Journal of Structural Biology 189 (2015) 81–86

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Journal of Structural Biology

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Structure of inorganic from Staphylococcus aureus reveals conformational flexibility of the ⇑ ⇑ Chathurada S. Gajadeera a, Xinyi Zhang b, Yinan Wei b, , Oleg V. Tsodikov a, a Department of Pharmaceutical Sciences, University of Kentucky, College of Pharmacy, 789 S. Limestone St., Lexington, KY 40536, United States b Department of Chemistry, University of Kentucky, 505 Rose St., Lexington, KY 40506, United States article info abstract

Article history: Cytoplasmic inorganic pyrophosphatase (PPiase) is an essential for survival of organisms, from Received 17 November 2014 bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Received in revised form 15 December 2014 Mg2+-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less Accepted 16 December 2014 understood family II PPiases are Mn2+-dependent and present only in some archaea, bacteria and prim- Available online 7 January 2015 itive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infec- tions, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel Keywords: antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn2+ at Pyrophosphorolysis 2.1 Å resolution. The active site contains two catalytic Mn2+ binding sites, each half-occupied, reconciling 2+ Phosphate metabolism the previously observed 1:1 Mn :enzyme stoichiometry with the presence of two divalent metal ion Novel drug target sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, Enzyme the two domains of SA PpaC form a closed active site, a conformation observed in structures of other fam- Phosphatase ily II PPiases only in complex with substrate or product mimics. A region spanning residues 295–298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprece- dented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissoci- ation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhib- itors of this enzyme. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction et al., 2001; Merckel et al., 2001). Family I includes hexameric archaeal and bacterial and dimeric eukaryotic PPiases Cytoplasmic inorganic pyrophosphatase (PPiase) is a ubiquitous that share a common catalytic fold (Baykov et al., 1999), whereas enzyme that plays a key role in phosphorus metabolism. PPiase family II are dimeric PPiases with a distinct fold present only in hydrolyzes inorganic pyrophosphate (PPi) generated upon nucleic some bacteria, archaea and primitive eukaryotes. Understanding acid synthesis and other numerous essential nucleotidyl transfer the structure and function of family II PPiases is biomedically reactions into two molecules of inorganic phosphate (Pi), thereby important because of their occurrence and essentiality in human providing a thermodynamic sink as well as eliminating inhibitory pathogens such as Staphylococcus aureus, Streptococcus agalactiae,

PPi product (Chen et al., 1990; Kornberg, 1962; Lundin et al., Streptococcus mutans and Bacillus anthracis. Expression of family 1991; Peller, 1976; Sonnewald, 1992). PPiases are divided into II PPiases is regulated depending on culture conditions (Lahti, two families based on their amino acid residue sequences 1983). The essentiality, the regulation and the uniqueness of family (Shintani et al., 1998; Young et al., 1998) and structures (Ahn II PPiases to specific bacteria make them attractive potential tar- gets for discovery and development of novel selective antibacterial agents. Abbreviations: SA, Staphylococcus aureus; PPiase, inorganic pyrophosphatase; Family II PPiases are two-domain with the active site PNP, imidodiphosphate. located at the domain interface. Based on the crystal structures ⇑ Corresponding authors. Fax: +1 (859) 323 1069 (Y. Wei), +1 (859) 257 7585 of PPiase II of Bacillus subtilis (Ahn et al., 2001; Fabrichniy et al., (O.V. Tsodikov). 2007), this interface is thought to be formed by a pivoting rotation E-mail addresses: [email protected] (Y. Wei), [email protected] (O.V. Tsodikov). of one domain relative to the other from a so-called open to a http://dx.doi.org/10.1016/j.jsb.2014.12.003 1047-8477/Ó 2014 Elsevier Inc. All rights reserved. 82 C.S. Gajadeera et al. / Journal of Structural Biology 189 (2015) 81–86 closed, active, conformation. This is inferred from the observation with shaking at 200 rpm, at 37 °C to attenuance of 0.2–0.3 at that a substrate analogue imidodiphosphate (PNP) is present in 600 nm, and then incubated at 16 °C for 1.5 h before induction the active site only in the closed conformation of this enzyme with IPTG (final concentration of 0.5 mM). The induced culture (Ahn et al., 2001; Fabrichniy et al., 2007; Merckel et al., 2001; was grown for an additional 16–18 h at 16 °C, 200 rpm. All purifi- Rantanen et al., 2007). The only reported structure of PPiase II that cation steps were carried out at 4 °C. The cells were harvested by is in the open conformations does not contain a bound substrate or centrifugation at 5000g for 10 min. The cell pellet was resus- a product (Fabrichniy et al., 2004). The catalytic mechanism of pended in lysis buffer (300 mM NaCl, 40 mM Tris–HCl pH 8.0, family II PPiases involves activation of a nucleophilic water by adjusted at room temperature, and 2 mM b-mercaptoethanol). metal ions, where three metal ions likely participate directly or The cells were disrupted by sonication on ice and the lysate was indirectly in the catalysis. The maximum catalytic turnover rate clarified by centrifugation at 40,000g for 45 min. The supernatant 2+ 2+ 1 in the presence of Mn or Co (kcat = 1700–3300 s ) is much was filtered through a 0.45 lm Millex-HV PVDF filter (Millipore) higher than that in the presence of Mg2+, the preferred catalytic and applied to a 1 mL Ni-IMAC HisTrap column (GE Healthcare) 1 ion for family I PPiases (kcat = 110–330 s )(Fabrichniy et al., equilibrated with lysis buffer. The column was washed with 2004; Kuhn et al., 2000; Parfenyev et al., 2001). This rate difference 20 mL of lysis buffer containing 50 mM imidazole. Then the 2+ is offset by the lower Km in the presence of Mg (Km = 10–30 lM) was eluted with 9 mL of lysis buffer containing 500 mM imidazole 2+ than that with Mn (Km = 90–160 lM) (Parfenyev et al., 2001), and 2 mM MnCl2 in 9 fractions of 1 mL each. The presence of MnCl2 making it unclear what physiological ion drives catalysis in vivo. was critical for obtaining a homogeneous dimerization state of SA However, it is thought that family II PPiases use Mn2+, because they PpaC. The fractions containing more than 95% pure desired protein, contain 1 nM-affinity Mn2+ and because bacteria con- as determined by SDS–PAGE, were pooled. The protein was further taining family II PPiases tend to accumulate Mn2+ (Charney et al., purified on a size-exclusion S-200 column (GE Healthcare) equili- 1951; Martin et al., 1986), through action of dedicated Mn2+ trans- brated with the gel filtration buffer (40 mM Tris–HCl pH 8.0, porters. Upon substrate binding, a high affinity metal site changes 100 mM NaCl, 2 mM MnCl2 and 2 mM b-mercaptoethanol). The its geometry from five-coordinated square pyramidal or trigonal protein-containing fractions were pooled and the protein was con- bipyramidal to six-coordinated octahedral (Fabrichniy et al., centrated using an Amicon Ultra (5000 MWCO) centrifugal filter 2007). Returning from the six-coordinated substrate-bound state device (Millipore) to 12 mg/mL. to the five-coordinated substrate-free state facilitates product release (Fabrichniy et al., 2004). Transition metal ions (Mn2+ or 2.2. Pyrophosphatase activity assay Co2+), but not Mg2+, which is nearly always six-coordinated (Harding, 2001), tolerate flexible coordination geometry. The SA PpaC activity was measured with sodium pyrophosphate First structures of family II PPiases from B. subtilis and S. mutans as a substrate, similarly to the previously described protocol showed two metal ions (M1 and M2) coordinating with protein (Baykov et al., 1988). A malachite green stock solution (0.12%, w/ ligands and another one (M3) that did not directly interact with v) was made by dissolving the dye in 3 M sulfuric acid. Fresh mala- the protein (Ahn et al., 2001; Merckel et al., 2001). A recent struc- chite green reagent was prepared prior to the assays by adding one tural study of PPiase II from B. subtilis in complex with PNP volume of 7.5% (w/v) ammonium molybdate into four volumes of revealed a fourth metal ion site occupied by protein-coordinated the malachite green stock solution followed by the addition of metal ion M4 (Fabrichniy et al., 2007). The trimetal center created Tween 20 to a final concentration of 0.2% (v/v). For activity mea- by M1, M2 and M4 allows proper substrate binding and positioning surements, purified SA PpaC (0.3 nM) was added into a freshly pre- of the nucleophilic water for a catalytic attack. The nucleophilic pared reaction mixture containing 1 mM sodium pyrophosphate water is positioned above the trimetal plane in the absence of sub- and 0.5 mM MnCl2, in a reaction buffer (25 mM Tris–HCl, 50 mM strate, and it crosses the plane upon substrate binding. Protein con- NaCl, pH 7.0) at 22 °C for 4 min. To stop the enzymatic reaction formational changes that occur in concert with binding and and determine the phosphate concentration of a sample, one vol- dissociation of metal ions, the substrate and the products in family ume of the malachite green reagent was mixed with four volumes II PPiases remain a substantial area of interest. of the enzymatic reaction mixture to be analyzed. The mixture Herein, we determine a crystal structure of the family II PPiase was incubated for 2 min, and the absorbance at 630 nm was mea- from an important pathogen S. aureus (SA PpaC), and compare it to sured with a BIOMATE 300 UV/vis spectrophotometer (Thermo Sci- other family II PPiases. entific). We confirmed that under these conditions, the were linear over time. To obtain relative activities, the 2. Materials and methods absorbance of the sample was corrected for non-enzymatic degra- dation of pyrophosphate and normalized against the maximum 2.1. Cloning, protein expression and purification absorbance of the sample containing wild-type PpaC and 0.8 mM sodium pyrophosphate. For construction of expression vector pET22-PpaC, the ppaC (locus tag: SAV1919) was amplified from the genomic DNA 2.3. Circular dichroism spectroscopy (CD) of SA strain Mu50 (ATCC #700699) by polymerase chain reaction and inserted between the NdeI and XhoI restriction sites in vector CD experiments were performed using a JASCO J-815 CD spec- pET22b. As a result, SA PpaC bears a C-terminal hexahistidine tag. trometer equipped with a Peltier temperature controller. Blank Site-directed mutagenesis was carried out by using the Quik- scans were collected from dialysis buffer and subtracted from the Change mutagenesis kit (Agilent Technologies) according to the spectra containing enzyme. Cuvettes of 1 mm or 1 cm pathlength kit manual. The construct sequences were confirmed by DNA were used for far UV and near UV scans, respectively. sequencing at the University of Kentucky DNA Sequencing Core. For protein purification, the pET22-PpaC plasmid was trans- 2.4. Protein crystallization formed into BL21 (DE3) chemically competent cells. A fresh colony from the transformation plate (Luria Bertani (LB) agar containing The initial crystallization conditions for SA PpaC were identified 100 lg/mL ampicillin) was grown in 5 mL of LB/ampicillin to the by the high-throughput screening service at the Hauptman- mid-log phase at 37 °C with shaking at 200 rpm and then used to Woodward Institute, NY (Luft et al., 2003), by a microbatch inoculate 4 L of LB/ampicillin medium. The culture was incubated method. After optimization, single rod-shaped crystals of SA PpaC C.S. Gajadeera et al. / Journal of Structural Biology 189 (2015) 81–86 83

0.07 0.07 0.2 mm in size were grown in 6–7 days by vapor dif- Table 1 fusion in hanging drops at 22 °C. In the drops, 1 lL of the concen- X-ray diffraction data collection and refinement statistics for SA PpaC. trated protein was mixed with 1 lL of the reservoir solution Data collection (1.32 M ammonium citrate tribasic) and equilibrated against Space group P61 22 Number of monomers per asymmetric unit 2 1 mL of the reservoir solution supplemented with 2 mM MnCl2. Unit cell dimensions The crystals were then gradually transferred into the cryoprotec- a, b, c (Å) 99.4, 99.4, 425.3 tant solution (1.32 mM ammonium citrate tribasic, 2 mM MnCl2, a, b, c (°) 90, 90, 120 15% glycerol), incubated for 30 min and rapidly frozen in liquid Resolution (Å) 50–2.1 2.14–2.10a nitrogen. Similarly, co-crystallization was attempted with 20, 50 I/rI 22.3 (2.9) and 100 mM Na PO , as well as with 1 mM PNP. In addition, - Completeness (%) 99.86 (99.48) 3 4 Redundancy 9.6 (9.8) dom factorial screening in the presence of PNP and fluoride was Rmerge (%) 8.5 (56.3) carried to crystallize SA PpaC–PNP complex in a crystal form differ- Number of unique reflections 70,222 ent from that of apo-SA PpaC. These experiments as well as soaking Structure refinement statistics the crystals in solutions containing phosphate or PNP did not yield Resolution (Å) 40–2.1 a bound phosphate or PNP in the active site. R (%) 22.1 Rfree (%) 24.9 Bond length deviation (rmsd) from ideal (Å) 0.005 2.5. Data collection and crystal structure determination Bond angle deviation (rmsd) from ideal (°) 1.022 Ramachadran plot statisticsb The X-ray diffraction data were collected at synchrotron beam- % of residues in most allowed regions 92 % of residues in additional allowed regions 8 line 21-ID-G (sector LS-CAT) at the Advanced Photon Source at % of residues in generously allowed regions 0.0 (0 residues) the Argonne National Laboratories (Argonne, IL). The data were % of residues in disallowed regions 0.0 (0 residues) indexed, integrated and scaled with HKL2000 (Otwinowski and a Numbers in parentheses indicate the values in the highest-resolution shell. Minor, 1997). The structure was determined by molecular replace- b Indicates PROCHECK (Laskowski et al., 1993) statistics. 98% of residues are in ment (MR) with program PHASER (McCoy et al., 2007) by using the favored regions and 1.8% are in the allowed regions according to RAMPAGE (Lovell structure of inorganic pyrophosphatase Bacillus subtilis ((Ahn et al., et al., 2003). 2001); PDB accession number: 1K23). The MR search was carried out in two steps: (1) with the N-terminal domain (up to residue 190), yielding a clear MR solution for this domain and (2) with 0.5 2+ the C-terminal domain (from residue 191 to the C-terminus), where dimer (+Mn ) the N-terminal domain was positioned as determined in step (1). 0.4 The structure building and refinement was then carried out itera- tively by using Coot (Emsley and Cowtan, 2004) and Refmac 0.3 (Murshudov et al., 1997) programs, respectively. The refined struc- ture contains a dimer in the asymmetric unit, with all annotated 0.2 residues in the protein sequence present in the structure of each monomer, except for the first Met residue. The data collection 0.1 monomer and refinement statistics are given in Table 1. The crystal structure Absorbance at 280 nm coordinates and structure factors for SA PpaC were deposited in the 0.0 with the PDB accession number 4RPA. 60 70 80 90 Time (min) 3. Results and discussion Fig.1. Size-exclusion chromatograms of SA PpaC in the absence of a divalent metal

3.1. The structure of PPiase II of S. aureus ion (the red trace) and in the presence of 2 mM MnCl2 (the black trace) in the buffer. The dimer and the monomer peaks are labeled. (For interpretation of the references A BLAST search (Altschul et al., 1997) yielded four structurally to colour in this figure legend, the reader is referred to the web version of this article.) characterized homologs of SA PpaC from bacteria with 46–57% sequence identity to SA PpaC and one from archaea (Methanococcus jannaschii) with 41% sequence identity (Supplementary Fig. 1). The The crystal structure of SA PpaC was determined by molecular residues critical for Mn2+ and substrate binding and its positioning replacement with individual domains of PPiase II of B. subtilis in the active site are conserved in SA PpaC, as expected. (57% identical in sequence) used sequentially as search models, SA PpaC was purified to homogeneity (Supplementary Fig. 2)in and refined at 2.1 Å resolution (Table 1). SA PpaC is a homodimer the presence of Mn2+. The protein was a mixture of dimers and of two 34 kDa subunits (Fig. 2A). The crystals contained a dimer monomers in the absence of a divalent metal ion, whereas only of PpaC per asymmetric unit. Strong electron density was observed dimers were observed in the presence of 2 mM MnCl2, as judged for the entire annotated polypeptide chain (residues 2–309) of by the size-exclusion chromatography (Fig. 1). A similar stabiliza- both monomers. As previously shown for other structures of family tion effect of Mn2+ on the dimeric, active form of the enzyme II PPiases (Ahn et al., 2001; Merckel et al., 2001), the N-terminal was observed in early studies of family II PPiases with Bacillus domain of SA PpaC (residues 1–190) consists of a six-stranded par- megaterium PPiase by the Arthur Kornberg’s group (Tono and allel b-sheet with extended loops on one side and helices on the Kornberg, 1967). This effect of Mn2+ is not direct or apparent in other side. The C-terminal domain contains a six-stranded b-sheet the structural sense (Merckel et al., 2001) and must be a conse- with helices flanking it on both sides. The SA PpaC dimer is formed quence of structural changes that propagate from the active site by adjoining of the two b-sheets of the N-terminal domains into to the dimerization interface. SA PpaC crystallized in the presence one b-sheet, where b-strands b6(Supplementary Fig. 1) from each of Mn2+, whereas no crystals were obtained at the same or other monomer interact with each other in a symmetrical fashion. In conditions without a divalent metal ion. This is only a second addition, the dimer is stabilized by predominantly hydrophobic report of a crystal structure of a family II PPiase in the apo-form. contacts between the loops of the two N-terminal domains and 84 C.S. Gajadeera et al. / Journal of Structural Biology 189 (2015) 81–86

AB

His9

M1

Asp75 Asp13 Asp15

M2 His97 Asp149

Fig.2. The structure of SA PpaC. (A) The dimer of SA PpaC with the two domains of each monomer colored differently (for monomer A in green and yellow and for monomer B in blue and grey). The domains are intimately interacting with each other, representing a closed conformation of the enzyme. The two Mn2+ ions and nucleophilic water are shown in spheres in red and cyan, respectively. (B) The active site of SA PpaC. The blue mesh shows the anomalous difference Fourier map contoured at 3.5r. The key residues involved in the catalysis are shown in sticks and the metal ion coordination as dashed lines. between a loop of the N-terminal domain of one monomer and the bound Mn2+ ion with the presence of two divalent metal ion bind- C-terminal helix of the other monomer. A similar dimeric interface ing sites in homologous apo-proteins (Halonen et al., 2005). These is also observed in other family II PPiases and in horse liver alcohol observations likely mean that in the absence of the substrate, bind- dehydrogenase (Colonna-Cesari et al., 1986; Eklund et al., 1976). ing of one Mn2+ ion to either of the two sites disfavors binding of Unexpectedly, even though no substrate or product is bound in the second ion to the other site. Substrate binding must stabilize the active site, the mutual disposition of the two domains of each simultaneous binding of both metal ions, since both M1 and M2 monomers corresponds to the closed conformation of family II PPi- ions are coordinated to the substrate oxygens, as observed in the ases, as seen in the published structures of the homologs only in crystal structure of B. subtilis PPiase in complex with PNP complexes with PNP, sulfate or chloride (Table 2). The closed con- (Fabrichniy et al., 2007). formation in these previously reported structures, thought to form The geometry of the active site (Fig. 2B) is similar to that of pre- upon substrate binding, ensures appropriate position of the resi- vious structures of PPiase family II enzymes. M1 and M2 metal ions dues of the N- and C-terminal domains for substrate binding and are positioned the same way as in the structures from S. mutans, S. catalysis, as explained below. gordonii and B. subtilis. Ions M1 and M2 are coordinated to four and five ligands, respectively (Fig. 2B). Ion M1 is coordinated with Ne of 3.2. The active site of SA PpaC His9, Od of Asp13, one of Od of Asp75 and a water molecule bridg- ing M1 and M2. Ion M2 is coordinated with Od of Asp15, Ne of The active site of SA PpaC is located at the interface between the His97, Od of Asp149, the other Od of Asp75 and with the bridging

N- and C-terminal domains (Fig. 2A). In the omit Fo–Fc electron water. Coordination with His residues has been noted as a property density map generated prior to including water or other ligands of Mn2+, whereas it is very rare for Mg2+ (Bock et al., 1999). In con- in the structure, two areas of strong density (>9r) in the active site trast to the closed conformation of substrate-bound PPiase of B. were observed that coincided with two strong density peaks in the subtilis, M3 and M4 metal ions were not present because of the anomalous difference Fourier map (>6r; Fig. 2B). No significant absence of a substrate or its analogue, which is normally involved peaks in the anomalous difference Fourier map were present else- in their coordination (Fabrichniy et al., 2007). where. These observations, together with the stabilizing effect of The conformations of Arg295, Lys296 and Lys297 differ dramat- Mn2+ on the dimerization of SA PpaC indicate that the electron ically from their counterparts in previously published structures of density peaks correspond to bound Mn2+ ions. However, the refine- PpaC homologs (Fig. 3, Supplementary Fig. 3). This region is far ment clearly demonstrated that each site is half-occupied, indicat- away from any crystal packing interface (Supplementary Fig. 4) ing that a Mn2+ ion occupies one or the other site in each monomer and; therefore, its conformation is not an artifact of dimer–dimer in the crystal with equal probabilities. The half-occupancy recon- interactions in the crystal. This region of SA PpaC (residues 295– ciles the previous observations of the 1:1 stoichiometry of a tightly 298) lacks secondary structure; its backbone and the side chains of Arg295 and Lys296 are flipped out of the active site and are sol- Table 2 vent-exposed (Fig. 3A). Arg295 forms a surface salt bridge with Active site conformations and ligands of structurally characterized family II Ppiases. Asp261, the residue which is conserved in B. subtilis (Supplemen- Bacterium PDB ID Conformation Bound ligands tary Fig. 1), suggesting that this conformation may be relevant for Staphylococcus aureus 4RPA Closed Mn2+ the B. subtilis homolog as well. In contrast to SA PpaC, in the struc- Bacillus subtilis 1K23 Open Mn2+ ture of B. subtilis PPiase in complex with PNP the respective region is 2+ 2 1WPM Closed Mg ,F ,SO4 a-helical, with the side chains of Arg295 and Lys296 pointing into 2+ 2HAW Closed Mg , PNP the active site ((Fabrichniy et al., 2007); Fig. 3B). The guanidinium 2IW4 Closed Mg2+,Mn2+,Fe3+, PNP Streptococcus agalactiae 2ENX Closed Mg2+ Mn2+, PNP group of Arg295 and the amino group of Lys296 form salt bridges 2+ 2+ 2 Streptococcus mutans 1I74 Closed Mn ,Mg ,SO4 with the phosphate groups of the bound PPi mimic, PNP. In other 2+ 2 Streptococcus gordonii 1K20 Closed Mn ,SO4 homologs, these two residues exhibit similar interactions with 2+ 2 1WPP Closed Zn ,SO4 PNP or inorganic phosphate to those observed for the B. subtilis PPi- 2+ a Methanococcus jannaschii 2EB0 Closed Mn ,Cl ase. Occasional exceptions from this rule is in that one of these two a ACl in this structure can be modeled based on the electron density map and residues forms a hydrogen bond with other active site residues to solvation bond lengths, as described in the text. ensure a proper active site conformation, instead of interacting C.S. Gajadeera et al. / Journal of Structural Biology 189 (2015) 81–86 85

Arg295 M2 M2 M1

PNP

M1 Asp261 Lys296

Arg295 Lys297 Lys297

Lys296 A B

Fig.3. Comparison of the closed forms of the active site in SA PpaC and B. subtilis PPiase. (A) The active site of SA PpaC. (B) The active site of B. subtilis PPiase (PDB ID: 2HAW). The color scheme is the same as in Fig. 2. The bound PNP and fluoride ion are shown in orange. The RKK motif is shown in dark grey. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1.2 25 B A 20 1.0

Wild-type -1 15 0.8 10 2 0.6 5 0 0.4 3 Activity (a. u.) -5 0.2 Mean residue ellipticity R295A (×10 deg cm dmol ) -10 0.0 -15 0.0 0.2 0.4 0.6 0.8 1.0 190 200 210 220 230 2040 25 260 Concentration of PP i (mM) Wavelength (nm)

Fig.4. The pyrophosphatase activity and secondary structural signature of the wild-type and the R295A mutant of SA PpaC. (A) Pyrophosphatase activity of the wild-type (filled circles) and R295A (open circles). The theoretical curves correspond to the best fit of the data to the Michaelis–Menten equation. (B) Circular dichroism spectra for wild-type (solid line) and R295A (dotted line) SA PpaC. with the substrate directly. For example, in PPiase from S. agalacti- consistent with a chloride ion forming a salt bridge with Lys204, ae, the side chain of Lys296 (SA PpaC residue numbering) points rather than a water molecule at this site, in agreement with the away from the substrate and its guanidinium group forms a hydro- closed conformation (Supplementary Fig. 5). The chloride site coin- gen bond with the carbonyl oxygen of His97 (Rantanen et al., 2007). cides exactly with the location of the bridging oxygen atom of a

The DHH motif (residues 96–98) including His97 was demonstrated bound PPi, as predicted by superposing this structure and struc- to be important for the function of phosphoesterase (Koonin and tures of PNP bound homologs. In addition, in place of Asp261, this Tatusov, 1994; Yamagata et al., 2002) and is involved in organizing homolog contains a glycine residue (bacterial homologs contain the active site for the catalysis. In the structures of Streptococcus either an aspartate or a glutamine; Supplementary Fig. 1), which 2 gordonii and S. mutans PPiases in complex with SO4 (mimicking may disfavor the flipped out conformation of the RKK motif seen 3 the enzyme bound to the product, PO4 ), Lys296 is pointing away in SA PpaC. We tested by mutagenesis whether conformational from the substrate binding site forming hydrogen bonds with car- flexibility of this substrate binding region provides this uncoupling. bonyl oxygen atoms of Val116 and His97 (in S. gordonii;(Ahn Indeed, mutation Arg295 to an alanine completely abolishes et al., 2001)) or water (S. mutans;(Fabrichniy et al., 2004)). enzyme activity (Fig. 4A). This observation suggests that Arg295 The structure of SA PPiase is the first example of uncoupling of and, through chemical constraints, Lys296, change their conforma- the closed conformation of the enzyme and substrate binding (no tion to bind PPi substrate to position it for catalysis. substrate or analogue is present in the active site, despite the CD spectra of wild-type SA PpaC and Arg295Ala mutant (Fig. 4B) closed form). In all other structures of bacterial family II PPiases, were consistent with mixed a/b fold of SA PpaC. The superimposed the open conformation of the active site is observed only in the spectra of the wild-type and the mutant proteins indicate that the absence of a substrate, product or their analogue, and, conversely, R295A mutation did not perturb the overall fold of the enzyme. the active site in the closed conformation invariably contains a Therefore, the observed loss of activity was not a result of protein ligand. The only structure of an archaeal representative, the puta- unfolding. tive family II PPiase from M. jannaschii (PDB ID: 2EB0, RIKEN Struc- tural Genomics Initiative), which exhibits the closed active site, 4. Conclusions was refined with a water located at the position of a bridging oxy- gen atom of the PPi substrate. Upon close inspection of the electron A crystal structure of the inorganic pyrophosphatase PpaC from density map, the electron density for this atom and short inter- S. aureus reported herein revealed that the conformation of the atomic distances to the nearby water molecules (2.4 Å) are more enzyme and the occupancy of its active site by a substrate or a 86 C.S. Gajadeera et al. / Journal of Structural Biology 189 (2015) 81–86 product can be uncoupled through an unprecedented conforma- Fabrichniy, I.P., Lehtio, L., Salminen, A., Zyryanov, A.B., Baykov, A.A., Lahti, R., tional change of the conserved substrate positioning motif RKK. Goldman, A., 2004. Structural studies of metal ions in family II : the requirement for a Janus ion. Biochemistry 43, 14403– Moreover, this structure strongly suggests that PPi binds to the 14411. closed state of SA PpaC, which is either fixed or is in equilibrium Fabrichniy, I.P., Lehtio, L., Tammenkoski, M., Zyryanov, A.B., Oksanen, E., Baykov, with the open state observed in the structure of B. subtilis PPiase A.A., Lahti, R., Goldman, A., 2007. A trimetal site and substrate distortion in a family II inorganic pyrophosphatase. J. Biol. Chem. 282, 1422–1431. in the apo-form (the only other structurally investigated apo- Halonen, P., Tammenkoski, M., Niiranen, L., Huopalahti, S., Parfenyev, A.N., enzyme of this family), rather than binding to the open conforma- Goldman, A., Baykov, A., Lahti, R., 2005. Effects of active site mutations on the tion and inducing its closing. Based on this and previously reported metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis. Biochemistry 44, 4004–4010. structures, we propose that the RKK motif likely undergoes an Harding, M.M., 2001. Geometry of metal–ligand interactions in proteins. Acta Cryst. induced-fit conformational change upon PPi binding. These mech- D 57, 401–411. anistic implications are yet to be tested. The observed conforma- Koonin, E.V., Tatusov, R.L., 1994. Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of with diverse tion where this motif is disengaged from the active site may specificity. Application of an iterative approach to database search. J. Mol. serve a target of inhibitors that would lock the enzyme in the inac- Biol. 244, 125–132. tive state. Kornberg, A., 1962. On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions. Academic Press, New York. Kuhn, N.J., Wadeson, A., Ward, S., Young, T.W., 2000. 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