulum. Acta Physiol. Scand. Suppl. 607, 141–150. 3. Lingrel, J.B. & Kuntzweiler, T. (1994) Na + ,K + -ATPase. J. Biol. Chem. 269, 19659–19662. 4.Collet,J.F.,Stroobant,V.&VanSchaftingen,E.(1999) Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases. J. Biol. Chem. 274, 33985–33990. 5. Rossmann, M.G., M oras, D . & Olsen, K.W. (1974) Chemical and biological evolution of a nucleotide-binding protein. Nature 250, 194–199. 6. Hisano, T., Hata, Y., Fujii, T., Liu, J.Q., Kurihara, T., Esaki, N. & Soda, K. (1996) Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp. YL. J. Biol. Chem. 271, 20322–20330. 7. Ridder, I.S. & Dijkstra, B.W. (1999) Identification of the Mg 2+ - binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator CheY. Biochem. J. 339, 223– 226. 8. Morais, M.C., Zhang, W., Baker, A.S., Zhang, G., Dunaway- Mariano, D. & A llen, K.N. (2000) The crystal structure of Bacillus cereus phosphonoacetald ehyd e hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily. Biochemistry 39, 10385–10396. 9. Argiriadi, M.A., Morisseau, C., Hammock, B.D. & Christianson, D.W. (1999) Detoxification of environmental mutagens and 3426 Y. Peeraer et al.(Eur. J. Biochem. 271) Ó FEBS 2004 carcinogens: structure, mechanism and evolution of liver epoxide hydrolase. Proc. Natl Acad. Sci. USA 96, 10637–10642. 10. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A ˚ resolution. Nature 405, 647–655. 11. Lahiri, S.D., Zhang, G., Dunaway-Mariano, D. & Allen, K.N. (2002) Caught in the act: the structure of phosphorylated b-pho sphoglu comutase from Lactococcus lactis. Biochemistry 41 , 8351–8359. 12. Cho, H., Wang, W., Kim, R., Yokota, H., Damo, S., Kim, S.H., Wemmer, D., Kustu, S. & Yan, D. (2001) BeF À 3 acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF À 3 complex with phosphoserine phosphatase. Proc.NatlAcad.Sci.USA98, 8525–8530. 13. Wang, W., Kim, R., Jancarik, J., Yokota, H. & Kim, S.H. (2001) Crystal structure of phosphoserine phosphatase from Methano- coccus jannaschii, a hyperthermophile, at 1.8 A ˚ resolution. Struc- ture 9, 65–71. 14. Kim, H.Y., Heo, Y .S., Kim, J.H., Park, M.H., M oon, J., Kwan, D., Yoon , J., Shin, D., Jeong, E., Park, S.Y., Lee, T.G., Jeon, Y.H., Ro, S., Cho, J.M. & Hwang, K.Y. (2002) Molecular basis for the local conformatio nal rearrangem ent of hum an pho spho- serine phosphatase. J. Biol. Chem. 227, 46651–46658. 15. Peeraer,Y.,Rabijns,A.,Verboven,C.,Collet,J.F.,VanSchaf- tingen, E . & De Ranter, C. (2003) High resolution structure of human p hosp hoserine phosphatase in open conformation. Acta Cryst. D59, 971–977. 16. Neuhaus, F.C. & Byrne, W.L. (1959) Metabolism o f phospho- serine. II. Purification and properties of O-phosphoserine phos- phatase. J. Biol. Chem. 234, 113–121. 17. Peeraer,Y.,Rabijns,A.,Verboven,C.,Collet,J.F.,VanSchaf- tingen, E. & De Ranter, C. (2002) P urification, crystallization a nd preliminary X-ray an alysis of human ph osphoserine p hosp hatase. Acta Cryst. D58, 133–134. 18. Fiske, C.H. & Subbarow, Y.P. (1925) The colorimetric determi- nation of phosphorus. J. Biol. Chem. 66, 375–400. 19. Wang,W.,Cho,H.,Kim,R.,Jancarik,J.,Yokota,H.,Nguyen, H.H., Grigoriev, I.V., Wemmer, D.E. & Kim, S.H. (2002) Struc- tural characterization of the reaction pathway in phosphoserine phosphatase: crystallographic ‘‘snapshots’’ of intermediate states. J. Mol. Biol. 319, 421–431. 20. Stokes, D.L. & Green, N.M. (2003) Structure and function of the calcium pump. Annu. Rev. Biophys. Biomol. Struct. 32, 445–468. 21. Yang, W., Lee, H.W., Hellinga, H. & Yang, J.J. (2002) Structural analysis, identification and design of calcium-bind ing sites in proteins. Proteins: Stuct. Func. Genet. 47, 344–356. 22. Shannon, R.D. (1976) Revised effective ionic radii and systematic studies of interatomic d istances in halides and chalcogenides. Acta Cryst. A36, 751–767. 23. Falke, J.J., Drake, S.K., Hazard, A.L. & Peersen, O.B. (1994) Molecular tuning of ion binding to calcium signaling proteins. Q Rev. Biophys. 27, 219–290. 24. Harding, M.M. (2001) Geometry of metal-ligand interactions in proteins. Acta Cryst. D57, 401–411. 25. Bellsollel, L., Prieto, J., Serr ano, L. & Coll, M. (1994) M agnesium binding to the bacterial chemotaxis protein CheY results in large conformational changes involving its functional surface. J. M ol. Biol. 238, 489–495. 26. Ridder, I.S., Rozeboom, H.J., Kalk, K.H., Janssen, D.B. & Dijkstra, B .W. (1997) Three-dim ensi onal structure o f L -2-haloacid dehalogenase from Xanthobacter autotrophicum GJ10 complexed with the substrate analogue formate. J. Biol. Chem. 272, 33015– 33022. 27. Needham, J.V., C hen, T.Y. & Falk e, J.J. (1993) Novel ion speci- ficity of a carboxylate cluster Mg(II) binding site: strong charge selectivity and weak size selectivity. Biochemistry 32, 3363–3367. 28. Romani, A. & Scarpa, A. (1992) Regulation of cell magnesium. Arch. Biochem. Biophys. 298, 1–12. Ó FEBS 2004 Inhibition of human phosphoserine phosphatase (Eur. J. Biochem. 271) 3427 . How calcium inhibits the magnesium-dependent enzyme human phosphoserine phosphatase Yves Peeraer 1 , Anja Rabijns 1 ,. Belgium The structure of the Mg 2+ -dependent enzyme human phosphoserine phosphatase (HPSP) was exploited to examine the structural and functional role of the