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Crystal Structure of the Hyperthermophilic Inorganic Pyrophosphatase from the Archaeon Pyrococcus horikoshii

Binbin Liu ^* , Mark Bartlam ^* , Renjun Gao , Weihong Zhou ^*, Hai Pang ^*, Yiwei Liu ^*, Yan Feng  and Zihe Rao ^* 

^* Laboratory of Structural Biology, Tsinghua University and National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China; and National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China

Correspondence: Address reprint requests to Zihe Rao, Laboratory of Structural Biology, Dept. of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, P. R. China. Tel.: 86-10-6277-1493; Fax: 86-10-6277-3145; E-mail: raozh{at}xtal.tsinghua.edu.cn.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88°C. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 Å resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia coli shows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential enzyme that specifically catalyzes the hydrolysis of the phosphoanhydride bond in inorganic phosphate (PPi) (Chen et al., 1990; Lahti, 1983). PPi is a central phosphorus metabolite and is a by-product of various reversible nucleoside 58-triphosphate–dependent reactions including tRNA charging and DNA and protein synthesis that utilize ATP in vivo. PPases from a wide variety of sources have been studied, but those from Saccharomyces cerevisiae and Escherichia coli are the most highly characterized, both biochemically and structurally. E. coli pyrophosphatase is a homohexamer with 175 amino acids per monomer (Avaeva et al., 1997), whereas S. cerevisiae pyrophosphatase is a homodimer with 286 amino acids per monomer (Heikinheimo et al., 2001).

PPases from archaebacterium exhibit different structural and catalytic properties (Hansen et al., 1999; Richter and Schafer, 1992). The archaeal PPases so far reported are relatively thermostable, especially in the presence of divalent metal cations (Ichiba et al., 1998). Understanding the structural basis for the enhanced stability of proteins from hyperthermophilic organisms relative to their mesophilic and thermophilic counterparts is a highly relevant but complex and challenging problem. Previous comparisons of high-resolution crystal structures of enzymes with the same fold and function in mesophiles, thermophiles and hyperthermophiles have revealed a number of potentially stabilizing features. A proper understanding of the molecular basis of thermal stability in proteins could have important consequences for their application in a range of biotechnological processes. For example, thermostable pyrophosphatases have common uses in cycle sequencing methods using thermostable DNA polymerases (Vander Horn et al., 1997). The crystal structures of PPases from thermophilic bacterium Thermus thermophilus (T-PPase; PDB ID 2PRD) (Teplyakov et al., 1994), thermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase; PDB ID 1QEZ) (Leppanen et al., 1999), and mesophile E. coli (E-PPase; PDB ID 1JFD) (Avaeva et al., 1997) have provided basic clues for the PPase catalytic mechanism and thermostability, but many important aspects remain to be resolved.

Microorganisms can be classified according to their optimal growth temperature, T_opt, into four groups: psychrophilic (0 < T_opt < 20°C), mesophilic (20 < T_opt < 50°C), thermophilic (50 < T_opt < 80°C), and hyperthermophilic (80 < T_opt < 120°C). Considerable efforts have been made during recent years to analyze the structural features that determine the extraordinary thermal stability of proteins from hyperthermophiles. Here we have isolated an inorganic pyrophosphatase (Pho-PPase) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, whose optimum growth temperature (95°C) is significantly higher than those of S. acidocaldarius (75–80°C), T. thermophilus (75–80°C) and E. coli (37°C). Pho-PPase showed a higher optimal activity at 88°C and an alkaline optimal pH of 10.3 (at 88°C). The enzyme has extreme thermostability and does not lose activity at 100°C. In addition, Pho-PPase is stable against various denaturants. All of these properties are different from those of other archaeal PPases: full details of the characterization of inorganic Pho-PPase will be reported elsewhere (Feng et al., unpublished results). To gain a more penetrating insight into its function, here we describe the structure determination of Pho-PPase and a comparison of the structure with its mesophilic and thermophilic counterparts in an attempt to understand the structural basis for thermal stability.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Crystallization and x-ray data collection
The preparation and preliminary characterization of Pho-PPase crystals have been described elsewhere (B. Liu X. Li, R. Gao, W. Zhou, G. Xie, M. Bartlam, H. Pang, Y. Feng, and Z. Rao, submitted). Briefly, the gene encoding inorganic pyrophosphatase from the archaea P. horikoshii was cloned into pET15b (Novagen) and expressed in E. coli strain BL21. After purification, the purified protein was concentrated to 20 mg/ml. Crystallization trials were set up using the hanging drop/vapor diffusion method with Crystal Screen reagent kits I and II (Hampton Research). Crystals suitable for diffraction were obtained after two weeks from the condition 3.8% PEG 4000, 0.1 M Na acetate, pH 5.0–5.2, 0.02 M MgCl₂. A set of data at 2.66 Å resolution were collected in house. All data were integrated using DENZO/HKL and scaled and merged with SCALEPACK (Otwinowski and Minor, 1997). Crystal lattice properties and data-collection statistics are listed in Table 1.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Monomeric structure
The current model of P. horikoshii inorganic pyrophosphatase includes residues 4–171 in chain A, 4–170 in chain B, 4–170 in chain C, and 93 water molecules. Pho-PPase is arranged in a globular form and belongs to the +ß class of protein folds. The Pho-PPase monomer structure is composed of nine ß-strands and two -helices arranged in a ß1-ß2-ß3-ß4-ß5-ß6-ß7-ß8-1-ß9-2 topology (Fig. 1). Pho-PPase shares 47% sequence identity with S-PPase (Fig. 2 A), and the two proteins have a similar core structure. Indeed, superposition of the Pho-PPase structure with S-PPase, T-PPase, and E-PPase shows that the four PPases are spatially homologous (Fig. 2 B). The RMSD between C atoms of the four PPases range from 0.83 to 1.14 Å. The mutual positions of the central ß-barrel structure and -helices are similar. However, the structure-based sequence alignment (Fig. 2 A) shows that Pho-PPase contains a single residue deletion in the loop formed by residues 25–30, two residues deleted in the loop formed by residues 111–114, and three residues deleted in the loop formed by residues 144–148.

View larger version (47K): [in this window] [in a new window]   FIGURE 1  (A) A ribbon representation of the Pho-PPase monomer structure. The structure is colored from blue at the N-terminal to red at the C-terminus. Secondary structure elements have been labeled. (B) Top and side views of the Pho-PPase hexamer. Three subunits are related by a noncrystallographic threefold axis to form a trimer. Two trimers are related by a crystallographic twofold axis to form a tightly packed hexamer.

View larger version (61K): [in this window] [in a new window]   FIGURE 2  (A) A structure-based sequence alignment between Pho-PPase and three related PPase structures from T. thermophilus (T-PPase; PDB ID 2PRD) (Teplyakov et al., 1994), S. acidocaldarius (S-PPase; PDB ID 1QEZ) (Leppanen et al., 1999) and E. coli (E-PPase; PDB ID 1JFD) (Avaeva et al., 1997). Secondary structure elements are shown for Pho-PPase. (B) A stereodiagram showing the superposition of the four PPase structures. Structures are represented as C backbone traces. The coloring is as follows: red, Pho-PPase; blue, S-PPase; green, T-PPase; magenta, E-PPase. RMSD between C atoms of the four PPases range from 0.83 to 1.14 Å.

From the crystallographic symmetry, the trimers are packed such as to form a tightly packed hexamer with twofold crystallographic symmetry. The intertrimer interactions in Pho-PPase are listed in Table 2. There are no large-scale distortions in monomeric structure, and the increased thermostability of Pho-PPase likely results from an increase in both hydrophilic and hydrophobic interactions. Intertrimer interactions are concentrated in strand ß3, helix 1, and the loop between ß8 and 1. The overall surface area buried in the hexamer is 2342 Ų, which is comparable to the 2430 Ų buried by the T-PPase hexamer and higher than the 2090 Ų buried by the E-PPase hexamer (Salminen et al., 1996).

The crystal structure of Pho-PPase reveals that K102, equivalent to residue E101 in E-PPase, is important for the active-site cavity and is located in the highly conserved region including the essential catalytic residues (numbered E97, D102, and K104 in E-PPase). Mutagenesis studies of E-PPase have shown that the enzyme activity of mutants E99D, D102E, and K104E almost disappeared or decreased rapidly (Hyytia et al., 2001). However, when E101 is replaced by a more negative aspartic acid residue, the enzyme activity of the mutant was increased by 10%. Alignment with other PPases shows that the residue located in the site is conserved as either an acidic or neutral residue. We propose that K102 is related to the alkaline optimal pH (10.3) of Pho-PPase, since only in such an alkaline environment would K102 not be positive. Further site-directed mutagenesis studies are in progress to investigate the role of K102.

Structural basis of thermostability in Pho-PPase
The free energy of stabilization of globular proteins is rather small. It lies in the range from 30 to 65 kJ/mol (Pfeil et al., 1986), which is equivalent to the energy contributed by a few hydrogen bonds, ion pairs, or hydrophobic interactions. The increase in free energy of stabilization observed for thermophilic proteins is of the same order of magnitude (Harris et al., 1980; Nojima et al., 1978). Recent structural studies also have identified several factors which are more often observed among thermophilic proteins and may account for their stability. These include an increased number of salt bridges or hydrogen bonds; optimized stability of helices, loops, and N- and C-termini; decreased solvent-exposed surface area; stronger interactions between the subunits in oligomers; and even an increased number of buried solvent molecules in hydrophilic cavities. We compare the present 2.66 Å resolution structure with three other PPases, to identify which factors may be important in stabilizing Pho-PPase.

Amino acid composition and thermostability
The amino acid composition of a protein has long been thought to be correlated with its thermostability. Compared with related PPase structures, the unusual amino acid composition of Pho-PPase would account for its extreme thermostability. The structure-based sequence alignment (Fig. 2) shows the amino acid sequences of the four PPases compared in this study. More charged residues were found in Pho-PPase, which is consistent with the idea that the number of ion pairs is an important determinant of protein thermostability. For instance, Pho-PPase contains a total number of eight arginine residues, which have a tendency to form multiple ion-pairs and H-bonds. Pho-PPase also contains 14 proline residues, which is the largest proportion in the four PPases studied here. Since proline residues affect local mobility of the chain by decreasing the conformational entropy of the unfolded state, the increased rigidity of the structure would be expected to increase the overall thermostability. Such stabilization by the introduction of proline residues into loop regions is a well-documented phenomenon (Matthews et al., 1987). A recent study of inorganic pyrophosphatase from thermophilic bacterium PS-3 was carried out in which proline residues were systematically replaced by alanines (Masuda et al., 2002). The authors found that most of the proline residues in PS-3 PPase play very important roles, and many of them are critical for the structural integrity of the protein. They also concluded that the thermostability of PS-3 PPase is profoundly related with its subunit structure.

The total number of hydrophobic residues (Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, and Pro) is also highest in Pho-PPase. Compositional differences between the PPases are more pronounced among exposed sites. Pho-PPase has an increased number of exposed hydrophobic residues, which are presumably involved in oligomer formation and stability.

Interestingly, previous site-directed mutagenesis of E-PPase has shown that aromatic residues play a very important role for the thermostability (Hyytia et al., 2001). There are an increased number of aromatic residues found in Pho-PPase compared to three other PPases. In particular, there is an increased frequency of phenylalanine and tyrosine residues in Pho-PPase, which are liable to form hydrophobic and aromatic interactions. Many of these aromatic residues are observed to form a cluster located at the bottom of the active site, and it is possible that stacking interactions involving the aromatic residues may contribute to enhanced thermostability of Pho-PPase.

The frequency of Asn (2) and Gln (0), which can be classed as thermolabile due to their tendency to undergo deamidation at high temperatures and therefore may be naturally discriminated against in thermostable proteins, is substantially reduced in Pho-PPase. Cysteine was also completely absent in Pho-PPase, which is easy to interpret since cysteine is highly sensitive to oxidation at high temperature. The frequency of glycine was not decreased, but changed in location. Interestingly, residue L83 of Pho-PPase is strictly conserved as a glycine in other PPases and is located in the short loop connecting ß-strands 5 and 6, thus increasing the rigidity of the Pho-PPase structure.

Ionic interactions and thermostability
The importance of ion-pairs as determinants of protein thermostability was first highlighted by Perutz and Raidt (Perutz and Raidt, 1975) while comparing ferredoxin and hemoglobin structures, and ion-pairs were subsequently proposed to be important for the stability of a number of other thermostable proteins (Korndorfer et al., 1995; Walker et al., 1980). Indeed, several reports of high resolution structures of hyperthermophilic proteins show the number of ion-pairs in most of the hyperthermophilic proteins is higher than in their mesophilic counterparts (DeDecker et al., 1996; Hennig et al., 1995; Yip et al., 1995). A total of 12 ionic interactions are formed per monomer in the E-PPase short c-axis crystal form, which is equivalent to the number in T-PPase and lower than S-PPase (17 ionic pairs). In contrast, there are 28 ionic interactions scattered throughout the Pho-PPase monomer (Table 3). It appears that Pho-PPase is more stabilized by ion-pairs than T-PPase, E-PPase, and S-PPase. Although the number of ion-pairs is increased, the multicenter ion interactions are decreased. Interestingly, two long ion-pair networks are observed in Pho-PPase. The first ion-pair network is located in the C-terminus 2 helix, which may stabilize the C-terminus against thermal denaturation. The second ion network is located in the active center and involves residues 112–120 and 127–139. There is also an increase in the number of intrasubunit ion-pairs in Pho-PPase, and their involvement in complex networks mirrors that observed in the structure of the hyperthermophilic Pyrococcus furiosus glutamate dehydrogenase (Yip et al., 1995). The presence of ion-pair networks has also been observed in Sulfolobus solfataricus indole-3-glycerol phosphate synthase (Hennig et al., 1995) and the TATA-box binding protein from P. furiosus (DeDecker et al., 1996). The presence of ion-pair networks may be energetically favorable due to the shared entropic cost upon ion-pair formation (Nakamura, 1996).

Oligomerization and thermostability
In the PPase family, the oligomerization state stabilizes the conformation of the enzyme that binds substrate and vice versa (Baykov et al., 1995). The oligomeric packing appears to provide a general strategy for enhancing the thermostability of the PPase family. Pho-PPase seems to be a more tightly packed hexamer. The total number of oligomeric hydrophilic contacts is listed in Table 2. There are notably more intermonomer hydrophilic interactions in Pho-PPase compared with the three other PPases, resulting in tighter twofold A-E and threefold A-B interfaces. This effect can be measured by the accessible surface area (ASA) buried per monomer on oligomerization, which is 13.6% higher than for S-PPase and 12.1% higher than for E-PPase. These increased hydrophilic interactions may provide the extra energy necessary for stabilization. Similar observations are found in several thermophilic protein structures. For example, thermostability was attributed to improved subunit interfaces in L-lactate dehydrogenase from Bacillus stearothermophilus (Kallwass et al., 1992), malate dehydrogenase from Thermus flavus (Kelly et al., 1993), and ornithine carbamoyltransferase from P. furiosus (Villeret et al., 1998). Other studies also indicate that multimer formation and subunit interactions are critical for thermal stability of, for example, hemocyanin from the ancient tarantula Eurypelma californicum (Sterner et al., 1995), phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima (Hennig et al., 1997), GluDH from the hyperthermophile P. furiosus (Vetriani et al., 1998), and chorismate mutase from the thermophilic archaeon Methanococcus jannaschii (MacBeath et al., 1998).

Helices and thermostability
It has been previously reported that the helical conformation is stabilized by oppositely charged ion-pair interactions (i.e., Glu-Lys, Glu-Arg, Asp-Lys, Asp-Arg) in the positions (i,i+4) or (i,i+3) (Scholtz et al., 1993). The 1 helix contains a single ion-pair between the nonconserved residues K127-D131. Examination of the C-terminus 2 helix sequence indicates that Pho-PPase contains a significant increase in the number of charged residues compared with the other PPases (Fig. 2 A). These residues—E157, R161, E162, R165, E168, K171—are ideally distributed in the sequence to allow the formation of intrahelix ion-pairs. The structure of Pho-PPase shows three intrahelical ion-pairs formed between residues E157-R161, E162-R165, and E169-K172 (Table 3), which are not conserved in other PPase structures. These additional ion-pairs may be responsible for the increased thermostability of Pho-PPase by stabilizing the C-terminus and increasing its resistance to denaturation.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Comparison of the structure of Pho-PPase with T-PPase, S-PPase, and E-PPase has identified a number of determining factors for the thermostability of PPase enzymes. Pho-PPase is the most thermostable of the four structures, and this is reflected by the following factors. First, shorter and more convergent loops in the active site imply that the two loops are important for enzyme-substrate or ion binding under high temperature, and this is consistent with the idea that shorter loops are more helpful for thermostable PPases to stabilize the conformation of the enzyme-substrate complex. Second, the basis of thermostability in PPases is generally believed to be related to the oligomer structure; Pho-PPase has an increase in the number of both intermonomer and intertrimer interactions, and this results in tighter packing of the hexamer. Third, an increase in the number of ion-pairs—in particular, two ion-pair networks—helps to stabilize the structure. One ion-pair network in the loop connecting strand ß8 and helix 1 may help to stabilize the conformation of the active site, while another network in helix 2 may stabilize the C-terminus and increase its resistance to thermal denaturation. The structure of Pho-PPase and the comparison between PPase structures should stimulate further kinetic and structural studies of enzymes adapted to extreme temperature and prove helpful for future biotechnology applications.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
We are grateful to Feng Gao for help with data collection.

This research was supported by the following grants: Project "973" G1999075602; Ministry of Science and Technology (MOST) 2002BA711A12.

	FOOTNOTES

 
Binbin Liu and Mark Bartlam contributed equally to this work.

Submitted on June 12, 2003; accepted for publication August 27, 2003.

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