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Protein Science (2005), 14:2562-2573. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Structural and functional features of an NDP kinase from the hyperthermophile crenarchaeon Pyrobaculum aerophilum

Jean-Denis Pédelacq1, Geoffrey S. Waldo1, Stéphanie Cabantous1, Elaine C. Liong2 and Thomas C. Terwilliger1

1 Bioscience Division and 2 Biophysics Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA

Reprint requests to: Jean-Denis Pédelacq, Bioscience Division, MS-M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; e-mail: jpdlcq{at}lanl.gov; fax: (505) 665-3024.

(RECEIVED June 21, 2005; FINAL REVISION July 7, 2005; ACCEPTED July 9, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Nucleoside diphosphate (NDP) kinases are ubiquitous enzymes that transfer {gamma}-phosphates from nucleoside triphosphates to nucleoside diphosphates via a ping-pong mechanism. The important role of this large family of enzymes in controlling cellular functions and developmental processes along with their crystallizability has made them good candidates for structural studies. We recently determined the structure of an evolved version of an NDP kinase from Pyrobaculum aerophilum, an extreme thermophile. This NDP kinase has similarity to the 42 other NDP kinases deposited in the Protein Data Bank (PDB) but differs significantly in sequence, structure, and biophysical properties. The P. aerophilum NDP kinase sequence contains two unique segments not present in other NDP kinases, comprising residues 66–100 and 156–165. We show that deletion mutants of the P. aerophilum NDP kinase lacking either or both of these inserts have an altered substrate specificity, allowing dGTP as the phosphate donor. A structural analysis of the evolved NDP kinase in conjunction with mutagenesis experiments suggests that the substrate specificity of the P. aerophilum NDP kinase is related to the presence of these two inserts.

Keywords: nucleoside diphosphate kinase; hyperthermophile; Pyrobaculum aerophilum; directed evolution; mutagenesis; X-ray crystallography

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051664205.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Nucleoside diphosphate (NDP) kinases were first characterized in 1973 (Fitz-Gibbon et al. 2002), and it is now well-established that in higher eukaryotes they catalyze nonsubstrate-specific conversion of nucleoside diphosphates to nucleoside triphosphates. Recently, attention has been drawn to this family of enzymes because of their involvement in a wide range of biological functions. Several reports established their role in the regulation of cell differentiation and proliferation (Keim et al. 1992; Okabekado et al. 1992). The membrane-associated NDP kinase is also implicated in the receptor-independent activation of G proteins (Wieland et al. 1992a, b; Piacentini and Niroomand 1996). More recently, Niroomand and collaborators (Lutz et al. 2001) identified a new signal transduction mechanism involving the human isoform Nm23-H2, which may be involved in the alteration of cAMP signaling during heart failure.

The first three-dimensional (3D) structure of an NDP kinase, from the slime mold Dictyostelium discoideum, was determined by X-ray crystallography in 1992 (Dumas et al. 1992). X-ray crystal structures of NDP kinases are now available from a wide variety of biological sources, from bacterium to human, and they all share an {alpha}{beta} domain comprising a four-stranded anti-parallel {beta}-sheet connected by two {alpha}-helices. Differences exist in the quaternary structure depending on whether they form tetramers or hexamers in solution and in the crystal state. A number of hexameric NDP kinase structures have been determined, including those from Drosophila melanogaster (Chiadmi et al. 1993) and Dictyostelium discoideum (Morera et al. 1994); two isoforms from Bovine taurus (Ladner et al. 1999); three from human (products of the Nm23-H1 [Min et al. 2002], Nm23-H2 [Morera et al. 1995; Webb et al. 1995], and Nm23-H4 [Milon et al. 2000] genes); and from Mycobacterium tuberculosis (Chen et al. 2002), Bacillus halodenitrificans (Chen et al. 2003), Pisum savitum (Johansson et al. 2004), Arabidopsis thaliana (Im et al. 2004), Oryza sativa (Huang et al. 2003), and Plasmodium falciparum (Protein Data Bank [PDB] code 1XIQ [PDB] ). The Myxococcus xanthus enzyme escapes this classification, as it forms tetramers (Williams et al. 1993).

In this paper, we discuss the X-ray structure of an NDP kinase from an extremophile organism, Pyrobaculum aerophilum (Fitz-Gibbon et al. 2002). This protein was insoluble when overexpressed in Escherichia coli cells. Our GFP-based technology (Waldo et al. 1999) was used to improve the solubility of the enzyme, and the structure of this variant was briefly reported earlier (Pédelacq et al. 2002). This variant contained six mutations relative to the wild-type enzyme: A10D, G33D, E40K, R71Q, S107N, and I117N. In a previous article (Pédelacq et al. 2002), we discussed the conservation of the central {alpha}{beta} sandwich and have made the point that none of the residues in the active site cavity have been mutated.

A significant difference in substrate specificity exists between the P. aerophilum NDP kinase and other members of this family of enzymes. The wild-type and evolved P. aerophilum enzymes are not active when using dGTP as the phosphate donor, while other members of this family of enzymes are typically most active with guanine nucleotide substrates (Lascu and Gonin 2000). Further, the sequence of the P. aerophilum NDP kinase differs from that of most members of this family by two inserts, residues 66–100 (I66–100) and 156–165 (I156–165). A detailed analysis of the 3D structure of the evolved P. aerophilum NDP kinase along with mutagenesis experiments suggests that the substrate specificity of this enzyme is related to the presence of these two inserts.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Structure of the SeMet evolved P. aerophilum NDP kinase
The crystal structure of the P. aerophilum selenomethionine-substituted NDP kinase was determined by the MAD method at 2.5 Å resolution (Pédelacq et al. 2002). The data collection and analysis statistics are summarized in Table 1Go. The final model was obtained by restrained refinement with atomic isotropic B-factors in REFMAC5 (Murshudov 1997). The N-terminal 13 residues are disordered. The protein core contains one four-stranded anti-parallel {beta}-sheet and two connecting {alpha}-helices, which forms the very common {alpha}{beta} sandwich, or ferredoxin fold (Fig. 1AGo).


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  		Table 1. Crystallographic statistics
 


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  		Figure 1. 3D structure of NDP kinases. Ribbon representation of the P. aerophilum (A) and the human Nm23-H2 (B) NDP kinase subunits, showing the {alpha}{beta} domain in blue, the extended C-terminal domain and the two inserts in yellow. Mutations in the P. aerophilum monomer (G33D, E40K, R71Q, S107N, and I117N) are shown as balls and sticks. Molecules present in the active site are also represented: Tris (T), PEG (P), and GDP. Figures were created using Molscript (Kraulis 1991) and Pymol (DeLano Scientific).

 
Structural comparison of the P. aerophilum NDP kinase with other NDP kinases structures in the PDB (42 structures deposited, which originate from 10 different biological sources) indicates that the root mean square deviations (rmsd) of corresponding residues varies from 1.0 Å with human isoform Nm23-H2 (Morera et al. 1995), Dictyostelium discoideum (Morera et al. 1994), and Drosophila melanogaster (Chiadmi et al. 1993) enzymes, to 1.5 Å with the Myxoccocus xanthus (Williams et al. 1993) enzyme. These values, which confirm the high overall structural similarity of this family of enzymes, do not reflect the high degree of variability in the length of the polypeptide chain. For example, only 137 out of 182 C{alpha} atoms from the P. aerophilum NDP kinase could be superimposed onto the human isoform Nm23-H2 in complex with GDP (Morera et al. 1995; Fig. 1BGo), with a sequence identity of 44%. Figure 2Go shows a backbone structure-based sequence alignment of the P. aerophilum NDP kinase with a set of 13 structural homologs. This alignment highlights differences in two distinct regions. The human NDP kinase M (Nm23-H4) possesses an N-terminal extension of 33 amino acids compared to the human NDP kinases A (Nm23-H1) and B (Nm23-H2). It was found that this extension causes the protein to form inclusion bodies in E. coli cells and that the full-length protein was not active after renaturation of the urea-denaturated sample (Milon et al. 2000). The crystal structure of the truncated form, missing the N-terminal 33 residues, was determined (Milon et al. 2000). There is a 12-amino-acid extension at the N terminus of the P. aerophilum NDP kinase relative to the M. xanthus enzyme. Differences in the C-terminal regions are also present, with a maximum difference of 17 amino acids observed between the NDP kinase from Mycobacterium tuberculosis and the human isoform Nm23-H4. The P. aerophilum NDP kinase has a short C-terminal extension.



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  		Figure 2. Sequence alignment of NDP kinases with known 3D structures. The P. aerophilum and human isoform NDKB_ HUMAN (Nm23-H2) structures were used for secondary structure assignment as determined using the program DSSP: {beta}-Strands are shown as arrows and {alpha}-helices as coils; TT is used to mark a {beta} turn. Sequence homologies are highlighted in red; sequence identities are shown as white letters on a red background. Residues in cyan at the N-terminal extremity were truncated for structural studies (NDKM_HUMAN). Mutated residues A10D, G33D, E40K, R71Q, S107N, and I117N in the evolved P. aerophilum enzyme are indicated by triangles. Residues important for making the deletion variants are shown in dark blue. PYRAE, Pyrobaculum aerophilum; MYCTU, Mycobacterium tuberculosis; MYXXA, Myxococcus xanthus; DROME, Drosophila melanogaster; PLAF7, Plasmodium falciparum (isolate 3D7); ARATH, Arabidopsis thaliana; ORYZA, Oryza sativa; DICDI, Dictyostelium discoideum; PEA, Pisum sativum; NDKA_HUMAN, Nm23-H1; NDKB_HUMAN, Nm23-H2; NDKM_HUMAN, Nm23-H4. The figure was created using ESPript (Gouet et al. 1999).

 
The structure of the variant of the P. aerophilum NDP kinase (Pédelacq et al. 2002) allows us to compare its sequence and structure to those of other NDP kinases. The P. aerophilum enzyme possesses two inserts compared to other members of the family (Fig. 2Go). The larger insert, residues 66–100 (I66–100) (Fig. 1AGo), is characterized by the presence of two new {alpha}-helices, {alpha}B and {alpha}C, and an extended helix {alpha}2. Helix {alpha}B, which comprises 18 residues, is the longest structural element found in an NDP kinase structure, and it is perpendicular to helix {alpha}2. The connection between {alpha}B and {alpha}2 is made by a short {alpha}-helix (from Pro88 to Ile92) flanked by two short segments. Lys91 is found in a tight turn, at the end of {alpha}C, where its main-chain nitrogen atom donates a hydrogen bond to the main-chain oxygen atom of Glu162 at the end of {alpha}D. Another insert, residues 156–165 (I156–165) (Fig. 1AGo), is located between helix {alpha}3' and strand {beta}4, at the C terminus of the K-pn loop (Sturtevant 1956; Fig. 2Go). This region starts with a Pro at position 157, where the direction of the backbone changes by more than 95°. Pro157 is also the first residue of the helix {alpha}D (Pro157–Glu163). At position 164, which corresponds to a glycine, the polypeptide chain takes another sharp turn. The resulting "U"-shape configuration is maintained by a hydrogen bond between the side chains of Asp155 and Arg165.

Quaternary structure of the P. aerophilum NDP kinase
To date, 42 crystal structures of wild-type and mutants of NDP kinase have been deposited in the PDB. Despite their various biological origins, all these structures, including the P. aerophilum NDP kinase, have in common quasi-identical dimers whose contacts can be attributed to adjacent strands {beta}2, which associate to create an extended eight-stranded anti-parallel {beta}-sheet. Except for the D. melanogaster enzyme (Chiadmi et al. 1993) for which the strands {beta}2 are considerably shorter than other members of the NDP kinase family, contributions from both strands involve the main chain C=O and NH groups from residues at corresponding positions (Lascu et al. 2000). Adjacent helices {alpha}1 also participate in dimer assembly. One key residue, Glu29 (Nm23-H2 numbering), is mostly invariant. The side-chain oxygen atoms of Glu29 make hydrogen bonds with the main-chain nitrogen atoms of residues Val21 and Gly22 (Morera et al. 1995; Webb et al. 1995) from the facing monomer. In the evolved P. aerophilum NDP kinase, Gly22 and Glu29 have been mutated into an Asp and a Lys, respectively (Fig. 2Go). As a consequence, the dimer interface of the evolved NDP kinase has a charge distribution more favorable for dimerization than the wild-type enzyme (Pédelacq et al. 2002).

The C terminus of NDP kinase constitutes the last major contributor to the dimer interface. This domain is relatively variable in length, short in the M. tuberculosis (Chen et al. 2002) and P. aerophilum enzymes, but more than 13 residues in the majority of eukaryotic NDP kinases. In the P. aerophilum enzyme, only Leu194 of this region contributes to the dimer interface, through nonpolar interactions with residues Met51 and Phe116. In the human Nm23-H4 isoform, polar interactions involve three residues: the side chain of Asp141 and the main-chain atoms of Lys143 and Cys145 (Milon et al. 2000). In the D. discoideum enzyme, the C-terminal domain plays a lesser role than in other NDP kinases. Polar interactions involving Leu144 and Tyr154 prevent the monomer from interacting with the neighboring twofold symmetry related subunit (Morera et al. 1994).

The P. aerophilum NDP kinase, along with nine out of the 10 of the other NDP kinases with known 3D structures, is hexameric. Of the NDP kinases with known 3D structures, only the NDP kinase from M. xanthus has been found to be in another quaternary form, a tetramer. In the P. aerophilum enzyme, I66–100 and I156–165 (Fig. 1AGo) are located at the periphery of the hexamer, between neighboring {beta}-sheets (Fig. 3AGo). Moreover, 1140 Å2 out of 9350 Å2 per subunit, or 12% of the total accessible surface, are buried in the hexamer interface. The interface contains nine potential hydrogen bonds or salt bridges and many hydrophobic contacts per subunit (Fig. 4AGo), compared to three in the Nm23-H2 hexamer (Fig. 4BGo). Polar interactions in the hexameric interface exclusively involve residues from I156–165 and the K-pn loop. We classified these residues into two groups according to their spatial distribution. One group of residues comprises Phe140, His141, Ala143, and Asp151. Main-chain oxygens of Phe140, His141, and Ala143 make potential hydrogen bonds to the side-chain NH of Lys133. Asp151 does so with the main-chain NH of Ala42. The second group comprises Ile154, Ser156, and Asp158. The Ile154 carboxylate and the side-chain oxygen of Ser156 are potentially bridged by the side chain of Asn125. Asp158 from one of the inserts is in a position to make a salt bridge with Arg126.



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  		Figure 3. The hexameric assembly. Ribbon diagram representation of the P. aerophilum (A) and the human Nm23-H2 (B) NDP kinases. The inserts and the longer C terminus are in blue. Drawn using Molscript (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

 


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  		Figure 4. The hexamer interface. Stereo views of the polar interactions at the interface of the P. aerophilum (A) and the human Nm23-H2 (B) NDP kinases. Molecules are presented in red and purple. The inserts and the longer C terminus are in blue. Drawn using Molscript (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

 
NDP kinase activity of mutants
Conserved residues important for NDP kinase activity have been identified in several X-ray structures with bound nucleotides (Janin et al. 2000). All of these previously identified catalytic residues are conserved in the P. aerophilum enzyme, and none are mutated in the evolved P. aerophilum enzyme (Fig. 5AGo). The orientations of their side chains in the structure of the evolved P. aerophilum enzyme are roughly the same as in the Nm23-H2 structure in complex with GDP (Fig. 5BGo). The NDP kinase residue that becomes phosphorylated during catalysis is His118 (Nm23-H2 numbering; Morera et al. 1995). Lys12 and Asn115 make polar interactions with the 2' and 3'-hydroxyl groups of the ribose, whereas the side chains of Thr94, Arg88, and Arg105 interact with the {beta}-phosphate (Fig. 5BGo). Tyr52, Ser120, and Glu129, which do not directly interact with the substrate, are predicted to play an important role in stabilizing the imidazole group in the correct position and orientation (Morera et al. 1995). Phe60, at the tip of {alpha}A-{alpha}2 hairpin, is a well-conserved residue that stacks its phenyl group onto the nucleotide base (Fig. 5BGo). Nevertheless, substitution by a Trp does not prevent the NDP kinase H122G-F64W mutant from D. discoideum from being active, and GTP has both the highest reactivity and the best affinity (Gallois-Montbrun et al. 2003). A structure of the H122G-N119S-F64W mutant from D. discoideum confirms the Trp stacking on the base (Gallois-Montbrun et al. 2002). The replacement of this Phe by an Ile in the wild-type P. aerophilum enzyme may have an effect on the positioning and stabilization of the base moiety.



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  		Figure 5. Stereo views of the nucleotide binding site. Active site of P. aerophilum NDP kinase with the Tris and PEG molecules (A) and human isoform Nm23-H2 with a bound GDP molecule (B). Residue side chains important for activity are labeled. Ligand atoms are colored according to atom type with carbon in yellow, oxygen in red, nitrogen in blue, and phosphate in purple. Figures were drawn with Pymol (DeLano Scientific) and rendered with Raster3D (Merritt and Bacon 1997).

 
No sign of activity of the P. aerophilum NDP kinase was detected when using dGTP as the phosphate donor (Pédelacq et al. 2002), a puzzling result given the preference of NDP kinases for the guanine nucleotides (Lascu and Gonin 2000). We modeled GDP in the active site of the structure of the evolved P. aerophilum NDP kinase, based on the structure of Nm23-H2 in complex with GDP (Fig. 6Go). We noticed that I156–165 could potentially block the positioning of the substrate in the active site cavity (Pédelacq et al. 2002). In particular, the C{alpha} atom of Val166 comes very close (0.8 Å) to the NH2 group of the modeled guanine base. In contrast, the wild-type and evolved P. aerophilum NDP kinase enzymes were active when using dCTP, dTTP, and dATP as phosphate donors (Pédelacq et al. 2002). To elucidate the role of the two inserts towards the stability and catalytic activity of the P. aerophilum enzyme, we constructed three variants resulting from the deletions of I66–100 ({Delta}1), I156–165 ({Delta}2), and I66–100 + I156–165 ({Delta}3) in the wild-type and evolved NDP kinases (see Materials and Methods).



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  		Figure 6. The molecular surface of the active site. Ribbon diagram of the human Nm23-H2 NDP kinase with dGTP in the active site (A) and P. aerophilum NDP kinase with dGTP modeled in the active site (B), colored from white to red according to normalized B-factor values (blue, 10 Å2; red, 40 Å2). The corresponding molecular surface, colored according to electrostatic potential, is shown. Blue and red colors indicate positive and negative charges, respectively. Rendered using Grasp (Nicholls et al. 1993).

 
Except for the evolved {Delta}2 variant, the variants were produced as inclusion bodies, and protein refolding screens from 8M urea-denatured protein samples were performed as previously described (Pédelacq et al. 2002). The urea-denatured proteins were refolded in the presence of 0.15 M Tris (pH 8.5), 0.15 M NaCl, 10% glycerol. For example, refolding 40 mg of washed wild-type NDP kinase inclusion bodies yielded ~1 mg of soluble protein after metal-affinity chromatography. As shown in Figure 7Go, analytical gel filtration data indicated that the fraction of soluble aggregates in the refolded protein solution varies from 20% (wild-type enzyme) to 100% (wild-type {Delta}3 variant). We note that the wild-type {Delta}2 and {Delta}3 variants have comparable enzymatic activities (Fig. 7Go), indicating that the aggregated character of the protein solution does not prevent the protein from being active.



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  		Figure 7. Gel filtration experiments and relative kinase activity as luminescent ATP measurement using a discontinuous luciferin-luciferase. aThe molecular assembly is given, as determined by analytical gel filtration. Relative uncertainty is ~5%. Deletion mutants originating from the bwild-type (WT) and cevolved (Ev) NDP kinases were analyzed, that partitioned into dhexamers (H) and/or esoluble aggregates (A).

 
At 25°C, the wild-type and evolved enzymes have the highest affinity for dCTP, followed by dATP, and then dTTP. No sign of activity was detected when using dGTP as the phosphate donor. The deletion variants had very different substrate specificities than the wild-type or evolved variant, with dCTP the poorest substrate among all the deletion variants and dGTP, a good substrate. The {Delta}1 deletion variant has a higher catalytic activity than any other deletion variants. Consistent with the fact that this enzyme is from a hyperthermophilic organism, we also observed higher kinase activity of the wild-type and evolved enzymes at 50°C relative to 25°C in the presence of dCTP, dTTP, and dATP. dCTP is the best phosphate donor at both 25°C and 50°C for the wild-type and evolved enzymes and is also one of the best substrates among all six deletion variants (Fig. 7Go).


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Overall structure of the P. aerophilum NDP kinase
We recently determined the X-ray structure of an NDP kinase from the hyperthermophile archaeon Pyrobaculum aerophilum, an organism that optimally grows at 100°C with a maximum temperature ~104°C (Fitz-Gibbon et al. 2002). The crystal structure of the P. aerophilum NDP kinase was determined by the multiwavelength anomalous diffraction (MAD) method at the selenium edge and was briefly described earlier (Pédelacq et al. 2002). Despite the presence of the conserved {alpha}{beta} sandwich domain (136 residues) found in all known NDP kinases, the P. aerophilum enzyme distinguishes itself by the presence of unique sequence elements not found in any of the ~130 identified NDP kinases. These sequence elements, which we call I66–100 and I156–165, are distant from the C and N termini (Fig. 2Go). Both inserts consist exclusively of {alpha}-helices: {alpha}B and {alpha}C in I66–100 and {alpha}D in I156–165.

NDP kinases are oligomeric species
Structural and biochemical studies have shown that most NDP kinases, including the P. aerophilum enzyme, form hexamers, but some bacterial enzymes are also tetrameric. In all of these oligomeric enzymes, the subunits are assembled as trimers of dimers or dimers of dimers. Two residues important for dimer assembly, G33 and E40 (Fig. 2Go), are mostly conserved throughout the range of known NDP kinase sequences. These residues are conserved in the wild-type P. aerophilum enzyme. In the evolved P. aerophilum enzyme, both residues have been mutated to the consensus amino acid types (G33D and E40K) which form a hydrogen bond across this dimer interface. The favorable hydrogen-bond network and charge distribution afforded by G33D–E40K probably enhance the stability of assembled dimers, thus suppressing the formation of nonspecific aggregates (Pédelacq et al. 2002).

As discussed by Lascu and colleagues (Lascu et al. 2000), sequence analysis in conjunction with the available structural information suggests the existence of at least two groups of NDP kinases. One group contains the hexameric proteins with conserved residues Lys31 and Pro101 (Nm-23-H2 numbering) and long C-terminal domains. Pro101 was shown to play a role in the stability of the hexameric assembly. Indeed, refolding of the urea-denaturated D. melanogaster (Awd) and D. discoideum NDP kinases containing the point mutation P101S could only generate inactive monomers (Lascu et al. 1992, 1993). In the human Nm23-H2 isoform, Lys31 makes polar interactions with the main-chain oxygens of two residues from the neighboring K-pn loop (Fig. 4BGo). Finally, as shown in Figure 4BGo, the long C-terminal domain in the human isoform Nm23-H2 stabilizes the K-pn loop of a neighboring dimer.

A second group contains the tetrameric NDP kinases with a shorter C-terminal domain. Residue 101 is not conserved in this group. The only identified representative of this group is the NDP kinase from M. xanthus. The shorter C-terminal extremity reinforces the dimer interface through nonpolar interactions. Nearly one-third of the total surface buried in the dimer interface originates from that region. A Glu replaces the Pro in position 101 (Fig. 2Go). This substitution locally affects the conformation of the K-pn loop. In a hypothetical Myxococcus hexamer, generated from a C{alpha} superimposition onto the Nm23-H2 hexamer, this glutamate side chain would come very close to a C{alpha} atom in the adjacent dimer (~0.77 Å). The side chain of E101 can still adopt a conformation that bends away from the K-pn region of the adjacent dimer, however.

The Pyrobaculum protein is atypical as it combines features from the two groups. Its C-terminal domain is short with no contribution towards stabilizing the hexameric interface as in the case of the Nm23-H2 (Fig. 4AGo) and other hexameric NDP kinases. Lys31 has been replaced by an alanine (Ala42) with its main-chain nitrogen hydrogen bonded to the carboxyl group of Asp151 from the neighboring dimer (Fig. 4AGo). In essence, Pro101 is the only distinguishing sequence feature in common with the other members of the hexameric group. The hexameric interface of the P. aerophilum NDP kinase also contains a dense network of salt bridges (E34–K41, R126–D158) and hydrogen bonds (R29–K41, A42–D151, N125–I154, N125–S156, K133–P140, K133–H141, K133–A143), as shown in Figure 4AGo. These intersubunit interactions may be involved in maintaining a stable structure, as shown for glutamate dehydrogenase (Rahman et al. 1998). None of the residues in thisinterface have been mutated in the evolved enzyme, so we can plausibly assume that the interface is similar in the wild-type NDP kinase.

The nucleotide binding site and enzymatic activity
We have explored the influence of the inserts I66–100 and I156–165 on the enzymatic activity of wild-type and evolved NDP kinases from P. aerophilum (Fig. 7Go). These experiments led to three important observations. First, unlike the wild-type and evolved enzymes, all the deletion variants are active in the presence of dGTP at 25°C. Although it is difficult to rationalize the absence of activity for the wild-type {Delta}1 (I66–100) construct at 50°C, overall, our structural predictions and measurements are in good agreement. At least one insert (I156–165) could potentially interfere with the correct positioning of the base moiety in the active site cavity of the wild-type and evolved enzymes (Pédelacq et al. 2002). As we expected, removing I156–165 increased dGTP-based activity. Surprisingly, the wild-type and evolved enzymes missing I66–100 were even more active than the enzymes missing I156–165 at 25°C in the presence of dGTP. Evidently both I156–165 and I66–100 have interactions that directly or indirectly affect substrate specificity.

Second, as expected for a hyperthermophile optimally growing at 100°C (Fitz-Gibbon et al. 2002), the enzymatic activity of the wild-type and evolved NDP kinases increases with temperature from 25°C to 50°C. Variants lacking both the I156–165 and I66–100 inserts are prone to aggregation, as shown in Figure 7Go. This may be part of the reason for lowered enzymatic activities of these two variants (Fig. 7Go). The importance of the inserts for overall stability is supported by the observation that the enzymatic activities of all the deletion variants decrease or remain approximately constant with increasing temperature from 25°C to 50°C, while those of the wild-type and evolved P. aerophilum NDP kinase without deletions increase at 50°C.

Finally, we note that there are more ion pairs, helical residues, and proline residues in the P. aerophilum NDP kinase relative to its mesophilic counterparts (Table 2Go), consistent with a role of each of these types of interactions and residues in stabilization of thermophilic proteins (Elcock 1998). Further, the P. aerophilum protein has a higher number of charged residues compared to the mesophilic homologs (Table 2Go). Twenty-nine percent of all the charged residues (Arg, Lys, Asp, and Glu) in the P. aerophilum NDP kinase are located in I66–100 and I156–165. One three-membered intramolecular ionic network involves residue side-chains at the C-terminal extremity of I156–165. Both NH groups of R165 are involved in three ion pairs with the C=O groups of D155 and E163. Removal of I156–165 would disrupt the ionic network around R165, and may have a destabilizing effect by accelerating the denaturation of the enzyme.


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  		Table 2. Examination of amino acid, secondary structures, and ion pairs differences between NDP kinases of the thermophilic P. aerophilum (Evolved Pa) and structural homologs from Mycobacterium tuberculosis (1K44), Myxococcus xanthus (2NCK), human Nm23-H4 (1EHW), Dictyostelium discoideum (1NPK), Bos taurus A (1BHN) and B (1NUE), and Drosophila melanogaster (1NDL)
 

   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Cloning, selenomethionine-substituted protein expression and purification, and crystallization
Protein engineering, cloning, and purification have been described (Pédelacq et al. 2002). The selenium-substituted protein was expressed in strain BL-21(DE3) using minimal medium supplemented with six amino acids, various salts, and sulfate (Van Duyne et al. 1993). A starter 50-mL preculture in LB medium was used to inoculate a liter of prewarmed minimal medium at 310 K. Leucine, isoleucine, valine, phenylalanine, lysine, and threonine were added at mid–log phase, and the culture was induced with 1 mM final IPTG.

The protein was dialyzed against 100 mM Tris (pH 8.5), 15 mM {beta}-mercaptoethanol, and concentrated to ~7 mg/mL. The optimized crystallization conditions were found to be 10% PEG 4000 (w/v) using the hanging drop vapor diffusion method at room temperature. Small plates appeared within 2 d, and reached their maximum size of 600 µm x 600 µm x 60 µm in 1 wk.

Data collection and phasing
Crystals belong to the monoclinic space group C2, with unit-cell dimensions a = 125 Å, b = 72 Å, c = 105 Å, and {beta} = 133.3°, as previously described (Matthews et al. 1968). MAD diffraction data were collected at 100 K from one SeMet crystal at the synchrotron beam line X8C at the National Synchrotron Light Source (NSLS; Brookhaven, NY). A fluorescence spectrum, recorded from the cryocooled crystal, was used to select the wavelengths at the selenium K absorption edge (0.9792 Å), at the peak (0.9791 Å), and at one remote wavelength on the high-energy side (0.9500 Å). Data were collected to a resolution of 2.4 Å on a 30-cm MAR Research image plate. Reflection intensities were processed using MOSFLM (Leslie 1992). The CCP4 suite of programs (Collaborative Computational Project Number 4 1994) was used to merge and scale these intensities and to compute the structure-factor amplitudes. All selenium sites were identified using Patterson map search procedures (DeLano Scientific) as implemented within the SOLVE package (Terwilliger and Berendzen 1999). Anomalously scattering-atom refinement and MAD phasing were conducted using all data between 40 and 3 Å and the absorption edge wavelength data set as a reference. Experimental phases were improved by density modification, including threefold averaging through matrices defined by the heavy-metal sites, using RESOLVE (Terwilliger 1999) and DM (Collaborative Computational Project Number 4 1994). The resulting electron density map was of good quality, and 135 of the 195 residues could be manually built using Turbo-Frodo (http://afmb.cnrs-mrs.fr/TURBO_FRODO/main.html). Subsequently, the model resolution was extended to 2.5 Å against data of the high-energy data set. Structure refinement was performed using CNS (Brunger et al. 1998), applying strict noncrystallographic constraints. The noncrystallographic symmetry restraints were relaxed during the later stages, and the final cycle was carried out with no restraints. The final model comprises 181 out of 195 residues in the recombinant protein and 158 water molecules. The crystallographic Rcryst and Rfree values were 0.187 and 0.26, respectively (Table 1Go). All residues except L170 are in the allowed regions of a Ramachandran plot, and 91% of them have the most favored backbone ({Phi},{Psi} values, as defined by PROCHECK (Laskowski et al. 1993). Structure factors and coordinates have been deposited in the RCSB PDB under accession number 1XQI [PDB] .

Mutagenesis experiments
The genes coding for the truncated versions {Delta}1 (I66–100), {Delta}2 (I156–165), and {Delta}3 (I66–100 + I156–165) of the P. aerophilum wild-type and evolved NDP kinases were amplified by conventional PCR (Pédelacq et al. 2002). For the {Delta}1 deletion variant, we used forward primer 5'-AGATATACATATGCATGCTATA AATATTGCTTTTTTCGC-3' and reverse prime 5'-CGGAC GGTCTTTCAGATCAATGTAAAATCTCTCTATTTC-3' to link residues 1–63 with the human isoform Nm23-H2 peptide coding for amino acids IDLKDRP (Fig. 2Go). A second set of primers using forward primer 5'-ATTGATCTGAAAG ACCGTCCGATTAAACGTAGTTTAGTT-3' and reverse primer 5'-AATTCGGATCCCTCTAAAACCTCCTCTTCTCTA AACCAA-3' was used to link the human peptide to amino acid residues 104–195. To create the {Delta}2 variant, forward primer 5'-AGATATACATATGCATGCTATAAATATTGCTTTTT TCGC-3' and reverse primer 5'-GTTAAAACCTACCTGA ATTGAGTAGTCGCCCC T-3' were used to link residues 1–154 with the peptide coding for amino acids QVG (Fig. 2Go). Forward primer 5'-TCAATTCAGGTAGGTTTTAACTT GGTCCACGCG-3' and reverse primer 5'-AATTCGGATC CCTCTAAAACCTCCTCTTCTCTAAACCAA-3' were used to link the human peptide to residues 168–195. Finally, the {Delta}3 was created using the {Delta}1 construct as a template and keeping the same oligonucleotides as for the {Delta}2 variant. The underlined codons represent the NdeI (CATATG) and BamH-I (GG ATCC) restriction sites. To facilitate purification, proteins were expressed with C-terminal hexahistidine tags. The resultant C-6HIS tagged proteins had the amino acid extension GSHHHHHH.

Analytical gel filtration
Analytical gel filtration chromatography was carried out on a Superdex HR 10/30 (Amersham Pharmacia Biotech) with a flow rate of 0.5 mL/min. Molecular weight standards (Bio-Rad gel filtration standards kit from Sigma) were independently chromatographed on the same column for estimation of the molecular weight of the species eluting from the column. The relative elution volume of different protein constructs was compared with that of standard markers (aprotinin, 6.5 kDa; cytochrome C, 12.4 kDa; carbonic anhydrase, 29 kDa; alcohol dehydrogenase, 150 kDa).

Activity experiments
NDP kinase catalytic activity was measured using a luciferin/luciferase assay kit from Sigma-Aldrich. The assays were performed at 25°C and 60°C in a 100-µL ATP generating reaction mix consisting of 0.1 M Tris (pH 8.5), 0.15 M NaCl, and 10 mM MgSO4, and equilibrated for 15 min with the protein. The reaction was started by the addition of a mix containing 2 mM of the phosphate donor (dGTP, dTTP, dCTP, dATP) and 2 mM ADP. The concentration of NDP kinase was maintained above 1.0 x 10–3 mg/mL to minimize wall absorption losses. One hundred (100) µL of a 25-fold dilution of ATP luciferin/luciferase assay solution was added to 100 µL of the ATP-generating reaction. Light emission was measured for 15 sec in a Turner Designs uminometer (model TD-20e).


   Acknowledgments
 
We thank Jeffrey H. Miller and Sorel Fitz-Gibbon of the University of California Los Angeles Molecular Biology Institute for giving us genomic DNA from Pyrobaculum aerophilum. We thank Joel Berendzen and Leon Flaks at the NSLS at Brookhaven National Laboratory for their assistance in performing data collection.


   References
TOP
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 Introduction
 Results
 Discussion
 Materials and methods
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