Cd 2+ -induced aggregation of Escherichia coli pyrophosphatase Yury V. Zimenkov 1 , Anu Salminen 2 , Irina S. Efimova 1 , Reijo Lahti 2 and Alexander A. Baykov 1 1 A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia; 2 Department of Biochemistry, University of Turku, Finland We report here that Escherichia coli pyrophosphatase aggregates in the presence of m illimolar Cd 2+ . This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd 2+ concentration. Aggregation was enhanced by Mg 2+ , the n atural cofactor of pyr ophosphatase, and Mn 2+ . Mutations at the intersubunit metal-binding site had no effect, whereas mutation a t Glu139, which is p art of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme m ole- cules, suppressed aggregation. These findings indicate that aggregation is affected by Cd 2+ binding to the peripheral metal-binding site, probably by strengthening intermole- cular Trp149–Trp149¢ stacking interactions. Keywords: aggregation; cadmium; inorgan ic pyrophospha- tase; site-directed mutagenesis. Protein aggregation is a common phenomenon, with important practical implications. A variety of diseases, including the amyloidoses and prion diseases, as w ell as other protein deposition disorders, involve protein aggre- gation [1]. In most cases, the proteins that aggregate are totally or partially unfolded, and the aggregation, which occurs via hydrophobic interactions, is almost completely irreversible [2,3]. Examples of proteins aggregating in their native state, other than salting out and isoelectric point precipitation, are l ess common, with the aggregation of t he mutant hemoglobin t hat causes s ickle-cell anemia b eing the best known example [4]. In addition, Zn 2+ and other divalent cations have been reported to aggregate native dodecameric g lutamine synthethase into tubular structures [5] and to have a role in amyloid formation [6,7]. Escherichia coli inorganic pyrophosphatase (PPase) is an essential enzyme that converts p yrophosphate, a byproduct of many biosynthetic reactions, into phosphate [8]. The native PPase molecule is formed by six identical subunits, of 20 kDa each, arranged in parallel layers of trimers [9,10]. Each subunit has an active site, resembling a large cavity, with subsites for two phosphate molecules and four divalent metal ions that can act as cofactors (Mg 2+ ,Mn 2+ )or inhibitors (Ca 2+ ). In the absence of pyrophosphate or phosphate, only two Mg 2+ ions are bound to the active site [11]. In addition, one Mg 2+ ion is bound at the intertrimeric interface, where it i s octahedrally associated with six water molecules, which in turn hydrogen bond to the Asn24 and Asp26 residues of the two interacting subunits [12,13]. PPase is a re adily soluble e nzyme that shows no tendency to aggregate in a variety of conditions. We s how here, however, that this enzyme reversibly aggregates in the presence of C d 2+ , a common polluting ion that is toxic to E. coli at millimolar concentrations [14], and we describe the mechanism behind this aggregation. Materials and methods Wild-type E. coli PPase and PPase variants were prepared and purified as described previously [15]. The final prepa- rations were homogeneous, according to SDS/PAGE. Enzyme aggregation was followed by measuring the absorbance of enzyme solution at 440 nm in a quartz cuvette of 1 cm path length. In kinetic experiments, an aliquot of 0.1 M cadmium acetate solution was added to 0.7 mL of enzyme solution (0.37 mgÆmL )1 ) containing 0.1 M Tris/HCl (pH 7.2), 1 m M MgCl 2 and 5 m M dithiothreitol; the contents of the cuvette were rapidly mixed and the absorbance was recorded on a Pharmacia-LKB Ultrospec Plus spectropho- tometer. In titration experiments, metal salt was added in 1.4 lL increments, the contents of the cuvette were stirred for 3 min, and the absorbance of the solution was measured. This time was selected on the basis of the observation that 3 min was sufficient for aggregation to reach > 85% of its equilibrium level under a v ariety of conditions. Each titration was repeated at least three times, and the SD value for the parameter describing the titration curve (c ½ )was calculated. Except where stated otherwise, aggregation of 0.37 mgÆmL )1 solutions of PPase was measured at 20 °C in a buffer at 0.1 M ionic s trength. The initial rates of PP i hydrolysis were measured by a continuous P i assay [16] in a reaction mixture containing 20 l M PP i ,20m M MgCl 2 ,0.15 M Tris/HCl (pH 7.2), and 0.2 m M EGTA. The reaction was initiated by adding enzyme (4–10 ngÆmL )1 final concentration) and was carried out for 3–4 min at 25 °C. Correspondence to R. Lahti, Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland. Fax: + 358 2333 6860, Tel.: + 358 2333 6845, E-mail: reijo.lahti@utu.fi; or A. A. Baykov, A. N. Belozersky I nstitute of Physico-Chemical Biology a nd Schoo l of Chemistry, Moscow State University, Moscow 119899, Russia. Fax: + 7095 9393181, Tel.: + 7095 9395541, E-mail: baykov@genebee.msu.su Abbreviations: c ½ , values for the parameter describing the titration curve; PPase, Escherichia coli inorganic pyrophosphatase. Enzyme: inorganic pyrophosphatase (EC 3.6.1.1). (Received 23 March 2004, revised 26 May 2004, accepted 1 June 2004) Eur. J. Biochem. 271, 3064–3067 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04239.x Results and discussion General characteristics of PPase aggregation Upon addition of 2 m M Cd 2+ , we noted the development of turbidity in the PPase solution, with a gradual increase in absorbance at 440 n m owing to light scattering by aggregate particles (Fig. 1). A fter sedimenting the aggregated protein in an Eppendorf microcentrifuge, less than 10% of PPase, as measured by activity and p rotein content, remained in the supernatant. The aggregated PPase could be easily solubi- lized, with a complete recovery of activity, by suspending the sediment in buffer c ontaining no cadmium salt, indicating that the native tertiary structure is regained when the Cd 2+ is removed. The dependence of aggregation on Cd 2+ concentration was characteristic of a highly cooperative process (Fig. 2). Virtually no aggregation w as observed at c oncentrations of <1 m M Cd 2+ ,whereas,at 1.5 m M Cd 2+ , the ag gregation was nearly complete. The theoretical curve, shown in Fig. 2, was constructed assuming that the aggregation depends on the 12th(!) power of Cd 2+ concentration. The position of this curve along the abscissa depended on reaction conditions and could be c haracterized by c ½ ,the Cd 2+ concentration corresponding to half the maximum A 440 (Fig. 2). The value of c ½ was determined manually and found to be 1.20 ± 0.06 m M under the standard conditions (0.37 mgÆmL )1 enzyme concentration, 0.1 M ionic strength, 20 °C) used for the experiment described in Fig. 2. Among the factors tested, ionic stren gth, which was adjusted bythe a ddition ofKCl, had the greatest effect on c ½ . When the ionic strength was increased to 0.16, 0.2 and 0.6 M , the c ½ increased to 2 .6 ± 0.1, 3.0 ± 0.2 and 1 1 ± 1 m M , respectively, indicating that increased ionic s trength d e- creased the tendency of the protein to aggregate. T his observation suggested that the aggregation is governed by ionic f orces, a finding supported b y the temperature dependence of this process, in th at c ½ decreased from 1.20 ± 0.06 m M at 20 °C to 0.8 ± 0.1 m M at 2 °C. The effect of PPase concentration on c ½ was m oderate, in that the latter increased to 1.70 ± 0.09 m M at 0.08 mgÆmL )1 PPase. Specificity of the aggregation Cd 2+ -induced aggregation was quite specific to E. coli PPase. No aggregation of the homologous yeast pyrophos- phatase was observed at Cd 2+ concentrations up to 11 m M [17,18], a finding confirmed by us (data not shown). Because of its specificity, the aggregation could be used as a simple and e fficient step in PPase purification. We found th at the addition of 4.5 m M cadmium acetate to an E. coli extract freed from nucleic acids [19] quantitatively precipitated PPase; the higher cadmium acetate concentration was required because of a low concentration of PPase in the extract. This precipitated PPase could be subsequently dissolved in Cd 2+ -free buffer. This aggregation step resulted in a sixfold purification of PPase, with an increase of specific activity from 26 to 150 IUÆmg )1 , and a yield of 90%. Thus, only small amounts of other proteins in the E. coli extracts were co-precipitated with PPase. PPase aggregation was also quite specific with respect to the divalent metal ion used. Of the other metal i ons tested, only C u 2+ and Z n 2+ induced aggregation, but their effective concentrations were much higher than that of Cd 2+ (Table 1). No aggregation was observed in the presence of the PPase cofactors Mg 2+ and M n 2+ and the PPase inh ibitor Ca 2+ , all of which bind to PPase. The nature of th e anionic counterion (acetate or chloride) in the metal salt had no effect on c ½ . Identification of the metal-binding site E. coli PPase possesses three types of metal-binding sites. Type 1 sites, of which there are two per monomer in the absence of su bstrate, are found in the active site cavity and are not involved in intersubunit or intermolecular contacts [12,13]. Type 2 sites, of which there are 0.5 per monomer, are found in the intersubunit c ontact region; each of these sites has ligands – Asn24 and Asp26 – from two subunits Fig. 1. Time-course of Escherichia coli inorganic pyrophosphatase (PPase) aggregation in the presence of 2 m M cadmium acetate. Fig. 2. Dependence of Escherichia coli inorganic pyrophosphatase (PPase) aggregation on cadmium acetate concentration. Absorbance values were measured 3 min after each metal salt addition. The line was obtained with the following equation: A 440 ¼ 1.03/(1 + 9.34/ [Cd 2+ ] 12 ). Ó FEBS 2004 Aggregation of pyrophosphatase (Eur. J. Biochem. 271) 3065 within the same hexamer [12,13]. Metal binding to type 2 sites strongly modulates trimer–trimer interactions in hexa- meric PPase [22]. Type 3 sites, of which there are six per hexamer, are found on the surface of the enzyme molecule and are formed by the Glu139 side-chain and backbone oxygens and the Val150 backbone oxygen [23] (Fig. 3). We ruled out the involvement of type 1 and type 2 sites in Cd 2+ -induced aggregation by testing the effects of Mg 2+ and Mn 2+ . Both of these divalent cations bind to type 1 and 2 sites, with dissociation constants ranging from 0.076 to 6.6 m M for Mg 2+ and from 0.006 to 0.35 m M for Mn 2+ [11]. Neither Mg 2+ nor Mn 2+ , however, binds to type 3 sites [12,13]. As neither Mg 2+ nor M n 2+ aggregated PPase (Table 1), they would be expected to increase the c ½ for Cd 2+ because of simple competition if the aggregating Cd 2+ ion binds to type 1 or type 2 sites. We actually observed t he opposite effect, in that c ½ ,whichwas 2.6 ± 0.1 m M in the absence of Mg 2+ and Mn 2+ (0.16 M ionic strength), decreased to 2.0 ± 0.1 m M in the presence of either 20 m M Mg 2+ or 20 m M Mn 2+ . Our finding, that Mg 2+ and Mn 2+ ions potentiated the effects of Cd 2+ , indicates t hat aggregation is caused by Cd 2+ binding to sites other than types 1 and 2. The lack of competition between Cd 2+ and the other metal ions in inducing aggregation suggested that aggrega- tion is governed by type 3 sites. This finding was supported by our results on PPase variants with specific mutations at the metal-binding sites (Table 2). A mutation at the peripheral type 3 site (E139Q) markedly increased c ½ (Table 2) and slowed down the aggregation (Fig. 1). By contrast, mutations at the intersubunit site (N24D, D26N and D26S) had no effect on c ½ or aggregation kinetics, although they stimulated (N24D) or eliminated (D26N and D26S) Mg 2+ binding to this site [22]. Based on these d ata, we conclude that Cd 2+ binding to type 3 sites is responsible for PPase aggregation. Possible mechanism One possible explanation of the Cd 2+ effect on PPase is t hat the Cd 2+ binds to peripheral metal-binding sites from different hexamers, i.e. Cd 2+ serves as a bridging atom. Structural analysis predicts, however, that although t his Cd 2+ ion is located on the protein surface, it may not be available to bind another hexamer without inducing signi- ficant structural alterations around the metal-binding site. A more probable a lternative is that Cd 2+ binding to the peripheral metal-binding site strengthens the intermolecular Trp149–Trp149¢ stacking interaction observed in PPase crystals [10]. Trp149 belongs to the most flexible segment (residues 147–153) of the loop shown in Fig. 3, as indicated by high B-factor values in s tructures that c ontain no bound metal ion in the peripheral binding site [9,10,12,13]. This segment also contains Val150, one of the metal ligands. Therefore, a bound metal ion would act to fix the 147–153 Fig. 3. A stereo view of the hexamer–hexamer contact obse rved in Escherichia coli inorganic pyrophosphatase (PPase) crystals [23]. The bound Ca 2+ ion is shown as a b lack sphe re, and its c oordin ation b onds are s hown as d ash ed line s. Unprim ed and prime d am ino ac id residue num bers an d metal ions refer to two different subunits from two n eighboring hexamers. Table 2. The effects of residue substitutions on c ½ for E scherich ia coli inorganic pyrophosphatase (PPase) aggregation by cadmium acetate. The v alues for the parameter describing the titration curve (c ½ )were measured as described in the legend of Fig. 2. PPase variant c ½ (m M ) WT 1.20 ± 0.06 N24D 1.16 ± 0.06 D26N 1.17 ± 0.06 D26S 1.17 ± 0.06 E139Q 2.2 ± 0.1 Table 1. Comparison of different metal ions in their ability to aggregate Escherichia c oli inorganic pyrophosphatase (PPase) and general chem- ical parameters. The most commonly occurring coordination numbers and respective ionic radii are separated by Ô/Õ. The preferred coordi- nation numbers are shown in bold. The values for the parameter describing the titration cu rve (c ½ ) were measured as described in Fig. 2. Metal salt c ½ (m M ) Cation coordination number [20] Cation ionic radius (A ˚ ) [21] CdCl 2 1.20 ± 0.06 4/5/6 0.78/0.87/0.95 ZnCl 2 6.5 ± 0.3 4/5/6 0.60/0.68/0.74 CuCl 2 13 ± 1 4/5/6 0.57/0.65/0.73 MgCl 2 > 100 6 0.72 CaCl 2 > 100 6/7/8 1.00/1.06/1.12 MnCl 2 > 100 4/5/6 0.66/0.75/0.83 3066 Y. V. Zimenkov et al. (Eur. J. Biochem. 271) Ó FEBS 2004 segment in space [23] and may adjust the position of Trp149 to allow its optimal interaction with its symmetrical partner in the other hexamer. Furthermore, the dependence of the aggregationon[Cd 2+ ] 12 in the millimolar range (Fig. 2), suggests that the corresponding Cd 2+ -binding constant is well above 1 m M and that a ggregate formation or growth requires that C d 2+ should be p resent in at least 1 2 peripheral sites in the interacting enzyme molecules. The effect of ionic strength on aggregation may thus be mediated by its effect on the Cd 2+ -binding constant. Further studies are, however, needed to explain the unusually strong Cd 2+ concentration dependence in structural terms. The above mechanism suggests that, in order to be effective at aggregating PPase, Cd 2+ wouldhavetobindto a type 3 binding site and properly position Trp149. These requirements c learly impose limitations on the c ation coordination number and ion ic radius. The preferred coordination number for all of the aggregating cations is four (Cd 2+ can equally well adopt six ligands), whereas the non-aggregating c ations have a coordination number of six or eight (Table 1). There is also a correlation between the value of c ½ and the ionic radius within the group of the aggregating cations. However, the high value of c ½ observed f or Cu 2+ , m ay also result from the fact that this cation favors square planar coordination rather than the tetrahedral coordination favored by Cd 2+ and Zn 2+ [20]. The i nability of Ca 2+ to aggregate PPase (Table 1), despite its presence i n the crystal structure [23], suggests that Ca 2+ does not ensure proper orientation of T rp149. Indeed, even in the crystal structure, where the neighboring protein molecules contribute to such an orientation, the Trp149 side-chain planes are not parallel in Ca 2+ PPase (Fig. 3). Acknowledgements The authors thank A. N. Parfenyev and I. P. Fabrichniy for help. 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(2001) The structures of E sch erichia coli inorganic pyrophosphatase complexed with Ca 2+ or CaPP i at atomic resolution and their mechanistic i mplications. J. M ol. Biol. 314, 633–645. Ó FEBS 2004 Aggregation of pyrophosphatase (Eur. J. Biochem. 271) 3067 . Time-course of Escherichia coli inorganic pyrophosphatase (PPase) aggregation in the presence of 2 m M cadmium acetate. Fig. 2. Dependence of Escherichia coli. mgÆmL )1 PPase. Specificity of the aggregation Cd 2+ -induced aggregation was quite specific to E. coli PPase. No aggregation of the homologous yeast pyrophos- phatase