Tài liệu Báo cáo khoa học: Active and regulatory sites of cytosolic 5¢-nucleotidase doc

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Tài liệu Báo cáo khoa học: Active and regulatory sites of cytosolic 5¢-nucleotidase doc

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Active and regulatory sites of cytosolic 5¢-nucleotidase Rossana Pesi 1 , Simone Allegrini 2 , Maria Giovanna Careddu 1,2 , Daniela Nicole Filoni 1 , Marcella Camici 1 and Maria Grazia Tozzi 1 1 Dipartimento di Biologia, Unita ` di Biochimica, Universita ` di Pisa, Pisa, Italy 2 Dipartimento di Scienze del Farmaco, Universita ` di Sassari, Sassari, Italy Introduction Cytosolic 5’-nucleotidase (cN-II) is a ubiquitous enzyme that catalyses either the hydrolysis or the transfer of phosphate esterified in the 5¢ position of 6-hydroxypu- rine monophosphate nucleosides [1]. The transfer of phosphate can lead to phosphorylation of inosine, guanosine and a number of their analogues [2]. There- fore, in addition to being involved in regulation of purine intracellular pool, the enzyme is also responsible Keywords cN-II active site; cN-II regulatory sites; cN-II structure; cytosolic 5¢-nucleotidase II Correspondence M. G. Tozzi, Dipartimento di Biologia, Via S. Zeno 51, Pisa, Italy Fax: +39 502211450 Tel: +39 502211457 E-mail: mtozzi@biologia.unipi.it (Received 19 July 2010, revised 10 September 2010, accepted 21 September 2010) doi:10.1111/j.1742-4658.2010.07891.x Cytosolic 5¢-nucleotidase (cN-II), which acts preferentially on 6-hydroxypu- rine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho- intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap 4 A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho- intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap 4 A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experi- ments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. Structured digital abstract l MINT-8011572: cN-II (uniprotkb:O46411) and cN-II (uniprotkb:O46411) bind (MI:0407)by dynamic light scattering ( MI:0038) l MINT-8011493, MINT-8011481: cN-II (uniprotkb:O46411) and cN-II (uniprotkb:O46411) bind ( MI:0407)bymolecular sieving (MI:0071) Abbreviations cN-II, cytosolic 5¢-nucleotidase; cN-III, cytosolic 5¢-nucleotidase III; cN-IA, cytosolic 5¢-nucleotidase IA; cN-IB, cytosolic 5¢-nucleotidase IB; cdN, cytosolic 5¢(3¢)-deoxyribonucleotidase; mdN, mitochondrial 5¢(3¢)-deoxyribonucleotidase. FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4863 for pro-drug activation and inactivation [3,4]. It has been demonstrated that the catalytic mechanism of cN-II requires formation of a covalent phospho-inter- mediate on an aspartate residue located in a conserved motif (motif I) [5]. This motif, together with three other conserved motifs, is shared among the members of the haloacid dehalogenase (HAD) superfamily, including the soluble 5¢-nucleotidase family: cytosolic 5¢-nucleotidases cN-II, cN-III, cN-IA and cN-IB, and both cytosolic and mitochondrial 5¢(3¢)-deoxyribonu- cleotidases [5]. Even though all soluble 5 ¢-nucleotidases share the same reaction mechanism and possess con- served structural motifs in the catalytic site, only two members of this family possess phosphotransferase activity, namely cN-II and cN-III. cN-II has several unique aspects, such as its complex regulation and the very high degree of primary sequence conservation during evolution [5]. These aspects indicate that this enzyme plays an important role. cN-II knockdown through RNAi causes apopto- sis in cultured cells [6]. Furthermore, over-expression of cN-II by more than 10-fold in HEK293 cells has proved impossible to achieve, probably because of an adverse effect on cell viability [7]. The reaction rates of both nucleotide hydrolysis and phosphate transfer catalysed by cN-II appear to be regulated by a number of phosphorylated compounds such as ADP, ATP, BPG, Ap 4 A and polyphosphates. These compounds act as allosteric activators. ATP causes an increase in V max of approximately 10-fold, with little effect on K m for the substrate IMP [8,9]. Free inorganic phosphate, on the other hand, acts as an allosteric inhibitor, causing a 20-fold increase in K m for the substrate IMP with little effect on V max . Inter- estingly, ATP partially counteracts the effect of phos- phate, by increasing V max ; however, it is unable to reverse the increase in K m [9]. cN-II has been described as a homotetramer with the ability to change its oligo- merization state in response to the presence of activa- tors or inhibitors. It has been suggested that the change in the oligomerization state is accompanied by a change in specific activity [10]. However, this simple model does not explain the kinetic evidence described above. Despite its cytosolic location, cN-II has particularly poor solubility. This is why it has been difficult to obtain the crystal structure of the whole protein. A truncated form of cN-II lacking the last 25 amino acids is significantly more soluble than the wild-type enzyme, and was recently crystallized [11]. The crystal- lographic model, constructed by ordering 487 residues (1-400 and 417-488) out of 561, indicates a homotetra- meric protein resulting from interaction through a specific interface (interface B) of two identical dimers that arise from interaction of two identical subunits through interface A. Because of the presence of adeno- sine bound to the crystallized protein, Wallde ´ n et al. [11] identified two putative effector sites, and suggested that effector site 1, close to interface A, might interact with BPG and Ap 4 A, while ATP and ADP might bind to effector site 2. In a previous paper, two active forms of cN-II were identified in extracts from different organs [12]. The two forms purified from calf thymus showed different behav- iour with activators. The heavier form (form A) has a high-affinity regulatory site for BPG, while ADP and ATP share a different site. The lighter form (form B) has three different sites for the three activators [5,12]. Although many papers have been published in the last few years describing structural and functional fea- tures of cN-II, fundamental aspects of how the enzyme functions remain to be unravelled, in particular the number and location of the interaction sites for the activators and inhibitors, the relationship between activity and enzyme oligomerization state, and, finally, the amino acid residue(s) responsible for phospho- transferase ability of cN-II. We have utilized a mecha- nistic approach in order to increase knowledge on these topics. Results Active site and phosphotransferase reaction Figure 1 shows the aligned sequences of motif I for the six intracellular 5¢-nucleotidases. Of the six enzymes, only cN-II and cN-III possess a Thr instead of a Val (position 56 of cN-II, boxed in Fig. 1) [11]. These are the only two enzymes for which phosphotransferase activity has been unquestionably ascertained, and Wallde ´ n et al. [11] suggested that the presence of T56 Fig. 1. Aligned conserved motif I of the six known intracellular human 5¢-nucleotidases: cN-II, cN-III, cN-IA, cN-IB, cytosolic 5¢(3¢)- deoxyribonucleotidase (cdN) and mitochondrial 5¢(3¢)-deoxyribonu- cleotidase (mdN). Residues 55 and 56 of cN-II are indicated in bold. Regulation of cytosolic 5¢-nucleotidase R. Pesi et al. 4864 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS might be important for this activity. However, we noted that there is another variable residue near T56. In position 55 of cN-II, a Tyr is present that is substi- tuted by Met in cN-III and by Ala or Gly in the other enzymes. Therefore, the two phosphotransferases have a bulkier amino acid in this position compared to the other four enzymes, which have amino acids with very small side chains. We decided to construct two mutants of motif I at positions 55 (Y55G) and 56 (T56V) in order to ascertain whether one of them is responsible for the phosphotransferase activity. Deter- mination of kinetic parameters showed that substitu- tion of Tyr55 by a smaller residue causes a dramatic increase in the ratio of nucleotidase to phosphotrans- ferase activity. The affinity for the substrates inosine and IMP remains unaltered, and the effect of the enzyme activators ATP and Mg 2+ is also unchanged (Table 1). Furthermore, by simultaneously measuring the rate of nucleoside and phosphate production from IMP and monophosphate synthesis from inosine (see Experimental procedures), we observed that, in the wild-type, the transfer of phosphate to inosine accounts for 53% of the phosphate produced from IMP, but only for 11% with the mutated Y55G enzyme under the same experimental conditions (Fig. 2). However, substituting Thr56 by Val produced an enzyme with a lower turnover but the same func- tional characteristics as the wild-type enzyme, included the nucleotidase ⁄ phosphotransferase ratio (Table 1). Regulatory sites Effector site 1 is located near subunit interface A, and is believed to interact with the activator Ap 4 A, by binding one adenosine moiety in each subunit (Fig. 3). We mutated Arg144, which has been proposed to bind the phosphate moiety of nucleotide activators, Ile152, which is thought to interact with the adenylic moiety of the activator, and Asn154, which probably interacts with the purine ring through a hydrogen bond [11], substituting these amino acids by negatively charged residues. For putative effector site 2, we mutated Phe127 and His428, as adenosine is presumably stacked between these two residues, and Met436, which forms a hydrogen bond with the purine ring through its carbonylic group [11] (Fig. 3). We substi- tuted the first two residues by a negatively charged res- idue to discourage stacking, and replaced Met436 with a bulkier amino acid (Trp). Putative effector site 1 None of the mutations produced had a significant effect on K m for the two principal substrates (Table 2). Table 1. Effect of point mutations on various kinetic parameters of recombinant bovine cN-II. Nucleotidase and phosphotransferase activi- ties were measured as described in Experimental procedures. Values are means ± SD of at least three independent assays. k cat refers to nucleotidase activity and is measured at saturating concentrations of IMP and sub-saturating concentrations of inosine. The K 50 for ATP was measured as phosphotransferase activity, while for Mg 2+ , it was measured as nucleotidase activity. cN-II Nucleotidase ⁄ phosphotransferase K m (inosine) (m M) K m (IMP) (m M) k cat (s )1 ) K 50 (Mg 2+ ) (m M) K 50 (ATP) (m M) Wild-type 1.8 ± 0.1 1.0 ± 0.2 0.1 ± 0.02 59.0 ± 6 2.0 ± 0.5 1.0 ± 0.3 Y55G 7.6 ± 2.1 1.3 ± 0.1 0.1 ± 0.03 323.0 ± 15 5.0 ± 2 0.40 ± 0.4 T56V 1.9 ± 0.1 1.0 ± 0.2 0.1 ± 0.02 6.0 ± 1 4.0 ± 1 0.80 ± 0.2 50 Rate of product formation (%) 100 0 Ino IMP P i Ino H 2 O E + IMP E + IMP E-P E + P i Ino Fig. 2. Rate of inosine, IMP and P i production catalysed by wild- type cN-II (black bars) or mutant Y55G (white bars) in the presence of 2 m M IMP and 1.4 mM inosine (Ino) as substrates. The assays were performed as described in Experimental procedures. For wild- type and Y55G, 100% activity corresponds to 32 and 18 UÆmL )1 , respectively. The upper scheme indicates the catalytic mechanism of cN-II. E, enzyme; P, phosphate. The products measured are boxed. R. Pesi et al. Regulation of cytosolic 5¢-nucleotidase FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4865 Mutant R144E showed an altered affinity for all the activators tested, N154D was normally activated by ATP and BPG but activation by ADP and Ap 4 A was completely prevented, as is the case for I152D, which showed a higher K 50 for ATP and BPG. Putative effector site 2 Study of cN-II crystallized in the presence of adeno- sine indicates that this nucleoside binds to this site, even though only the amino acid residues involved in the binding of the purine base were identified, while the ribose moiety was completely disordered [11]. Mutation of two residues involved in the binding of adenine (F127E and M436W) had no effect on the functional characteristics of the enzyme, except for an increase in the K 50 for Ap 4 A for mutant M436W. The mutations did not alter the activatory capacity of all the compounds tested. The mutant H428D was almost insensitive to all known activators of cN-II (Table 2). CN-II subunit oligomerization Using ‘FirstGlance in Jmol’ (http://molvis.sdsc.edu/ fgij/), we analysed the cN-II crystal structure described by Wallde ´ n et al. [11]. We mutated three amino acids at interface A (Phe36, Tyr115 and Asp396) and two at interface B (Lys311 and Gly319). Interface A In the model proposed on the basis of the crystal struc- ture, interface A is involved in formation of a dimeric structure (Fig. 3). Fifty-three amino acids contribute to interaction of the two monomers by forming both hydrogen bonds and salt bridges. Some of these residues are near to effector site 1. We designed our mutants in Fig. 3. Model of the homotetrameric quaternary structure of cN-II showing interfaces A and B and the Mg 2+ site. The inset shows the tertiary structure of each subunit. Effector sites 1 and 2 and the active site are shown. Table 2. Effect of point mutations on various kinetic parameters of recombinant bovine cN-II. Nucleotidase and phosphotransferase activi- ties were measured as described in Experimental procedures. Values are means ± SD of at least three independent assays. K 50 values for P i , ATP, ADP and Ap 4 A were measured as phosphotransferase activity, while those for BPG and Mg 2+ were measured as nucleotidase activ- ity. The extent of activation, when present, was between 5- and 10-fold. (1) Mutation in putative effector site 1; (2) mutation in putative effector site 2. NA, no activation. cN-II K m (IMP) (m M) K m (inosine) (m M) K 50 (Mg 2+ ) (m M) K 50 (P i ) (m M) K 50 (BPG) (m M) K 50 (ATP) (m M) K 50 (ADP) (m M) K 50 (Ap 4 A) (m M) Wild-type 0.10 ± 0.02 1.0 ± 0.2 2.0 ± 0.5 2.0 ± 0.3 0.3 ± 0.06 1.0 ± 0.3 2.2 ± 0.5 0.1 ± 0.05 R144E (1) 0.10 ± 0.05 1.0 ± 0.2 2.0 ± 1.0 6.5 ± 1.0 5.0 ± 1.50 30.0 ± 5.0 33.0 ± 6.0 1.6 ± 1.00 N154D (1) 0.1 ± 0.04 2.5 ± 0.5 0.3 ± 0.3 4.0 ± 1.0 0.3 ± 0.07 0.5 ± 0.2 NA NA I152D (1) 0.2 ± 0.05 0.7 ± 0.2 0.9 ± 0.8 3.5 ± 1.2 5.0 ± 2.00 20.0 ± 4.0 NA NA F127E (2) 0.1 ± 0.03 1.0 ± 0.3 0.9 ± 0.7 1.5 ± 0.5 0.7 ± 0.10 1.5 ± 1.0 2.5 ± 0.7 0.2 ± 0.03 M436W (2) 0.1 ± 0.02 0.9 ± 0.3 1.5 ± 1.0 1.5 ± 0.5 0.5 ± 0.06 1.5 ± 1.0 1.5 ± 1.0 1.0 ± 0.50 H428D (2) 0.3 ± 0.05 0.6 ± 0.5 1.5 ± 1.0 0.4 ± 0.3 NA NA NA NA Regulation of cytosolic 5¢-nucleotidase R. Pesi et al. 4866 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS an attempt to interfere with monomer aggregation via interface A by introducing or deleting charged residues or altering molecular hindrance. One of the mutated amino acids (Tyr115) is located very close to Arg144, which is part of effector site 1 (Fig. S1). The mutant F36R was inactive, while Y115A behaved normally and D396A was only activated by BPG (Table 3). Interface B The structure described by Wallde ´ n et al. [11] indicates that the active tetramer is formed by interaction between two identical dimers via interface B: this interface con- tains 28 aminoacid residues, of which 8 make hydrogen bonds (Fig. 3). We constructed two mutants by introdu- cing a negative charged residue or substituting a residue with a non polar group (K311A), thus forming both hydrogen and van der Waals interactions. The two mutants showed kinetic characteristics similar to those of the wild-type (Table 3). FPLC gel-filtration chroma- tography of purified recombinant wild-type cN-II indi- cated that this enzyme exists in solution as a tetramer (molecular mass 260 kDa) (Fig. 4). Actually, the chro- matogram showed two peaks, but the first was inactive. Immunoblotting of both peaks indicated cross-reactivity with specific antibodies against cN-II, but the first inac- tive peak disappeared when Escherichia coli cells expressing cN-II were grown at 20 °C instead of 37 °C (data not shown). A dimeric active form (130 kDa) was present in addition to the tetramer for mutants K311A and G319D (mutations at interface B) (Fig. 4). Finally, we investigated the chromatographic behav- iour of recombinant wild-type enzyme in the presence of ATP and Mg 2+ as an activator and P i as an inhibitor. Figure 5 shows that the enzyme exists and functions as a tetramer irrespectively of the presence of activator or inhibitor. We also performed light-scattering measure- ments of the enzyme alone or in the presence of its effec- tors. There was no change in molecular mass in the presence of enzyme activators or inhibitors (Table 4). Discussion Active site The soluble 5¢-nucleotidase family shares four con- served motifs with the HAD superfamily that are involved in the reaction mechanism [5]. Resolution of the crystal structure of some family members indicated that the active site contains all the conserved motifs, whose role in catalysis was determined through a mechanistic approach [5,11,13]. Other than these con- served motifs, cN-IA, cN-IB, cN-II, cN-III and the cytosolic and mitochondrial 5¢(3¢)-deoxyribonucleotid- ases differ considerably in their primary structure. Despite this poor similarity, there is much evidence to indicate that all members of soluble 5¢-nucleotidase family share the same reaction mechanism, proceeding through a covalent phospho-enzyme intermediate [13,14]. Phospho-cN-II has been isolated, and the phosphate has been found to be localized to a con- served aspartate residue (D52) in the first of the four conserved motifs [13]. Formation of a phospho-inter- mediate suggests the possibility that the enzyme cataly- ses transfer of phosphate to a suitable acceptor [15]. A number of HAD superfamily members are able to catalyse a phosphotransferase reaction, including at least two soluble nucleotidases (cN-II and cN-III). The aligned sequence of motif I of soluble nucleotidases indicates that both nucleotidases with phosphotransfer- ase activity had a Thr instead of Val in the fifth posi- tion after the phosphorylated Asp, and a relatively bulky residue in the fourth position instead of Gly, which is present in all the other nucleotidases. On this basis, we mutated two residues in motif I, Thr56 and Tyr55. Thr56 was substituted by Val, and Tyr55 was substituted by Gly. Our results indicate that the pres- ence of Thr or Val in position 56 is not relevant for the nucleotidase ⁄ phosphotransferase ratio. However, the increase in flexibility very close to Asp54, which Table 3. Effect of point mutations on various kinetic parameters of recombinant bovine cN-II. Nucleotidase and phosphotransferase activi- ties were measured as described in Experimental procedures. K 50 values for P i , ATP, ADP and Ap 4 A were measured as phosphotransferase activity, while those for BPG and Mg 2+ were measured as nucleotidase activity. The extent of activation, when present, was between 5- and 10-fold. Values are means ± SD of at least three independent assays. (A) Mutation in interface A; (B) mutation in interface B. NM, not measurable. NA, no activation. cN-II K m (IMP) (m M) K m (inosine) (m M) K 50 (Mg 2+ ) (m M) K 50 (P i ) (m M) K 50 (BPG) (m M) K 50 (ATP) (m M) K 50 (ADP) (m M) K 50 (Ap 4 A) (m M) Wild-type 0.10 ± 0.02 1.0 ± 0.2 2.0 ± 0.5 2.0 ± 0.3 0.3 ± 0.06 1.0 ± 0.3 2.2 ± 0.5 0.10 ± 0.05 F36R (A) NM NM NM NM NM NM NM NM Y115A (A) 0.06 ± 0.05 2.0 ± 0.5 1.0 ± 0.7 1.1 ± 0.5 0.3 ± 0.05 2.0 ± 0.4 NA NA D396A (A) 0.10 ± 0.03 0.5 ± 0.5 0.2 ± 0.06 2.6 ± 0.6 0.5 ± 0.05 NA NA NA K311A (B) 0.04 ± 0.03 0.2 ± 0.5 0.7 ± 0.5 2.0 ± 0.4 0.5 ± 0.04 7.3 ± 1.5 1.00 ± 0.6 0.25 ± 0.07 G319D (B) 0.05 ± 0.04 0.5 ± 0.5 2.5 ± 0.6 1.6 ± 0.7 0.5 ± 0.03 3.0 ± 1.0 2.00 ± 0.5 0.25 ± 0.05 R. Pesi et al. Regulation of cytosolic 5¢-nucleotidase FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4867 participates in binding of the phosphate [13], obtained with the Y55G mutant, causes an increase in turnover for the nucleotidase reaction, and, as a consequence, a large increase in the nucleotidase ⁄ phosphotransferase ratio. Regulatory sites cN-II is activated by BPG, a number of triphosphate and diphosphate nucleosides (ATP and ADP being the best activators), Ap 4 A and polyphosphates. Con- versely, orthophosphate has an inhibitory effect. Kinetic studies have indicated that ATP (and presum- ably other phosphorylated compounds) causes stabil- ization of an enzyme form with a high k cat , without substantial alteration of the K m for the substrates, while orthophosphate stabilizes a form at high K m with no effect on k cat . If ATP and phosphate are present at the same time, an enzyme form with high K m and high k cat is observed [9]. Therefore, depending on the effec- tor, cN-II may be present as one of two structures . . . . . . . . . . . . . . . . . . . . . . 670 kDa 210 kDa 150 kDa 443 kDa Activity (Arbitrary units) (–) 840 72 Retention time (min) A B C Abs 254 nm (Arbitrary units) (–) . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fig. 5. FPLC profiles of wild-type cN-II alone (A), or in the presence of 5 m M ATP and 10 mM MgCl 2 (B), or in the presence of 5 mM P i and 10 mM MgCl 2 (C). The column, prepared and eluted as described in Experimental procedures, was loaded with approxi- mately 150 lg of purified protein, and the activity was measured as the rate of IMP production in the presence of inosine (phospho- transferase activity) as described in Experimental procedures. Arrows indicate the molecular mass of the marker proteins thyro- globulin (670 kDa), apoferritin (443 kDa), b-amylase (210 kDa) and alcohol dehydrogenase (150 kDa). . . . . . . . . . . . . . . . . . . . . . . 670 kDa 210 kDa 150 kDa 443 kDa Activity (Arbitrary units) (–) 840 72 Retention time (min) Abs 254 nm (Arbitrary units) (–) WT G319D K311A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • Fig. 4. FPLC profiles of wild-type cN-II, and two mutants in inter- face B: G319D and K311A. The column, prepared and eluted as described in Experimental procedures, was loaded with approxi- mately 150 lg of purified protein, and the activity was measured as the rate of phosphate production in the presence of IMP (phospha- tase) as described in Experimental procedures. Arrows indicate the molecular mass of the marker proteins thyroglobulin (670 kDa), apoferritin (443 kDa), b-amylase (210 kDa) and alcohol dehydroge- nase (150 kDa). Regulation of cytosolic 5¢-nucleotidase R. Pesi et al. 4868 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS with high and low K m . In addition, a low and high k cat may be associated with each structure. It has also been suggested, on the basis of kinetic characterization, that the enzyme has at least three effector sites, one for ATP, one for ADP and one for BPG [5,12]. On the basis of the results obtained from crystallization of a truncated form of cN-II [11], we constructed point mutants for a number of amino acid residues located in putative effector sites 1 and 2 and involved in bind- ing of adenylic nucleotides and BPG. Our results partially confirm the molecular model- ling, suggesting that effector site 1 is the binding site for Ap 4 A and that ADP binds this site as well. Ap 4 A binds between two subunits with one adenosine moiety in each subunit [11], but ADP may possibly fill the whole site in each subunit and attain the same result. Mutant N154D shows a decrease in the value of K 50 for Mg 2+ . Moreover, the mutated enzyme showed some activity even in the absence of added Mg 2+ , whereas all the other active mutants, as well as the wild-type enzyme, are completely dependent on addi- tion of the metal. This behaviour may be due to the proximity of Asn154 to the active site (Fig. S2), and, in particular, a modification in the interaction of this resi- due with Asp351 (directly involved in Mg 2+ binding) and His352 both belonging to motif III. Remarkably, mutant D396A, in which the mutation is located in interface A, very close to effector site 1 and the active site, shows a similar increase in affinity for Mg 2+ . Our results also indicate that ATP and BPG probably bind to a different site and that this is effector site 2. Point mutations at this site result in either generalized impair- ment of enzyme regulation or characteristics very simi- lar to those of the wild-type. As virtually all purine and pyrimidine triphosphates have some activatory effect on cN-II, the interaction site is likely to be more selec- tive for the phosphorylated sugar moiety than for the base. Therefore, binding of adenosine to effector site 2, as demonstrated by the cN-II crystal structure [11], may not be indicative of the location of the nucleoside triphosphate effector site. CN-II subunit oligomerization CN-II has been purified from various sources and has always been described as a homotetramer [16,17]. The crystal structure suggests that the tetramer arises from interaction of two dimers through interface B. Muta- tions in interface A, through which two monomers interact, and which is very close to the effector site 1, either resulted in a completely inactive enzyme or strongly interfered with activation by ADP and Ap 4 A. This indirectly confirms that effector site 1 is specific for these compounds. Mutation of amino acid residues located in interface B generated proteins for which an active dimeric form was detected in addition to the tet- ramer. However, stabilization of the dimeric form had no effect on the catalytic capacity of the enzyme. Our results show that the monomer is probably inactive, and the dimer is the smallest active cN-II quaternary structure. FPLC analysis of purified recombinant enzymes shows a heavier protein (720 kDa) in addition to the proteins at the expected molecular mass. This protein was an unusual oligomerization state of a pro- tein that was identified by immunoblotting as cN-II but completely inactive. E. coli produces a small amount of cN-II that is correctly folded and a large amount of incorrectly folded and insoluble protein. It is conceivable that a cN-II protein that is incorrectly folded but still soluble is also produced. A decrease in growth temperature resulted in disappearance of the heavier inactive peak. Furthermore, FPLC analysis of the recombinant wild-type cN-II showed the presence of the tetrameric active form irrespective of the pres- ence of effectors. This result disagrees with other results obtained on recombinant human cN-II [10], which is almost identical to our bovine enzyme. To support our finding, we used light scattering, a tech- nique that, unlike gel-filtration chromatography, does not require protein dilution. However, light-scattering experiments confirmed that enzyme activation or inhi- bition is not followed by a change in enzyme subunit oligomerization. In conclusion, our results confirm that there is indeed an activatory site specific for Ap 4 A located at the inter- face between two subunits (interface A). This site may also accommodate ADP at lower affinity, but resulting in the same level of activation as Ap 4 A. ATP binds to a different site; however, it was not possible to confirm this as effector site 2 indicated by the crystal structure obtained in the presence of adenosine. In contrast to Table 4. Variation of the protein molecular mass in the presence of specific effectors as estimated by the ratio of the scattered light intensity. Values are means ± SD of at least three measurements. Sample Ratio a cN-II in 20 mM Tris ⁄ HCl, pH 8, +0.2 M NaCl (control) (control), +5 m M ATP 1.08 ± 0.08 (control), +5 m M ⁄ 10 mM ATP ⁄ Mg 2+ 1.16 ± 0.06 (control), +10 m M ATP 1.13 ± 0.09 (control), +10 m M ⁄ 10 mM ATP ⁄ Mg 2+ 1.15 ± 0.08 (control), +5 m M P i 1.08 ± 0.07 (control), +5 m M ⁄ 10 mM P i ⁄ Mg 2+ 0.95 ± 0.07 (control), +100 l M AP 4 A 1.11 ± 0.10 a Ratio of molecular mass in the presence of specific effectors to that of the free protein. R. Pesi et al. Regulation of cytosolic 5¢-nucleotidase FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4869 the suggestion by Wallde ´ n [11], we demonstrated that BPG does not bind effector site 1. Finally, mutations at the interfaces and gel-filtration experiments indicated that the tetramer is the major quaternary structure of cN-II, irrespectively of the presence of effectors, but that the dimer may also be stable and active. Experimental procedures Materials DpnI was provided by New England Biolabs (Ipswich, MA, USA) and Pfu DNA polymerase by Promega (Madison, WI, USA). Ni-NTA agars was purchased from Qiagen (Valencia, CA, USA). [8- 14 C]-inosine and [8- 14 C]-IMP were obtained from Sigma-Aldrich (St Louis, MO, USA). Opti- phase ‘HiSafe’ 3 scintillation liquid was obtained from Per- kin-Elmer (Waltham, MA, USA). Superdex-200 was purchased from GE Healthcare (Piscataway, NJ, USA). All other chemicals were of reagent grade. Site-directed mutagenesis The point mutants F36R, Y115A, F127E, I152D, R144E, N154D, K311A, G319D, D396A and M436W were obtained using the PCR-based site-directed mutagenesis method described by Fisher and Pei [18], and the point mutants Y55G, T56V and H428D were produced as described in the QuikChange Ò site-directed mutagenesis kit manual (Stratagene, La Jolla, CA, USA). The primers used are listed in Table 5. Expression and purification of the recombinant proteins Expression of the recombinant mutants was performed as previously described [19]. The 6· His-tagged proteins were purified using the Ni-NTA agar method as described in the QIAexpressionistÔ handbook (Qiagen). The protein con- centration was determined using the Bradford method [20], with BSA as the standard. Enzyme assays The nucleotidase activity of cN-II and its mutants was mea- sured as the rate of [8- 14 C]-inosine formation from 2 mm [8- 14 C]-IMP in the presence of 1.4 mm inosine, 20 mm MgCl 2 , 4.5 mm ATP and 5 mm dithiothreitol, as previously described [9]. Phosphotransferase activity was measured as the rate of [8- 14 C]-IMP formation from 1.4 mm [8- 14 C]-ino- sine, in the presence of 2 mm IMP, 20 mm MgCl 2 , 4.5 mm ATP and 5 mm dithiothreitol, as previously described [9]. For determination of kinetic parameters (K m and k cat ), the concentration of the labelled substrates ranged from 0.02 to 4mm. A plot of the dependence of the rate of phospho- transferase activity on MgCl 2 , ATP, ADP and BPG con- centration was used to determine the value of K 50 for these compounds. Under these experimental conditions, the accu- mulation of radiolabelled inosine (nucleotidase activity) rep- resents the sum of the phosphatase and the phosphotransferase activities. It has been previously reported that, at a concentration close to the K m value (1.4 mm), inosine reduces phosphatase activity to 50% without affecting the V max for both reactions [9]. Thus, the expected value of 2 was determined for the ratio between nucleotidase and phosphotransferase activities under the experimental conditions used for the wild-type recombinant cN-II assay. Accordingly, an alteration of this ratio for a mutant was considered as caused either by an alteration of the K m value for one of the two substrates or by a variation of the k cat value for one of the two activities. When required, the rate of phosphate formation (phosphatase activity) was measured as described by Chifflet et al. [21]. One unit of enzyme activity is the amount of enzyme Table 5. Primers used for site-directed mutagenesis. Mutant Forward primer (5¢ to 3¢) Reverse primer (5¢ to 3¢) F36R GCGCGTGAACCGGAGTT ACCCGATGATAGGCTTC Y115A CGCTGGAAACCTCTTGG GCATCAACTTTCAAAAGAT F127E CGAGATAAGGGGACCAG TTAAATCCATGTGCACAG R144E AGAAGATGACACTGAAAG TGAATAAATTTATTTGGATAC I152D CGATCTGAACACACTATTC TAAAATCTTTCAGTGTCAT N154D GGACACACTATTCAACCT AGAATGTAAAATCTTTCAGT K311A CGCGCTGAAAATTGGTAC CCAGTTTTAGTATCCACC G319D GGACCCCTTACAGCA GTGTAGGTACCAATTTTC D396A GGCTATTTTCTTGGCTGA AAGCTCTGAAGCTCTTC M436W GTGGATGGGGAGCCTG CCGTAGCACATGTCCA H428D AAGAAAGTAACTGACGACATGGACATGTG CACATGTCCATGTCGTCAGTTACTTTCTT Y55G AGTGTTTTGGGTTTGACATGGATGGCACACTTGCTG CAGCAAGTGTGCCATCCATGTCAAACCCAAAACACT T56V AGTGTTTTGGGTTTGACATGGATTATGTGCTTGCTG CAGCAAGCACATAATCCATGTCAAACCCAAAACACT Regulation of cytosolic 5¢-nucleotidase R. Pesi et al. 4870 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS required to convert 1 lmol of substrate to product per min- ute under the assay conditions. Gel-filtration chromatography The gel-filtration chromatography was performed on a FPLC system utilizing a Superdex-200 column (1.2 · 32 cm). Purified wild-type or mutant cN-II (150 lg) was loaded onto the column, and the chromatography was performed at a flow rate of 0.3 mLÆmin )1 using 50 mm Tris ⁄ HCl, pH 7.4, with addition of 200 mm NaCl. Frac- tions of 0.1 mL were collected. Light scattering The intensity of light scattered at 90° from the incident beam was measured using a spectrofluorometer (Fluoro- max-4; Horiba, Edison, NJ, USA). The wavelength of the incident light was 350 nm, and the band-pass used in the excitation and emission monochromators was 1.0 nm. The sample cell (fluorescence cell 1 · 1cm 2 cross-section) was cleaned using water filtered through 0.22 lm pore mem- brane. Protein and additive solution were passed through identical filters. All reagents were dissolved in 20 mm Tris ⁄ HCl + 0.2 m NaCl, pH 8, and the enzyme concentra- tion ranged between 0.09 and 0.14 mgÆmL )1 . Scattering intensities were compared between samples that had the same protein concentration and refractive index. Under these conditions, their ratio, according to Parr and Ham- mes [22], is proportional to the ratio of molecular mass. Acknowledgements We would like to thank Dr Giovanni Strambini and Dr Margherita Gonelli of the Institute of Biophysics (National Research Centre, Pisa, Italy) for the light- scattering analysis. We would also like to thank Dr Adrian Wallwork for careful language revision of the manuscript. This work was supported by a grant from the Ministero dell’Istruzione, dell’Universita ` e della Ricerca and by local funds from the University of Pisa. References 1 Tozzi MG, Camici M, Pesi R, Allegrini S, Sgarrella F & Ipata PL (1991) Nucleoside phosphotransferase activ- ity of human colon carcinoma cytosolic 5¢-nucleotidase. Arch Biochem Biophys 291, 212–217. 2 Banditelli S, Baiocchi C, Pesi R, Allegrini S, Turriani M, Ipata PL, Camici M & Tozzi MG (1996) The phosphotransferase activity of cytosolic 5¢-nucleotidase; a purine analog phosphorylating enzyme. Int J Biochem Cell Biol 2, 711–720. 3 Hunsucker SA, Mitchell BS & Spychala J (2005) The 5¢-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol Ther 107, 1–30. 4 Galmarini CM, Jordheim L & Dumontet C (2003) Role of IMP-selective 5¢-nucleotidase (cN-II) in haematologi- cal malignancies. Leuk Lymphoma 44, 1105–1111. 5 Allegrini S, Scaloni A, Careddu MG, Cuccu G, D’Am- brosio C, Pesi R, Camici M, Ferrara L & Tozzi MG (2004) Mechanistic studies on bovine cytosolic 5¢-nucle- otidase II, an enzyme belonging to the HAD superfam- ily. Eur J Biochem 271, 4881–4891. 6 Careddu MG, Allegrini S, Pesi R, Camici M, Garcia- Gil M & Tozzi MG (2008) Knockdown of cytosolic 5¢-nucleotidase II (cN-II) reveals that its activity is essential for survival in astrocytoma cells. Biochim Biophys Acta 1783, 1529–1535. 7 Rampazzo C, Gazziola C, Ferraro P, Gallinaro L, Johansson M, Reichard P & Bianchi V (1999) Human high-K m 5¢-nucleotidase: effects of overexpression of the cloned cDNA in cultured human cells. Eur J Biochem 261, 689–697. 8 Spychala J, Madrid-Marina V & Fox IH (1988) High K m soluble 5¢-nucleotidase from human placenta. Prop- erties and allosteric regulation by IMP and ATP. J Biol Chem 263, 18759–18765. 9 Pesi R, Turriani M, Allegrini S, Scolozzi C, Camici M, Ipata PL & Tozzi MG (1994) The bifunctional cytosolic 5¢-nucleotidase: regulation of the phosphotransferase and nucleotidase activities. Arch Biochem Biophys 312, 75–80. 10 Spychala J, Chen V, Oka J & Mitchell BS (1999) ATP and phosphate reciprocally affect subunit association of human recombinant high Km 5¢-nucleotidase. Role for the C-terminal polyglutamic acid tract in subunit associ- ation and catalytic activity. Eur J Biochem 259, 851– 858. 11 Wallden K, Stenmark P, Nyman T, Flodin S, Graslund S, Loppnau P, Bianchi V & Nordlund P (2007) Crystal structure of human cytosolic 5¢-nucleotidase II: insights into allosteric regulation and substrate recognition. J Biol Chem 282, 17828–17836. 12 Pesi R, Baiocchi C, Allegrini S, Moretti E, Sgarrella F, Camici M & Tozzi MG (1998) Identification, separation and characterisation of two forms of cytosolic 5¢-nucle- otidase ⁄ nucleoside phosphotransferase in calf thymus. Biol Chem 379, 699–704. 13 Allegrini S, Scaloni A, Ferrara L, Pesi R, Pinna P, Sgarrella F, Camici M, Eriksson S & Tozzi MG (2001) Bovine cytosolic 5¢-nucleotidase acts through the forma- tion of an aspartate 52-phosphoenzyme intermediate. J Biol Chem 276, 33526–33532. 14 Baiocchi C, Pesi R, Camici M, Itoh R & Tozzi MG (1996) Mechanism of the reaction catalysed by cytosolic 5¢-nucleotidase ⁄ phosphotransferase: formation of a phosphorylated intermediate. Biochem J 317, 797–801. R. Pesi et al. Regulation of cytosolic 5¢-nucleotidase FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4871 15 Fersht A (1999) Structure and Mechanism in Protein Science – A Guide to Enzyme Catalysis and Protein Folding, 2nd edn. W.H. Freeman and Company, New York, NY. 16 Zimmermann H (1992) 5¢-nucleotidase: molecular struc- ture and functional aspects. Biochem J 285, 345–365. 17 Itoh R (1993) IMP–GMP 5¢-nucleotidase. Comp Biochem Physiol B 105, 13–19. 18 Fisher CL & Pei GK (1997) Modification of a PCR- based site-directed mutagenesis method. BioTechniques 23, 570–571. 19 Allegrini S, Pesi R, Tozzi MG, Fiol CJ, Johnson RB & Eriksson S (1997) Bovine cytosolic IMP ⁄ GMP-specific 5¢-nucleotidase: cloning and expression of active enzyme in Escherichia coli. Biochem J 328, 483–487. 20 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein uti- lizing the principle of protein–dye binding. Anal Bio- chem 72, 248–254. 21 Chifflet S, Torriglia A, Chiesa R & Tolosa S (1988) A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases. Anal Biochem 168, 1–4. 22 Parr GR & Hammes GG (1975) Subunit dissociation and unfolding of rabbit muscle phosphofructokinase by guanidine by hydrochloride. Biochemistry 14, 1600– 1605. Supporting information The following supplementary material is available: Fig. S1. Proximity between effector site 1 and inter- face A. Fig. S2. Proximity between effector site 1 and the active site. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Regulation of cytosolic 5¢-nucleotidase R. Pesi et al. 4872 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS . (MI:0071) Abbreviations cN-II, cytosolic 5¢-nucleotidase; cN-III, cytosolic 5¢-nucleotidase III; cN-IA, cytosolic 5¢-nucleotidase IA; cN-IB, cytosolic 5¢-nucleotidase IB; cdN, cytosolic. Active and regulatory sites of cytosolic 5¢-nucleotidase Rossana Pesi 1 , Simone Allegrini 2 , Maria

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