Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

The crystal structure of shrimp nucleoside diphosphate kinase in binary complex with dCDP, dADP and ADP shows conserved contacts independent of the kind of nucleotide bound.

Abstract

Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012 ▶), J. Bioenerg. Biomembr. 44, 325–331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2′-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.

1. Introduction  

Nucleoside diphosphates from either a de novo or a salvage pathway require a final phosphorylation stage that leads to substrates for DNA polymerase. Nucleoside diphosphate kinase (EC 2.7.4.6; NDK) is responsible for catalyzing the final phosphorylation reaction of nucleoside diphosphates. This reaction is a reversible phosphate transfer to both ribonucleoside and deoxyribonucleoside diphos­phates using NTP as a phosphate donor to provide adequate nucleosides for RNA or DNA synthesis (Gonin et al., 1999 ▶). The reaction catalyzed by NDK starts with the transfer of a phosphate from ATP to His117; ADP is then released and the acceptor nucleoside diphosphate is bound and phosphorylated. Nucleotide phosphates are coordinated by a network of basic residues, Arg94, Arg9 and His59, and the polar Thr98 (from the Dictyostelium discoideum amino-acid sequence; Cherfils et al., 1994 ▶). All NDKs follow a ping-pong enzymatic mechanism involving a phosphorylated histidine residue in the active site (Moréra et al., 1995 ▶).

NDKs are known to have relaxed substrate specificity, since they phosphorylate both deoxyribonucleotides and ribonucleotides (Bilitou et al., 2009 ▶; Deville-Bonne et al., 2010 ▶). This enzyme has been characterized from several prokaryotic and eukaryotic sources, and has been described as an important housekeeping enzyme that maintains and controls the pool of intracellular nucleotides (Bernard et al., 2000 ▶).

Crystallographic and biochemical studies have indicated that most NDKs are hexameric (Dumas et al., 1992 ▶; Lascu et al., 2000 ▶); exceptions are the dimeric NDK from Halomonas sp. 593 (Tokunaga et al., 2008 ▶) and the tetrameric NDKs from Escherichia coli (Moynié et al., 2007 ▶), Aquifex aeolicus (Boissier et al., 2012 ▶) and Myxoccocus xanthus (Williams et al., 1993 ▶).

Tetramers of NDKs are formed through a dimer of dimers, while the hexameric arrangement is achieved either by a trimer of dimeric units or dimer of trimeric units (Gonin et al., 1999 ▶; Lascu et al., 2000 ▶). Hexameric D. discoideum NDK is one of the most well characterized enzymes from this family. Like other NDKs, it shows a characteristic fold with a βαββαβ motif, similar to the ferredoxin fold, where α-helices pack onto a β-sheet motif, creating a highly conserved hydrophobic central core (Janin et al., 2000 ▶). A further two typical features that complete the folding of the NDK monomer are the Kpn loop (named after the Drosophila Killer of prune mutant) and the C-terminus. The Kpn loop is a small compact structure comprised of residues 96–113 in human NDK. The C-terminal fragment is composed of the last 20 residues in hexameric NDKs and this small domain interacts with neighbouring subunit residues. This interaction is associated with loss of enzymatic activity and quaternary stabilization of hexameric NDK. In the tetrameric M. xanthus NDK the C-terminus is shorter by ten residues and is not implicated in function (Williams et al., 1993 ▶). In most of the NDK crystal structures in complex with a nucleoside diphosphate, the active site presents a phosphate moiety bound to the side of the β-sheet and the purine or pyrimidine base is located in a hydrophobic pocket formed by a conserved Phe and Ile/Val. The complex also indicates that the active site has a preformed rigid core with very little change in protein conformation after nucleotide binding and in the covalent phosphorylated histidine intermediate.

NDK from the marine shrimp (Crustacea, Decapoda) Litopenaeus vannamei (LvNDK) contains 151 residues and shows high amino-acid identity (>75%) to the hexameric human and D. discoideum NDKs and only ∼42% identity to the tetrameric NDKs from M. xanthus (Williams et al., 1993 ▶) and E. coli (Moynié et al., 2007 ▶). In general, the sequence of the shrimp NDK is highly conserved among eukaryotic and bacterial NDKs (40 and 60% identity, respectively).

We have studied shrimp NDK in solution: it is a homotrimer of ∼57 kDa and binds and phosphorylates nucleoside diphosphates (Quintero-Reyes et al., 2012 ▶). The aim of this work is to experimentally characterize the structural interaction of an invertebrate NDK with either acceptor (dADP and dCDP) or donor (ADP) phosphate substrates in their binary complexes. Since this enzyme appears to be key in viral infection by White spot syndrome virus (Leu et al., 2011 ▶), it may provide clues towards novel antiviral strategies.

2. Materials and methods  

2.1. LvNDK binary-complex co-crystallization with different nucleoside diphosphates  

Recombinant nucleoside diphosphate kinase from L. vannamei (GenBank DQ907945) was obtained from a synthetic gene optimized for expression in E. coli and its purification has been described elsewhere (Quintero-Reyes et al., 2012 ▶). A screening of crystallization conditions of LvNDK was performed using Crystal Screen from Hampton Research (Aliso Viejo, California, USA) by the microbatch method in Greiner plates. Three different nucleotide complexes were obtained by incubating LvNDK (20 mg ml⁻¹) with ADP, dCDP or dADP at 20 mM concentration in solution consisting of 100 mM NaCl, 40 mM MgCl₂, 4 mM DTT. Each nucleotide complex was incubated for 30 min at 277 K before the crystallization trials. The drops were set up by mixing 1 µl nucleotide–enzyme complex solution with 1 µl Crystal Screen crystallization solution (50 solutions) and were covered with 10 µl paraffin oil. The plates were incubated at 289 K and monitored every 3 d until crystals appeared.

After a week, crystals of all of the LvNDK–nucleotide complexes could be grown using three different crystallization buffers. Specifically, LvNDK binary-complex crystals were obtained using the following conditions: conditions 6 [0.2 M magnesium chloride hexahydrate, 0.1 M Tris–HCl pH 8.5, 30%(w/v) PEG 4000] and 22 [0.2 M sodium acetate trihydrate, 0.1 M Tris–HCl pH 8.5, 30%(w/v) PEG 4000] gave crystals for all nucleotides (LvNDK–dADP, LvNDK–dCDP and LvNDK–ADP), while condition 19 [0.2 M ammonium acetate, 0.1 M Tris–HCl pH 8.5, 30%(v/v) 2-propanol] only gave crystals of the dCDP binary complex. Before cooling, the crystals were transferred into a cryocooling solution containing 30%(v/v) PEG 400 substituted for PEG 4000 in the mother liquor, except for condition 19 where 2-propanol acted as a cryoprotectant. Cryoprotectant solutions were supplemented with the appropriate ligands to give the same final concentration as in the crystallization drops. 24 single crystals, including all of the binary complexes, were mounted using CryoLoops according to their size, flash-cooled in liquid N₂ and stored at 100 K.

2.2. X-ray data collection and structure determination  

LvNDK crystals were transported to the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), Upton, USA for data collection. Diffraction experiments were performed on beamline X6A using an ADSC Quantum 270 detector. A data set for each binary complex was collected from a single crystal at a wavelength of 0.979 Å (12 672 eV) at 100 K. 200 images were collected for each crystal with an oscillation range of 1.0° in all cases. The crystal-to-detector distance was 250, 290 and 230 mm for the LvNDK–ADP, LvNDK–dCDP and LvNDK–dADP crystals, respectively. Data sets were reduced and scaled using XDS (Kabsch, 2010 ▶). Data-collection statistics are listed in Table 1 ▶. Initial analysis was performed with the LvNDK–dADP data, and the phase problem was solved by molecular replacement using the NDK crystal structure from bovine retina as a search model (PDB entry 1be4; Abdulaev et al., 1998 ▶). The identity between the shrimp and bovine NDK amino-acid sequences was 78%. Phases were obtained by molecular replacement using Phaser in the CCP4 suite (Winn et al., 2011 ▶; McCoy et al., 2005 ▶). The LvNDK–dADP structure was refined and used as a starting model in the refinement of the other LvNDK binary complexes. Several refinement cycles were performed using PHENIX (Adams et al., 2010 ▶) and Coot for manual building (Emsley et al., 2010 ▶). Ligands were added manually by visual inspection of the electron density at the active site using a 2F _o − F _c map at 1.5σ. All figures were produced using CCP4mg (McNicholas et al., 2011 ▶).

Table 1. Data reduction and refinement statistics of LvNDK binary-complex structures.

Values in parentheses are for the highest resolution shell.

Data set	LvNDK–ADP (PDB entry 4uoh)	LvNDK–dADP (PDB entry 4uof)	LvNDK–dCDP (PDB entry 4uog)
X-ray source	NSLS X6A	NSLS X6A	NSLS X6A
Detector	Quantum 270 CCD	Quantum 270 CCD	Quantum 270 CCD
Wavelength (Å)	0.979	0.979	0.979
Space group	C2221	C2221	C2221
Unit-cell parameters (Å)	a = 69.8, b = 120.8, c = 103.3	a = 70.1, b = 134.3, c = 104.7	a = 70.1, b = 135.3, c = 104.8
No. of residues	430	453	453
Monomers per asymmetric unit	3	3	3
Matthews coefficient (Å3 Da−1)	2.1	2.4	2.4
Solvent content (%)	42.5	49.5	49.5
Resolution range (Å)	34.8–2.0 (2.1–2.0)	19.8–2.10 (2.2–2.1)	19.3–2.3 (2.4–2.3)
Total reflections	242640 (23658)	207177 (10259)	176768 (18322)
Unique reflections	29462 (2912)	26320 (2111)	21500 (2199)
R merge	0.145 (0.735)	0.142 (0.621)	0.171 (0.702)
Completeness (%)	99.6 (96.4)	91.2 (95.1)	95.2 (97.7)
〈I/σ(I)〉	16.7 (3.4)	15.4 (2.7)	13.4 (3.5)
Wilson plot B value (Å2)	24.01	20.36	22.50
Multiplicity	8.2 (8.1)	7.8 (5.4)	8.2 (8.3)
R work/R free †	0.178 (0.241)/0.217 (0.298)	0.189 (0.243)/0.248 (0.299)	0.198 (0.296)/0.256 (0.368)
Content of the asymmetric unit
Protein atoms	3371	3579	3579
Ligand atoms	56	81	75
Water molecules	422	481	221
R.m.s.d. from ideal
Bond lengths (Å)	0.012	0.013	0.015
Bond angles (°)	1.27	1.87	1.55
Mean overall B value (Å2)
Protein	24.7	21.7	21.8
Ligand	27.2	25.7	33.9
Solvent	31.1	26.8	25.0
Ramachandran plot, residues in
Most favoured regions	421 [98%]	439 [97.0%]	439 [97%]
Additionally allowed regions	6 [1.3%]	9 [2.1%]	10 [2.3%]
Outliers	3 [0.7%]	4 [0.9%]	3 [0.7%]

^† R _merge = , where I_i(hkl) and 〈I(hkl)〉 represent the intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements. R _free was caculated using 5% of the reflections.

3. Results and discussion  

3.1. Overall LvNDK binary-complex structures  

To achieve the nucleotide phosphorylation reaction, the active site of LvNDK has a nucleotide-binding site and a catalytic histidine nucleophile (His117), which is part of a phosphorelay that transfers a phosphoryl group obtained from ATP to the nucleoside diphos­phate (Janin et al., 2000 ▶). Enzymatic activity and isothermal titration calorimetry assays of LvNDK have been reported (Quintero-Reyes et al., 2012 ▶).

As we have previously shown, recombinant LvNDK is active and tightly binds nucleoside diphosphates. The dissociation constant (K _d) was determined using ITC: the K _d for dADP was 14.6 µM (Quintero-Reyes et al., 2012 ▶) and the K _d for ADP was 17 µM (unpublished data). The binding of dCDP is atypical, because although LvNDK phosphorylated the nucleotide no heat of binding was observed by ITC (Quintero-Reyes et al., 2012 ▶).

Crystallization plates were set up with LvNDK pre-equilibrated with dADP, dCDP or the ribonucleotide ADP, in all cases including Mg²⁺. All crystals obtained belonged to space group C222₁ with similar unit-cell parameters and highest resolution limits in the range 1.8–2.3 Å. The Matthews coefficient indicates three monomers per asymmetric unit (V _M = 2.4 ų Da⁻¹ and 49.5% solvent content for LvNDK–dADP; Matthews, 1968 ▶; see Table 1 ▶ for details). This is consistent with our previous data showing that LvNDK is a trimer in solution by size-exclusion chromatography studies (Quintero-Reyes et al., 2012 ▶), as previously reported for B. anthracis trimeric NDK (Misra et al., 2009 ▶). Crystal-packing analysis performed with PISA (Protein Interfaces, Surfaces and Assemblies (http://pdbe.org/pisa/; Krissinel & Henrick, 2007 ▶) suggested that a hexamer is actually the biologically significant molecule and therefore our experimental evidence suggests that a dimer of trimers is present instead of a trimer of dimers as observed elsewhere (Lascu et al., 2000 ▶).

The overall structure of each LvNDK monomer shows a typical α/β fold, including six α-helices with a central core formed by a four-stranded antiparallel β-sheet (see Fig. 1 ▶). This β-sheet, a small flexible loop (55–59 residues) and the Kpn loop participate in the nucleotide-binding site. Additionally, the Kpn loop located at the centre of the trimer plays an important role in stabilizing the quaternary structure (Lascu et al., 2000 ▶). Likewise, the C-terminus of each monomer makes intersubunit contacts with neighbouring side chains, providing structural stability to the enzyme (Karlsson et al., 1996 ▶).

Figure 1.

Ribbon representation of the asymmetric unit content of an LvNDK structure with one substrate molecule bound to each monomer. Each subunit is shown in a different colour (yellow, blue and green) and dADP–Mg²⁺ is presented as spheres coloured by atom type.

The C^α-atom root-mean-square deviation (r.m.s.d.) of each monomer within the complete structure is less than 0.6 Å. In this work, the nucleotide–Mg²⁺ complex occupies each active site. The catalytic histidine (His117) and other amino-acid residues that interact with the phosphate group (Arg87) and the sugar moiety (Lys11 and Asn114) and Phe59 and Val111 that stabilize the nitrogen base of the nucleoside diphosphate substrate are invariant and no conformational changes were observed upon ligand binding (r.m.s.d. of 0.22 Å).

One striking feature of NDK is the lack of specificity towards the nucleoside base. The positions of a purine (ADP) and a pyrimidine (dCDP) are superimposable and invariant, as are the phosphate-binding pocket and ribose contacts (Lys11 and Asn114; Fig. 2 ▶). The nitrogenous bases are contacted by a hydrophobic sandwich comprised of Phe59 and Val111, which are conserved among all known NDKs.

Figure 2.

Superposition of the LvNDK active site in a binary complex with dCDP–Mg²⁺ (protein structure in yellow and nucleotide in red) and with ADP–Mg²⁺ (protein structure in blue and nucleotide in green). Distances are in Å and are labelled in black for the NDK–dCDP complex and in blue for the NDK–ADP complex.

3.2. LvNDK nucleotide-binding site: comparison of ribonucleoside and deoxyribonucleoside diphosphates  

To understand the structural details of nucleotide binding, LvNDK was co-crystallized with both ribonucleoside (LvNDK–ADP) and deoxyribonucleoside (LvNDK–dADP and LvNDK–dCDP) diphos­phates.

Fig. 3 ▶ shows a superposition of the active site in the LvNDK binary complexes with dADP and ADP. The positions of the amino-acid residues Arg87 and Lys11, Phe59, Val111 and His117 (not shown) are identical (r.m.s.d. < 0.22 Å). The hydrophobic ‘sandwich’ made by the side chains of Phe59 and Val111 with the purine ring is remarkable, so the electron density strongly supports the ligands modelled in two different structures.

Figure 3.

The specific interaction of Lys11 with both dADP and ADP is mediated by one water molecule in the LvNDK–nucleotide binary-complex structures. Hydrogen bonds are shown between Arg87 and Lys11 and the nucleotide substrate. The electron-density map corresponds to dADP and is displayed as a 2F _o − F _c map at a 3σ contour level (grey mesh). NDK is shown in binary complexes with dADP–Mg²⁺ (protein structure in white and nucleotide in magenta) and with ADP–Mg²⁺ (protein structure in blue and nucleotide in green). Distances are in Å and are labelled in black for the NDK–dADP complex and in blue for the NDK–ADP complex.

The LvNDK binary complex with ADP (Fig. 3 ▶, ligand in green) has two hydrogen bonds between Lys11 and the 2′-OH and 3′-OH ribose groups. These contacts are consistent with those reported by Cherfils et al. (1994 ▶) in the crystal structure of NDK–ADP from D. disco­ideum and also by other authors (Janin et al., 2000 ▶; Souza et al., 2011 ▶). When dADP is co-crystallized with LvNDK (Fig. 3 ▶, ligand in magenta), the position of the ribose is identical apart from the lack of the 2′-OH group. Nonetheless, ‘replacing’ the ribose hydroxyl with a water interacting with Lys11 conserves the hydrogen-bonding pattern. The same water–Lys11 hydrogen bonding was also observed in the complex of LvNDK with dCDP. Lys11 and Asn114 are well conserved among all NDK kinases, creating a preformed rigid pocket that includes an ordered water molecule as needed (Cherfils et al., 1994 ▶). With regard to the lack of heat of dCDP binding as reported previously (Quintero-Reyes et al., 2012 ▶), in the crystal structure we observed a larger B factor for dCDP (33.9 Ų) compared with 25.7 Ų for dADP or 27.2 Ų for ADP.

Although all of the structures reported here appear to be very similar, the main conformational change of NDKs occurs during the binding of nucleoside monophosphates to the ligand-free structures, as reported by Souza et al. (2011 ▶). We conclude that the broad substrate recognition of LvNDK is achieved through a hydrophobic interaction between the Phe59 and Val111 residues and the heterocyclic base and that the binding of deoxynucleosides is assisted through a hydrogen bond to a water molecule. This suggests that invertebrate NDKs may phosphorylate nucleoside analogues that could act as chain terminators for DNA viruses such as White spot syndrome virus (Liu et al., 2009 ▶).

Supplementary Material

PDB reference: LvNDK, complex with dCDP, 4uog

PDB reference: complex with ADP, 4uoh

PDB reference: complex with dADP, 4uof