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GPPA Phosphatase from Aquifex aeolicus in Complex with the Alarmone ppGpp
doi:10.1016/j.jmb.2007.11.073
J. Mol. Biol. (2008) 375, 1469–1476
Available online at www.sciencedirect.com
Structure of the PPX/GPPA Phosphatase from Aquifex aeolicus in Complex with the Alarmone ppGpp Ole Kristensen⁎, Birthe Ross and Michael Gajhede Biostructural Research, Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received 19 September 2007; received in revised form 21 November 2007; accepted 27 November 2007 Available online 4 December 2007 Edited by I. Wilson
The crystal structure of the prototype exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family member from Aquifex aeolicus in complex with the intracellular second messenger guanosine tetraphosphate was determined at 2.7-Å resolution. The hydrolytic base is identified as E119. The dual specificity established for the Escherichia coli homolog is shown to be compatible with a common active site for guanosine pentaphosphate and polyphosphate hydrolysis. Distinct and different degrees of closure between the two domains of the enzyme are associated with substrate binding. The arginines R22 and R267, residing in different domains, are crucial for guanosine pentaphosphate specificity as they interact with the unique 3′-ribose phosphorylation. © 2007 Elsevier Ltd. All rights reserved.
Keywords: stringent response; exopolyphosphatase; guanosine pentaphosphate; Aquifex aeolicus
Introduction Metabolism and accumulation of inorganic polyphosphate (polyP) are in bacteria closely connected to a cellular defense mechanism referred to as the stringent response. It is developed to adjust to various stresses and to ensure survival, when the supply of such nutrients as amino acids is abruptly deprived. 1 Guanosine tetraphosphate (ppGpp) functions as a signaling molecule or an alarmone under such circumstances to coordinate changes in transcription, protein synthesis and degradation.2–4 Stringent response and polyP metabolism have been extensively studied in Escherichia coli with respect to all three issues. Transcription is modulated by ppGpp binding to RNA polymerase.2,5 With respect to protein synthesis, limiting the amount of amino acids will increase chances that uncharged transfer RNA molecules associate with ribosomes. In turn, this *Corresponding author. E-mail address: [email protected]. Abbreviations used: polyP, polyphosphate; ppGpp, guanosine tetraphosphate; SF, stringent factor; pppGpp, guanosine pentaphosphate; GPPA, guanosine pentaphosphate phosphohydrolase; PPX, exopolyphosphatase; PPK, polyphosphate kinase; PDB, Protein Data Bank; smPPK, 3′-pyrophosphokinase purified from Streptomyces morookaensis culture medium.
causes activation of the ribosome-bound protein “stringent factor” (SF or RelA).6,7 SF catalyzes a pyrophosphoryl transfer from ATP to the 3′-hydroxyl group of GTP, forming guanosine pentaphosphate (pppGpp).8,9 The E. coli gppA gene product [guanosine pentaphosphate phosphohydrolase (GPPA)] regulates the conversion of pppGpp to the alarmone ppGpp.10,11 Increased levels of ppGpp are known to regulate the RNA polymerase and to inhibit exopolyphosphatase (PPX) activity.11,12 Reduced PPX activity shifts the equilibrium between polyP breakdown and accumulation.3,4 Synthesis of polyP is catalyzed by polyP kinase (PPK). Subsequently, association of polyP with the Lon protease causes degradation of free ribosomal protein to compensate for the lack of amino acids in the cell. The ppk and ppx genes constitute a polyP operon in E. coli.13 Crystal structures of the E. coli PPX protein were determined recently, providing insight into polyP degradation.12,14 Different suggestions were presented in these studies concerning the bifunctional aspect of PPX/GPPA (i.e., pppGpp and polyP hydrolysis); one study suggested two separate active sites,14 and the other was in favor of a common active site.12 Additional information on the stringent response is available from the extremophile Aquifex aeolicus. This organism completely lacks PPK-coding genes13 and only has a single two-domain PPX/GPPA protein family member.15,16 This suggests that the
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Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
primary function of the A. aeolicus PPX/GPPA protein is to provide GPPA activity. We have previously determined crystal structures of the apoenzyme from different crystal forms.16 Here we present the crystal structure of the A. aeolicus PPX/ GPPA enzyme in complex with its reaction product, ppGpp.
Results and Discussion Overall structure of the Aquifex PPX/GPPA enzyme The crystal structure of the A. aeolicus PPX/GPPA mutant Y19N–ppGpp complex was determined at 2.7-Å resolution (see Table 1). The Y19N mutant was chosen for this study based on sequence comparisons (Fig. 1a). These showed that asparagine is found most frequently at this position in orthologue enzymes. Two molecules (A and B) were contained in the asymmetric unit of the crystal. Molecule A represents a ligand-bound form of the enzyme, whereas molecule B has no ligand bound in the
Table 1. Data reduction and refinement statistics PPX/GPPA (Y19N)–ppGpp Data collection X-ray source, wavelength (Å) Space group Cell dimensions (Å) Monomer per asymmetric unit Resolution range (Å) Unique reflections Average multiplicity Completeness (%) 〈I/σI〉 Rmergeb (%) Refinement Rwork/Rfreec (%) No. of atoms Protein Ligand Water Average B-factor (Å2) Protein Ligand Water rmsd Bond lengths (Å) Angles (°) Ramachandran plotsd Most favored regions (%) Additional allowed regions (%) a
I711/MaxLab, 1.085 P3221 a = b = 70.5, c = 223.7 2 74.5–2.71 (2.86–2.71)a 17,675 (2285) 4.9 (4.3) 96.8 (88.9) 10.5 (3.2) 11.3 (44.5) 21.2/29.6 4792 36 170 43.6 65.5 39.7 0.007 1.4 87.7 10.9
Values in parentheses refer to the highest-resolution bin. Rmerge = ∑hkl(∑i(|Ihkl, i − 〈Ihkl〉|)) / ∑hkl, i〈Ihkl〉, where Ihkl, i is the intensity of an individual measurement of the reflection with Miller indices h, k, and l and 〈Ihkl〉 is the mean intensity of that reflection. c Rwork = ∑hkl(‖Fobs, hkl|−|Fcalc, hkl‖) /|Fobs, hkl|, where |Fobs, hkl| and |Fcalc, hkl| are the observed and calculated structure factor amplitudes, respectively. Rfree is equivalent to Rwork but is calculated with 2.5% of the reflections omitted from the refinement process. d Values from PROCHECK.17 b
active site. The two molecules are both in an “open” conformation; however, molecule B has a slightly more closed configuration. The conformation of the ligand-bound molecule A is very similar to what we have observed previously in studies of apo-PPX/ GPPA [Protein Data Bank (PDB) codes 1t6c and 1t6d (molecule A)]. The structure is shown in cartoon representation in Fig. 1b. The structure is composed of two domains, each with a fold that contains a mixed five-stranded β-sheet with helical segments located on one side of the sheet. This architecture characterizes the actin-like ATPase domain superfamily. Within the superfamily of actin-like ATPase domain proteins, domain movements up to 30° are observed to be closely linked to the catalytic function of the enzymes.21,22 Such flexibility was also noted in the previous structure determinations of the A. aeolicus PPX/GPPA protein.16 Here, rotational movement of the two domains around a single hinge region (Fig. 1b) indicated that access to the active site, which is located at the interface between the two domains, could be achieved through a “butterfly-like” cleft opening. Comparisons of E. coli PPX and A. aeolicus PPX/GPPA structures Crystal structures of the E. coli PPX were determined recently from two crystal forms.12,14 The structure presented by Rangarajan et al.14 (PDB code 2FLO) was crystallized in the absence of potential ligands, while the structure obtained by Alvarado et al.12 (PDB code 1U6Z) contained a number of bound sulfate ions. The two E. coli structures are highly similar with an rmsd of 0.9 Å, including Cα atoms of 479 identical residues. The four-domain E. coli PPX enzyme forms a strong dimer. Domains I and II display close structural similarity to the A. aeolicus PPX/GPPA enzyme.12,14 However, in the crystal structures of E. coli PPX, these two domains are close together, and Rangarajan et al. referred to this as the “closed” state,14 as compared with the “open”-state conformation revealed by the A. aeolicus PPX/GPPA crystal structures.16 The significance of such structural flexibility of the E. coli PPX domains I and II was recognized by Rangarajan et al.14 but apparently not noted by Alvarado et al.12 The E. coli PPX enzyme has been shown to possess both PPX and GPPA activities.10–12 Both studies share similar interpretations of the PPX phosphatase activity and polyP substrate recognition. However, the bifunctional aspect of pppGpp hydrolysis was linked to the presence of two active sites in one study12 but was in favor of a common active site in the other.14 Alvarado et al.12 argued by comparison with the ATP-binding protein FtsA that the E. coli PPX side chains (residues N21, C169 and R267) would clash with the ribose and purine bases of a bound nucleotide. The authors concluded that the domain I/domain II interface specifically allows polyP
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
1471
Fig. 1. (a) Sequence of A. aeolicus PPX/GPPA. Twenty of the most similar orthologue PPX/GPPA sequences were aligned by CLUSTAL W.16,18 Residues boxed and shown in black are fully conserved in all 20 sequences, and those shown in dark gray represent other highly conserved positions. The extremophile A. aeolicus has unusual residues at some positions. The identities of the most dominant amino acids at these positions are indicated below the sequence. (b) Overall structure of the A. aeolicus PPX/GPPA enzyme. The structure is composed of two domains that form a cleft around the active site. The ligand-bound form (molecule A) of the protein is shown in cartoon representation. The ligand ppGpp (sticks) is shown inside a mesh of bias-reduced electron density19 and contoured at a 1.3-σ level. The flexible hinge region (residues 121–124) as identified by the program DYNDOM20 is shown in red on the central helix, and the mutated residue Y19N is shown in blue.
binding as well as occludes nucleotide binding and implied two active sites. However, this is in conflict with the finding that (p)ppGpp is an inhibitor of PPX activity.11,12 The structure of the A. aeolicus PPX/GPPA alarmone complex provides support to evaluate likely conformational changes in the E. coli PPX
enzyme and consequences for nucleotide binding. As in the FtsA–PPX comparison mentioned above, superposition of domain I of the A. aeolicus and E. coli structures reveals that clashes would prevent (p)ppGpp binding given a static conformation of the enzymes (Fig. 2a). However, as shown previously,16 A. aeolicus PPX/GPPA has an inherent flexibility
1472
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
Fig. 2. (a) Suggested nucleotide occlusion at the PPX domain I/domain II interface. Structures of ligand-bound A. aeolicus PPX/GPPA (red) and E. coli PPX (blue)14 have been superimposed using Cα positions of domain I. A large conformational domain I/domain II shift is observed between the two structures; the ligand-bound conformation (red) represents an “open” state, whereas the E. coli structure is found in a “closed” state. Steric clashes (black, E. coli residues N21, C169 and R267) indicate that the closed state is incompatible with nucleotide binding. The A. aeolicus side chain of N19, corresponding to N21 in E. coli, is shown in red. (b) Flexibility allows (p)ppGpp binding to the E. coli PPX domain I/domain II active site. Superposition of the individual E. coli PPX domains I and II (blue) on the A. aeolicus alarmone complex structure reveals that the two proteins share a highly similar fold. A relative rotational movement (22.5°) of the E. coli PPX domains I and II is sufficient to switch the structure from the closed to the open state. The open conformation of E. coli PPX seems to allow (p)ppGpp binding without steric conflicts.
characterized by a simple relative hinge motion of its two domains (Fig. 1b) and only the “open”-state conformation is compatible with (p)ppGpp ligand binding (Fig. 2b). Also, superposition of each of the E. coli PPX domains I and II on the ligand-bound A. aeolicus structure is perfectly compatible with similar conformational flexibility around a common hinge region (Fig. 2b), as suggested by Rangarajan et al.14 The rotational movement of domains I and II required to shift the E. coli PPX structure from the “closed” to the “open” state was found to be 22.5°. The “open” state of the PPX enzyme allows (p)ppGpp ligand binding without apparent clashes. Domain III of E. coli PPX The six-helix domain III is structurally similar to proteins of the HD phosphohydrolase superfamily, which is characterized by a conserved metal-binding histidine:aspartate residue pair involved in catalysis. The structural analysis by Alvarado et al. identified domain III as a second PPX active site responsible for pppGpp hydrolysis.12 Rangarajan et al. also noted that domain III belongs to this superfamily but pointed to a lack of conservation of the catalytically important residues in a comparison with the related SpoT protein, suggesting that domain III in PPX does not exhibit enzymatic activity.14 Our study supports that the E. coli PPX enzyme accommodates alarmone hydrolysis in the domain I/domain II active site cleft. The contradicting studies both implicate domain III as the major contributor to dimerization and assign it a prominent role in aqueduct formation, polyP channeling and enzymatic processivity. Thus, there are
several reasons why this domain has been conserved through evolution also in the absence of any associated enzymatic activity. Hydrolysis of pppGpp The bifunctional aspect of PPX/GPPA enzymes implicates phosphatase activity, whether it acts on polyP or on pppGpp substrates. In this setup, it is not surprising that the mechanism for both activities could share some features and a common active site. Both activities are most thoroughly investigated in E. coli but remain to be functionally verified in A. aeolicus. Mutational data and PPX activity analysis of E. coli PPX was presented only recently,12 while similar analysis of mutants on (p)ppGpp activity has not been performed. Alanine substitutions at positions E121, D143 or E150 in E. coli PPX resulted in substantial loss of PPX activity, and the E121A mutant was most seriously affected. The E121 position corresponds to E119 in A. aeolicus, and we previously predicted it to be a key residue in catalysis.16 In the PPX/GPPA–ppGpp structure, the side chain of E119 is located approximately 6 Å from the 5′-β-phosphate of the bound nucleotide, providing sufficient space for the 5′-γ-phosphate of the pppGpp substrate to be positioned properly for hydrolytic attack by E119 either directly or via a water molecule that could potentially be activated by the same residue (Fig. 3). In contrast to the previous structures of the A. aeolicus PPX/GPPA enzyme, no metal ion is observed bound to the active site glycine-rich loop, despite that calcium was deliberately added. That calcium is bound neither to the nucleotide-bound
1473
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
Fig. 3. (a) Surface representation of the ppGpp nucleotide bound to the A. aeolicus active site. The nucleotide is trapped by the “open” conformation of the enzyme in a cleft at the domain I/domain II interface. There is sufficient space at the bottom of the binding site to accommodate the 5′-γ-phosphate of the cognate substrate pppGpp. (b) Surface representation of the modeled substrate pppGpp in the PPX/ GPPA active site. The substrate is positioned with its terminal 5′-γphosphate fitted into a pocket at the bottom of the binding site. (c) Stereo view of the ppGpp alarmone bound to the A. aeolicus PPX/GPPA active site. The 3′-pyrophosphate specificity anchor is chelated between the side chains of R22 and R267 and recognized by hydrogen bonding to the G144 and G211 main chain amides. (d) Modeling of the substrate pppGpp in the active site shown in stereo view. All parts of the ligand were fixed at the positions indicated by the PPX/GPPA– ppGpp complex structure except for the 5′-triphosphate moiety, which was allowed to move freely under the restraint of the applied force field. The active site accommodates the 5′-γ-phosphate of the substrate by positioning it close to the anticipated active site base E119.
molecule A nor to the apo form of molecule B in the present structure suggests that divalent cations required for optimal activity23,24 may be involved in a more transient manner during catalysis or possibly be chelated by unbound nucleotide. Substrate specificity by 3′-ribose phosphorylation The previous structures of the apoenzyme 16 indicated by the presence of bound chloride ions a likely position of the 3′-phosphate moiety of the substrate in the active site. This suggestion is confirmed by structure determination of the ppGpp enzyme complex (Fig. 3). It can also be seen that the arginine residues R22 and R267, located at opposite sides of the active site cleft, are intimately involved
in binding the 3′-pyrophosphate. This shows that a certain domain arrangement of the enzymes is required for recognition of this hallmark substrate feature. Further conformational adaptability to substrate binding is provided by the two glycinerich segments. Backbone amides of G144, which is part of the central Walker B loop region,16 and G211 contribute direct hydrogen bonds to the terminal 3′-phosphate. A glycine residue is also found at position 210, thereby further relaxing conformational restraints upon interaction with the alarmone 3′-specificity tail. Interactions with the nucleotide base R266 stacking over the nucleotide base is supported by locking its position through hydrogen
1474
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
bonding to the 2′-oxygen of the ribose (Fig. 3c); similar geometrical arrangements are often observed in nucleotide binding proteins.25 Y19 was mutated to the superfamily consensus residue (Fig. 1a) as a wild-type ppGpp complex could not be obtained (data not shown). Other highly conserved positions in the PPX/GPPA protein family that happen to be occupied by unusual residues in the A. aeolicus sequence are located outside the active site region. The N19 side chain is positioned to participate in direct interactions with N7 of the base. Occurrence of a tyrosine at position 19 in A. aeolicus PPX/GPPA appears to be sterically unfavorable for ligand binding at ambient temperature, but it is possible that active site geometry is slightly different at the normal growth temperature of this extremophile organism. Furthermore, the PPX/GPPA–ppGpp complex provides insight into the question of selective processing of purine pentaphosphate substrates. Adenine and guanine bases differ at the N6 position by having amino group and carbonyl substituents, respectively. However, in the structure of the ppGpp complex, the N6 carbonyl is solvent exposed with no obvious discriminating interaction, thus making it unlikely to contribute to specific base recognition. Also, the N2 positions of the two purine bases differ in the sense that guanine carries an exocyclic amine group at this position. The side chain of E264 is located at an angle to the guanine N2 amino group and could possibly participate in hydrogen bonding,26 albeit at a distance (3.8 Å) that suggests a very weak interaction. However, it is possible that a guanine base could have stronger electrostatic interactions compared with adenine due to the difference at the N2 position. Differentiation may not be important, however, since, at least in E. coli, hyperphosphorylated adenine nucleotides do not appear to accumulate under stringent response.27
Conclusions The present structure determination of the A. aeolicus PPX/GPPA product complex reveals the nucleotide position and indicates how the 5′-γphosphate of the pppGpp substrate can be properly accommodated for hydrolytic attack involving the key residue E119. This is in agreement with previous predictions based on analysis of the apoenzyme.16 It is shown that a simple hinge movement of domains I and II is sufficient to bring the E. coli PPX enzyme into an “open” state that is compatible with polyP and pppGpp hydrolysis at a common active site. The importance of the 3′-pyrophosphate tail and that of purine base recognition in substrate specificity were discussed. Modeling of the extracellular portion of human nucleoside triphosphate diphosphohydrolase 328 was based on our previous apo-PPX/GPPA structures,16 and these efforts may also benefit from the current insight.
Materials and Methods Preparation of ppGpp ATP nucleotide 3′-pyrophosphokinase was purified from the medium of a Streptomyces morookaensis culture (smPPK).29 smPPK was used to prepare ppGpp according to a published procedure.30 A 10-ml reaction (125 μl of smPPK, 1 ml of 1 M glycine, pH 10, 200 μl of 1 M MgCl2, 2 ml of 100 mM GDP, 1 ml of 150 mM ATP, 125 μl of [γ-32P] ATP and 5.55 ml of H2O) was incubated for 1 h at 37 °C. Crude ppGpp was purified by diethylaminoethyl chromatography (buffer: 0.01–1.5 M gradient in triethylamine bicarbonate buffer saturated with CO2), and the ppGppcontaining fractions were identified by detection of radioactivity and thin-layer chromatography. The ppGpp preparation was freeze-dried and dissolved in water. Concentrations of ppGpp were estimated assuming that the UV properties were identical with those of a GDP reference solution. Expression and purification Expression and purification of the A. aeolicus PPX/GPPA enzyme have been described previously.16,31 In summary, the gene was amplified by PCR from genomic DNA15 and cloned as an N-terminal hexahistidine-tagged construct in the vector pET28a (Novagen). A mutated variant (Y19N) including residues Pro7–Asn310 was prepared using PCR (primers: 5′-GCTTAGCTGGAAGCATATGCCAATTATGAGGGTGGCGTCCATAGACATAGGCTCCAACTCC-3′ and 5′-GCTTAGCTGGAAGCGGCCGCTTATTAATTTTCCTTTAATACTT-3′). The protein used in this study (Y19N variant) was purified on nickel-affinity resin followed by hexahistidine tag removal (thrombin) and gel filtration (Superdex 200: 5 mM Tris–HCl, pH 8.0, 100 mM NaCl and 1 mM DTT). Calcium chloride and ppGpp were added to the sample at concentrations of 0.5 and 3 mM, respectively. The sample was concentrated to 2 mg/ml prior to crystallization. Crystallization and data collection The A. aeolicus PPX/GPPA mutant Y19N–ppGpp complex was crystallized using the hanging-drop vapor diffusion method by mixing 2 μl of protein and 2 μl of reservoir solutions. The reservoir solution contained 200 mM sodium acetate (pH 6.4), 100 mM 4-morpholineethanesulfonic acid (pH 5.7) and 50% methyl-2,4pentanediol. Diffraction data were collected at MaxLab (Lund, Sweden) with beamline I711 (Table 1) and processed using the program XDS.32 Various CCP4 programs were used to prepare the structure factor files.33 Structure determination and refinement The structure was solved by molecular replacement using the CNS software34 and the apo-PPX/GPPA structure (PDB code 1T6C) as a search model. The CNS program was used at all stages of refinement (Table 1). Initially, the automated refinement procedure implemented in the program LAFIRE35 was used, followed by alternating rounds of minimization and manual model building using the software Coot.36 The PRODRG server37 was used to generate ligand parameters. The Bias Removal Server was used to produce electron density
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex maps with reduced model bias.19 The program DYNDOM was used to analyze structural flexibility.20 Figures were prepared using PyMOL38 and ALSCRIPT.39 9. Modeling A tentative position for the terminal 5′-γ-phosphate of the substrate was obtained by modeling using the location of the product ppGpp as a guide. First, GRID40 phosphate probe calculations were performed to map favorable phosphate positions. The 5′-triphosphate tail was manually moved to match the GRID results. Parameter and topology data were obtained from the PRODRG server,37 and the structure of the PPX/GPPA–pppGpp complex was refined using the program CNS.34 The ligand ribose, base and 3′-pyrophosphate parts as well as all protein atoms were fixed in the conjugate gradient minimization, while no harmonic restraint was applied to the 5′-tail. PDB accession number
10.
11.
12.
13.
Crystallographic coordinates and structure factors have been deposited to the PDB with accession code 2j4r.
14.
Acknowledgements
15.
This work was supported by DANSYNC, the Lundbeck Foundation, the Danish Natural Science Research Council and the Danish Medical Research Council. We appreciate the support in data collection provided by the staff at MaxLab (beamline I711, Lund, Sweden).
16.
17.
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1475 Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. J. Bacteriol. 183, 570–579. Hogg, T., Mechold, U., Malke, H., Cashel, M. & Hilgenfeld, R. (2004). Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response. Cell, 117, 57–68. Keasling, J. D., Bertsch, L. & Kornberg, A. (1993). Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase. Proc. Natl Acad. Sci. USA, 90, 7029–7033. Kuroda, A., Murphy, H., Cashel, M. & Kornberg, A. (1997). Guanosine tetra- and pentaphosphate promote accumulation of inorganic polyphosphate in Escherichia coli. J. Biol. Chem. 272, 21240–21243. Alvarado, J., Ghosh, A., Janovitz, T., Jauregui, A., Hasson, M. S. & Sanders, D. A. (2006). Origin of exopolyphosphatase processivity: fusion of an ASKHA phosphotransferase and a cyclic nucleotide phosphodiesterase homolog. Structure, 14, 1263–1272. Zhang, H., Ishige, K. & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proc. Natl Acad. Sci. USA, 99, 16678–16683. Rangarajan, E. S., Nadeau, G., Li, Y., Wagner, J., Hung, M. N., Schrag, J. D. et al. (2006). The structure of the exopolyphosphatase (PPX) from Escherichia coli O157: H7 suggests a binding mode for long polyphosphate chains. J. Mol. Biol. 359, 1249–1260. Deckert, G., Warren, P. V., Gaasterland, T., Young, W. G., Lenox, A. L., Graham, D. E. et al. (1998). The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature, 392, 353–358. Kristensen, O., Laurberg, M., Liljas, A., Kastrup, J. S. & Gajhede, M. (2004). Structural characterization of the stringent response related exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family. Biochemistry, 43, 8894–8900. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Reddy, V., Swanson, S. M., Segelke, B., Kantardjieff, K. A., Sacchettini, J. C. & Rupp, B. (2003). Effective electron-density map improvement and structure validation on a Linux multi-CPU web cluster: The TB Structural Genomics Consortium Bias Removal Web Service. Acta Crystallogr., Sect. D: Biol. Crystallogr. 59, 2200–2210. Hayward, S. & Berendsen, H. J. C. (1998). Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme. Proteins: Struct. Funct. Genet. 30, 144–154. Bork, P., Sander, C. & Valencia, A. (1992). An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc. Natl Acad. Sci. USA, 89, 7290–7294. Schuler, H. (2001). ATPase activity and conformational changes in the regulation of actin. Biochim. Biophys. Acta, 1549, 137–147. Akiyama, M., Crooke, E. & Kornberg, A. (1993). An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J. Biol. Chem. 268, 633–639.
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Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
24. Hara, A. & Sy, J. (1983). Guanosine 5′-triphosphate, 3′-diphosphate 5′-phosphohydrolase. Purification and substrate specificity. J. Biol. Chem. 258, 1678–1683. 25. Flocco, M. M. & Mowbray, S. L. (1994). Planar stacking interactions of arginine and aromatic side-chains in proteins. J. Mol. Biol. 235, 709–717. 26. Fabiola, F., Bertram, R., Korostelev, A. & Chapman, M. S. (2002). An improved hydrogen bond potential: impact on medium resolution protein structures. Protein Sci. 11, 1415–1423. 27. Nishino, T., Gallant, J., Shalit, P., Palmer, L. & Wehr, T. (1979). Regulatory nucleotides involved in the Rel function of Bacillus subtilis. J. Bacteriol. 140, 671–679. 28. Ivanenkov, V. V., Meller, J. & Kirley, T. L. (2005). Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling. Biochemistry, 44, 8998–9012. 29. Oki, T., Yoshimoto, A., Ogasawara, T., Sato, S. & Takamatsu, A. (1976). Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase. Arch. Microbiol. 107, 183–187. 30. Oki, T., Yoshimoto, A., Sato, S. & Takamatsu, A. (1975). Purine nucleotide pyrophosphotransferase from Streptomyces morookaensis, capable of synthesizing pppApp and pppGpp. Biochim. Biophys. Acta, 410, 262–272. 31. Kristensen, O., Laurberg, M. & Gajhede, M. (2002). Crystallization of a stringent response factor from Aquifex aeolicus. Acta Crystallogr., Sect. D: Biol. Crystallogr. 58, 1198–1200.
32. Kabsch, W. (1993). Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800. 33. Collaborative Computational Project, Number 4. (1994). The CCP4 Suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760–763. 34. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 54, 905–921. 35. Yao, M., Zhou, Y. & Tanaka, I. (2006). LAFIRE: software for automating the refinement process of protein-structure analysis. Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 189–196. 36. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132. 37. Schuttelkopf, A. W. & van Aalten, D. M. F. (2004). PRODRG: a tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 1355–1363. 38. DeLano, W. L. (2002). The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA. 39. Barton, G. J. (1993). ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37–40. 40. Goodford, P. J. (1985). A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. J. Med. Chem. 28, 849–857.
J. Mol. Biol. (2008) 375, 1469–1476
Available online at www.sciencedirect.com
Structure of the PPX/GPPA Phosphatase from Aquifex aeolicus in Complex with the Alarmone ppGpp Ole Kristensen⁎, Birthe Ross and Michael Gajhede Biostructural Research, Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received 19 September 2007; received in revised form 21 November 2007; accepted 27 November 2007 Available online 4 December 2007 Edited by I. Wilson
The crystal structure of the prototype exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family member from Aquifex aeolicus in complex with the intracellular second messenger guanosine tetraphosphate was determined at 2.7-Å resolution. The hydrolytic base is identified as E119. The dual specificity established for the Escherichia coli homolog is shown to be compatible with a common active site for guanosine pentaphosphate and polyphosphate hydrolysis. Distinct and different degrees of closure between the two domains of the enzyme are associated with substrate binding. The arginines R22 and R267, residing in different domains, are crucial for guanosine pentaphosphate specificity as they interact with the unique 3′-ribose phosphorylation. © 2007 Elsevier Ltd. All rights reserved.
Keywords: stringent response; exopolyphosphatase; guanosine pentaphosphate; Aquifex aeolicus
Introduction Metabolism and accumulation of inorganic polyphosphate (polyP) are in bacteria closely connected to a cellular defense mechanism referred to as the stringent response. It is developed to adjust to various stresses and to ensure survival, when the supply of such nutrients as amino acids is abruptly deprived. 1 Guanosine tetraphosphate (ppGpp) functions as a signaling molecule or an alarmone under such circumstances to coordinate changes in transcription, protein synthesis and degradation.2–4 Stringent response and polyP metabolism have been extensively studied in Escherichia coli with respect to all three issues. Transcription is modulated by ppGpp binding to RNA polymerase.2,5 With respect to protein synthesis, limiting the amount of amino acids will increase chances that uncharged transfer RNA molecules associate with ribosomes. In turn, this *Corresponding author. E-mail address: [email protected]. Abbreviations used: polyP, polyphosphate; ppGpp, guanosine tetraphosphate; SF, stringent factor; pppGpp, guanosine pentaphosphate; GPPA, guanosine pentaphosphate phosphohydrolase; PPX, exopolyphosphatase; PPK, polyphosphate kinase; PDB, Protein Data Bank; smPPK, 3′-pyrophosphokinase purified from Streptomyces morookaensis culture medium.
causes activation of the ribosome-bound protein “stringent factor” (SF or RelA).6,7 SF catalyzes a pyrophosphoryl transfer from ATP to the 3′-hydroxyl group of GTP, forming guanosine pentaphosphate (pppGpp).8,9 The E. coli gppA gene product [guanosine pentaphosphate phosphohydrolase (GPPA)] regulates the conversion of pppGpp to the alarmone ppGpp.10,11 Increased levels of ppGpp are known to regulate the RNA polymerase and to inhibit exopolyphosphatase (PPX) activity.11,12 Reduced PPX activity shifts the equilibrium between polyP breakdown and accumulation.3,4 Synthesis of polyP is catalyzed by polyP kinase (PPK). Subsequently, association of polyP with the Lon protease causes degradation of free ribosomal protein to compensate for the lack of amino acids in the cell. The ppk and ppx genes constitute a polyP operon in E. coli.13 Crystal structures of the E. coli PPX protein were determined recently, providing insight into polyP degradation.12,14 Different suggestions were presented in these studies concerning the bifunctional aspect of PPX/GPPA (i.e., pppGpp and polyP hydrolysis); one study suggested two separate active sites,14 and the other was in favor of a common active site.12 Additional information on the stringent response is available from the extremophile Aquifex aeolicus. This organism completely lacks PPK-coding genes13 and only has a single two-domain PPX/GPPA protein family member.15,16 This suggests that the
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
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Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
primary function of the A. aeolicus PPX/GPPA protein is to provide GPPA activity. We have previously determined crystal structures of the apoenzyme from different crystal forms.16 Here we present the crystal structure of the A. aeolicus PPX/ GPPA enzyme in complex with its reaction product, ppGpp.
Results and Discussion Overall structure of the Aquifex PPX/GPPA enzyme The crystal structure of the A. aeolicus PPX/GPPA mutant Y19N–ppGpp complex was determined at 2.7-Å resolution (see Table 1). The Y19N mutant was chosen for this study based on sequence comparisons (Fig. 1a). These showed that asparagine is found most frequently at this position in orthologue enzymes. Two molecules (A and B) were contained in the asymmetric unit of the crystal. Molecule A represents a ligand-bound form of the enzyme, whereas molecule B has no ligand bound in the
Table 1. Data reduction and refinement statistics PPX/GPPA (Y19N)–ppGpp Data collection X-ray source, wavelength (Å) Space group Cell dimensions (Å) Monomer per asymmetric unit Resolution range (Å) Unique reflections Average multiplicity Completeness (%) 〈I/σI〉 Rmergeb (%) Refinement Rwork/Rfreec (%) No. of atoms Protein Ligand Water Average B-factor (Å2) Protein Ligand Water rmsd Bond lengths (Å) Angles (°) Ramachandran plotsd Most favored regions (%) Additional allowed regions (%) a
I711/MaxLab, 1.085 P3221 a = b = 70.5, c = 223.7 2 74.5–2.71 (2.86–2.71)a 17,675 (2285) 4.9 (4.3) 96.8 (88.9) 10.5 (3.2) 11.3 (44.5) 21.2/29.6 4792 36 170 43.6 65.5 39.7 0.007 1.4 87.7 10.9
Values in parentheses refer to the highest-resolution bin. Rmerge = ∑hkl(∑i(|Ihkl, i − 〈Ihkl〉|)) / ∑hkl, i〈Ihkl〉, where Ihkl, i is the intensity of an individual measurement of the reflection with Miller indices h, k, and l and 〈Ihkl〉 is the mean intensity of that reflection. c Rwork = ∑hkl(‖Fobs, hkl|−|Fcalc, hkl‖) /|Fobs, hkl|, where |Fobs, hkl| and |Fcalc, hkl| are the observed and calculated structure factor amplitudes, respectively. Rfree is equivalent to Rwork but is calculated with 2.5% of the reflections omitted from the refinement process. d Values from PROCHECK.17 b
active site. The two molecules are both in an “open” conformation; however, molecule B has a slightly more closed configuration. The conformation of the ligand-bound molecule A is very similar to what we have observed previously in studies of apo-PPX/ GPPA [Protein Data Bank (PDB) codes 1t6c and 1t6d (molecule A)]. The structure is shown in cartoon representation in Fig. 1b. The structure is composed of two domains, each with a fold that contains a mixed five-stranded β-sheet with helical segments located on one side of the sheet. This architecture characterizes the actin-like ATPase domain superfamily. Within the superfamily of actin-like ATPase domain proteins, domain movements up to 30° are observed to be closely linked to the catalytic function of the enzymes.21,22 Such flexibility was also noted in the previous structure determinations of the A. aeolicus PPX/GPPA protein.16 Here, rotational movement of the two domains around a single hinge region (Fig. 1b) indicated that access to the active site, which is located at the interface between the two domains, could be achieved through a “butterfly-like” cleft opening. Comparisons of E. coli PPX and A. aeolicus PPX/GPPA structures Crystal structures of the E. coli PPX were determined recently from two crystal forms.12,14 The structure presented by Rangarajan et al.14 (PDB code 2FLO) was crystallized in the absence of potential ligands, while the structure obtained by Alvarado et al.12 (PDB code 1U6Z) contained a number of bound sulfate ions. The two E. coli structures are highly similar with an rmsd of 0.9 Å, including Cα atoms of 479 identical residues. The four-domain E. coli PPX enzyme forms a strong dimer. Domains I and II display close structural similarity to the A. aeolicus PPX/GPPA enzyme.12,14 However, in the crystal structures of E. coli PPX, these two domains are close together, and Rangarajan et al. referred to this as the “closed” state,14 as compared with the “open”-state conformation revealed by the A. aeolicus PPX/GPPA crystal structures.16 The significance of such structural flexibility of the E. coli PPX domains I and II was recognized by Rangarajan et al.14 but apparently not noted by Alvarado et al.12 The E. coli PPX enzyme has been shown to possess both PPX and GPPA activities.10–12 Both studies share similar interpretations of the PPX phosphatase activity and polyP substrate recognition. However, the bifunctional aspect of pppGpp hydrolysis was linked to the presence of two active sites in one study12 but was in favor of a common active site in the other.14 Alvarado et al.12 argued by comparison with the ATP-binding protein FtsA that the E. coli PPX side chains (residues N21, C169 and R267) would clash with the ribose and purine bases of a bound nucleotide. The authors concluded that the domain I/domain II interface specifically allows polyP
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
1471
Fig. 1. (a) Sequence of A. aeolicus PPX/GPPA. Twenty of the most similar orthologue PPX/GPPA sequences were aligned by CLUSTAL W.16,18 Residues boxed and shown in black are fully conserved in all 20 sequences, and those shown in dark gray represent other highly conserved positions. The extremophile A. aeolicus has unusual residues at some positions. The identities of the most dominant amino acids at these positions are indicated below the sequence. (b) Overall structure of the A. aeolicus PPX/GPPA enzyme. The structure is composed of two domains that form a cleft around the active site. The ligand-bound form (molecule A) of the protein is shown in cartoon representation. The ligand ppGpp (sticks) is shown inside a mesh of bias-reduced electron density19 and contoured at a 1.3-σ level. The flexible hinge region (residues 121–124) as identified by the program DYNDOM20 is shown in red on the central helix, and the mutated residue Y19N is shown in blue.
binding as well as occludes nucleotide binding and implied two active sites. However, this is in conflict with the finding that (p)ppGpp is an inhibitor of PPX activity.11,12 The structure of the A. aeolicus PPX/GPPA alarmone complex provides support to evaluate likely conformational changes in the E. coli PPX
enzyme and consequences for nucleotide binding. As in the FtsA–PPX comparison mentioned above, superposition of domain I of the A. aeolicus and E. coli structures reveals that clashes would prevent (p)ppGpp binding given a static conformation of the enzymes (Fig. 2a). However, as shown previously,16 A. aeolicus PPX/GPPA has an inherent flexibility
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Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
Fig. 2. (a) Suggested nucleotide occlusion at the PPX domain I/domain II interface. Structures of ligand-bound A. aeolicus PPX/GPPA (red) and E. coli PPX (blue)14 have been superimposed using Cα positions of domain I. A large conformational domain I/domain II shift is observed between the two structures; the ligand-bound conformation (red) represents an “open” state, whereas the E. coli structure is found in a “closed” state. Steric clashes (black, E. coli residues N21, C169 and R267) indicate that the closed state is incompatible with nucleotide binding. The A. aeolicus side chain of N19, corresponding to N21 in E. coli, is shown in red. (b) Flexibility allows (p)ppGpp binding to the E. coli PPX domain I/domain II active site. Superposition of the individual E. coli PPX domains I and II (blue) on the A. aeolicus alarmone complex structure reveals that the two proteins share a highly similar fold. A relative rotational movement (22.5°) of the E. coli PPX domains I and II is sufficient to switch the structure from the closed to the open state. The open conformation of E. coli PPX seems to allow (p)ppGpp binding without steric conflicts.
characterized by a simple relative hinge motion of its two domains (Fig. 1b) and only the “open”-state conformation is compatible with (p)ppGpp ligand binding (Fig. 2b). Also, superposition of each of the E. coli PPX domains I and II on the ligand-bound A. aeolicus structure is perfectly compatible with similar conformational flexibility around a common hinge region (Fig. 2b), as suggested by Rangarajan et al.14 The rotational movement of domains I and II required to shift the E. coli PPX structure from the “closed” to the “open” state was found to be 22.5°. The “open” state of the PPX enzyme allows (p)ppGpp ligand binding without apparent clashes. Domain III of E. coli PPX The six-helix domain III is structurally similar to proteins of the HD phosphohydrolase superfamily, which is characterized by a conserved metal-binding histidine:aspartate residue pair involved in catalysis. The structural analysis by Alvarado et al. identified domain III as a second PPX active site responsible for pppGpp hydrolysis.12 Rangarajan et al. also noted that domain III belongs to this superfamily but pointed to a lack of conservation of the catalytically important residues in a comparison with the related SpoT protein, suggesting that domain III in PPX does not exhibit enzymatic activity.14 Our study supports that the E. coli PPX enzyme accommodates alarmone hydrolysis in the domain I/domain II active site cleft. The contradicting studies both implicate domain III as the major contributor to dimerization and assign it a prominent role in aqueduct formation, polyP channeling and enzymatic processivity. Thus, there are
several reasons why this domain has been conserved through evolution also in the absence of any associated enzymatic activity. Hydrolysis of pppGpp The bifunctional aspect of PPX/GPPA enzymes implicates phosphatase activity, whether it acts on polyP or on pppGpp substrates. In this setup, it is not surprising that the mechanism for both activities could share some features and a common active site. Both activities are most thoroughly investigated in E. coli but remain to be functionally verified in A. aeolicus. Mutational data and PPX activity analysis of E. coli PPX was presented only recently,12 while similar analysis of mutants on (p)ppGpp activity has not been performed. Alanine substitutions at positions E121, D143 or E150 in E. coli PPX resulted in substantial loss of PPX activity, and the E121A mutant was most seriously affected. The E121 position corresponds to E119 in A. aeolicus, and we previously predicted it to be a key residue in catalysis.16 In the PPX/GPPA–ppGpp structure, the side chain of E119 is located approximately 6 Å from the 5′-β-phosphate of the bound nucleotide, providing sufficient space for the 5′-γ-phosphate of the pppGpp substrate to be positioned properly for hydrolytic attack by E119 either directly or via a water molecule that could potentially be activated by the same residue (Fig. 3). In contrast to the previous structures of the A. aeolicus PPX/GPPA enzyme, no metal ion is observed bound to the active site glycine-rich loop, despite that calcium was deliberately added. That calcium is bound neither to the nucleotide-bound
1473
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
Fig. 3. (a) Surface representation of the ppGpp nucleotide bound to the A. aeolicus active site. The nucleotide is trapped by the “open” conformation of the enzyme in a cleft at the domain I/domain II interface. There is sufficient space at the bottom of the binding site to accommodate the 5′-γ-phosphate of the cognate substrate pppGpp. (b) Surface representation of the modeled substrate pppGpp in the PPX/ GPPA active site. The substrate is positioned with its terminal 5′-γphosphate fitted into a pocket at the bottom of the binding site. (c) Stereo view of the ppGpp alarmone bound to the A. aeolicus PPX/GPPA active site. The 3′-pyrophosphate specificity anchor is chelated between the side chains of R22 and R267 and recognized by hydrogen bonding to the G144 and G211 main chain amides. (d) Modeling of the substrate pppGpp in the active site shown in stereo view. All parts of the ligand were fixed at the positions indicated by the PPX/GPPA– ppGpp complex structure except for the 5′-triphosphate moiety, which was allowed to move freely under the restraint of the applied force field. The active site accommodates the 5′-γ-phosphate of the substrate by positioning it close to the anticipated active site base E119.
molecule A nor to the apo form of molecule B in the present structure suggests that divalent cations required for optimal activity23,24 may be involved in a more transient manner during catalysis or possibly be chelated by unbound nucleotide. Substrate specificity by 3′-ribose phosphorylation The previous structures of the apoenzyme 16 indicated by the presence of bound chloride ions a likely position of the 3′-phosphate moiety of the substrate in the active site. This suggestion is confirmed by structure determination of the ppGpp enzyme complex (Fig. 3). It can also be seen that the arginine residues R22 and R267, located at opposite sides of the active site cleft, are intimately involved
in binding the 3′-pyrophosphate. This shows that a certain domain arrangement of the enzymes is required for recognition of this hallmark substrate feature. Further conformational adaptability to substrate binding is provided by the two glycinerich segments. Backbone amides of G144, which is part of the central Walker B loop region,16 and G211 contribute direct hydrogen bonds to the terminal 3′-phosphate. A glycine residue is also found at position 210, thereby further relaxing conformational restraints upon interaction with the alarmone 3′-specificity tail. Interactions with the nucleotide base R266 stacking over the nucleotide base is supported by locking its position through hydrogen
1474
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
bonding to the 2′-oxygen of the ribose (Fig. 3c); similar geometrical arrangements are often observed in nucleotide binding proteins.25 Y19 was mutated to the superfamily consensus residue (Fig. 1a) as a wild-type ppGpp complex could not be obtained (data not shown). Other highly conserved positions in the PPX/GPPA protein family that happen to be occupied by unusual residues in the A. aeolicus sequence are located outside the active site region. The N19 side chain is positioned to participate in direct interactions with N7 of the base. Occurrence of a tyrosine at position 19 in A. aeolicus PPX/GPPA appears to be sterically unfavorable for ligand binding at ambient temperature, but it is possible that active site geometry is slightly different at the normal growth temperature of this extremophile organism. Furthermore, the PPX/GPPA–ppGpp complex provides insight into the question of selective processing of purine pentaphosphate substrates. Adenine and guanine bases differ at the N6 position by having amino group and carbonyl substituents, respectively. However, in the structure of the ppGpp complex, the N6 carbonyl is solvent exposed with no obvious discriminating interaction, thus making it unlikely to contribute to specific base recognition. Also, the N2 positions of the two purine bases differ in the sense that guanine carries an exocyclic amine group at this position. The side chain of E264 is located at an angle to the guanine N2 amino group and could possibly participate in hydrogen bonding,26 albeit at a distance (3.8 Å) that suggests a very weak interaction. However, it is possible that a guanine base could have stronger electrostatic interactions compared with adenine due to the difference at the N2 position. Differentiation may not be important, however, since, at least in E. coli, hyperphosphorylated adenine nucleotides do not appear to accumulate under stringent response.27
Conclusions The present structure determination of the A. aeolicus PPX/GPPA product complex reveals the nucleotide position and indicates how the 5′-γphosphate of the pppGpp substrate can be properly accommodated for hydrolytic attack involving the key residue E119. This is in agreement with previous predictions based on analysis of the apoenzyme.16 It is shown that a simple hinge movement of domains I and II is sufficient to bring the E. coli PPX enzyme into an “open” state that is compatible with polyP and pppGpp hydrolysis at a common active site. The importance of the 3′-pyrophosphate tail and that of purine base recognition in substrate specificity were discussed. Modeling of the extracellular portion of human nucleoside triphosphate diphosphohydrolase 328 was based on our previous apo-PPX/GPPA structures,16 and these efforts may also benefit from the current insight.
Materials and Methods Preparation of ppGpp ATP nucleotide 3′-pyrophosphokinase was purified from the medium of a Streptomyces morookaensis culture (smPPK).29 smPPK was used to prepare ppGpp according to a published procedure.30 A 10-ml reaction (125 μl of smPPK, 1 ml of 1 M glycine, pH 10, 200 μl of 1 M MgCl2, 2 ml of 100 mM GDP, 1 ml of 150 mM ATP, 125 μl of [γ-32P] ATP and 5.55 ml of H2O) was incubated for 1 h at 37 °C. Crude ppGpp was purified by diethylaminoethyl chromatography (buffer: 0.01–1.5 M gradient in triethylamine bicarbonate buffer saturated with CO2), and the ppGppcontaining fractions were identified by detection of radioactivity and thin-layer chromatography. The ppGpp preparation was freeze-dried and dissolved in water. Concentrations of ppGpp were estimated assuming that the UV properties were identical with those of a GDP reference solution. Expression and purification Expression and purification of the A. aeolicus PPX/GPPA enzyme have been described previously.16,31 In summary, the gene was amplified by PCR from genomic DNA15 and cloned as an N-terminal hexahistidine-tagged construct in the vector pET28a (Novagen). A mutated variant (Y19N) including residues Pro7–Asn310 was prepared using PCR (primers: 5′-GCTTAGCTGGAAGCATATGCCAATTATGAGGGTGGCGTCCATAGACATAGGCTCCAACTCC-3′ and 5′-GCTTAGCTGGAAGCGGCCGCTTATTAATTTTCCTTTAATACTT-3′). The protein used in this study (Y19N variant) was purified on nickel-affinity resin followed by hexahistidine tag removal (thrombin) and gel filtration (Superdex 200: 5 mM Tris–HCl, pH 8.0, 100 mM NaCl and 1 mM DTT). Calcium chloride and ppGpp were added to the sample at concentrations of 0.5 and 3 mM, respectively. The sample was concentrated to 2 mg/ml prior to crystallization. Crystallization and data collection The A. aeolicus PPX/GPPA mutant Y19N–ppGpp complex was crystallized using the hanging-drop vapor diffusion method by mixing 2 μl of protein and 2 μl of reservoir solutions. The reservoir solution contained 200 mM sodium acetate (pH 6.4), 100 mM 4-morpholineethanesulfonic acid (pH 5.7) and 50% methyl-2,4pentanediol. Diffraction data were collected at MaxLab (Lund, Sweden) with beamline I711 (Table 1) and processed using the program XDS.32 Various CCP4 programs were used to prepare the structure factor files.33 Structure determination and refinement The structure was solved by molecular replacement using the CNS software34 and the apo-PPX/GPPA structure (PDB code 1T6C) as a search model. The CNS program was used at all stages of refinement (Table 1). Initially, the automated refinement procedure implemented in the program LAFIRE35 was used, followed by alternating rounds of minimization and manual model building using the software Coot.36 The PRODRG server37 was used to generate ligand parameters. The Bias Removal Server was used to produce electron density
Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex maps with reduced model bias.19 The program DYNDOM was used to analyze structural flexibility.20 Figures were prepared using PyMOL38 and ALSCRIPT.39 9. Modeling A tentative position for the terminal 5′-γ-phosphate of the substrate was obtained by modeling using the location of the product ppGpp as a guide. First, GRID40 phosphate probe calculations were performed to map favorable phosphate positions. The 5′-triphosphate tail was manually moved to match the GRID results. Parameter and topology data were obtained from the PRODRG server,37 and the structure of the PPX/GPPA–pppGpp complex was refined using the program CNS.34 The ligand ribose, base and 3′-pyrophosphate parts as well as all protein atoms were fixed in the conjugate gradient minimization, while no harmonic restraint was applied to the 5′-tail. PDB accession number
10.
11.
12.
13.
Crystallographic coordinates and structure factors have been deposited to the PDB with accession code 2j4r.
14.
Acknowledgements
15.
This work was supported by DANSYNC, the Lundbeck Foundation, the Danish Natural Science Research Council and the Danish Medical Research Council. We appreciate the support in data collection provided by the staff at MaxLab (beamline I711, Lund, Sweden).
16.
17.
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Crystal Structure of a PPX/GPPA Phospatase–Ligand Complex
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