structural communications
Structure of Mycobacterium tuberculosis nucleoside diphosphate kinase R80N mutant in complex with citrate
aInstitut de Biochimie et de Génétique Cellulaires, UMR 5095, CNRS, 1 Rue Camille Saint-Saëns, 33077 Bordeaux, France, and bInstitut de Biochimie et de Génétique Cellulaires, UMR 5095, Bordeaux University, 1 Rue Camille Saint-Saëns, Bordeaux, France
*Correspondence e-mail: fgeorges@biochem.mpg.de, a.dautant@ibgc.cnrs.fr
The Mycobacterium tuberculosis at 2.6 Å resolution revealed that the intersubunit salt bridge Arg80–Asp93 contributes to the thermal stability of the hexamer (Tm = 76°C). On mutating Asp93 to Asn to break the salt bridge, the thermal stability dramatically decreased by 27.6°C. Here, on mutating Arg80 to Asn, the thermal stability also significantly decreased by 8.0°C. In the X-ray structure of the R80N mutant solved at 1.9 Å resolution the salt bridge was replaced by intersubunit hydrogen bonds that contribute to the thermal stability of the hexamer. A citrate anion from the crystallization buffer was bound at the bottom of the nucleotide-binding site via electrostatic and hydrogen-bonding interactions with six conserved residues involved in nucleotide binding. Structural analysis shows that the citrate is present at the location of the nucleotide phosphate groups.
of the wild-type nucleoside diphosphate kinase from3D view: 4ane
PDB reference: nucleoside diphosphate kinase, R80N mutant, 4ane
1. Introduction
Nucleoside diphosphate kinase (NDPK) is an essential enzyme for the synthesis of non-adenine nucleoside (and deoxynucleoside) 5′-triphosphates. NDPK catalyzes the reversible transfer of the γ-phosphate of nucleoside triphosphates to nucleoside diphosphates (Janin & Deville-Bonne, 2002). NDPKs form hexamers or tetramers (Moynié et al., 2007). 3′-Phosphorylated (Schneider et al., 1998) and cAMP analogues (Strelkov et al., 1995; Anciaux et al., 1997) are known inhibitors of NPDK activity. Although NDPK can function as a protein kinase (Wagner & Vu, 1995), it has been convincingly demonstrated in Escherichia coli that phosphotransferase activity is mediated by ADP (Levit et al., 2002).
The Mycobacterium tuberculosis NDPK (Mt-NDPK; PDB entry 1k44 ) revealed the presence of an intersubunit salt bridge, Arg80–Asp93, which was missing in NDPKs from other organisms (Chen et al., 2002). To check the significance of this salt bridge in the high thermal stability of the hexamer, Arg80 or Asp93 was mutated to a neutral asparagine. Previously, studies using temperature and chaotropic reagents showed that the D93N mutation dramatically decreases the stability of the hexamer. In the crystal structures of the D93N mutant (PDB entries 4anc and 4and ), Arg80 and Asn93 were not involved in any intersubunit interactions (Georgescauld et al., 2013).
of the wild-typeHere, the R80N mutation was introduced. Unexpectedly, the hexamer stability was very different on mutating one or the other of the amino acids forming the bridge. In the 4ane ), the side chain of Asn80 forms two strong intersubunit hydrogen bonds. This new explains why the wild-type (WT) and R80N Mt-NDPK hexamers display higher stability compared with the D93N hexamer. A citrate anion was bound in the active site, making ten interactions with protein ligands, including six direct interactions. Although inhibition of NDPK by citrate has never been reported in vivo, such a possibility cannot be excluded.
of the R80N mutant refined at 1.9 Å resolution (PDB entry2. Materials and methods
2.1. Macromolecule production
The experimental procedures have been described elsewhere (Georgescauld et al., 2013).
2.2. Crystallization
The protein solution was dialysed against 20 mM Tris–HCl pH 7.5 buffer containing 20 mM MgCl2 and concentrated to 11 mg ml−1. The drops were prepared by mixing equal amounts (200 + 200 nl) of the protein and reservoir solutions using a Honeybee 961 robot (Cartesian Technology). Crystals of the R80N mutant grew at 20°C in a few hours using condition No. 86 of The Classics Suite (Qiagen) consisting of 30%(w/v) PEG 4000, 0.2 M ammonium acetate, 0.1 M trisodium citrate pH 5.6. The crystals were cryoprotected in mother liquor supplemented with 20%(v/v) glycerol and flash-cooled in liquid nitrogen.
2.3. Data collection and processing
Diffraction data were collected at 107 K on beamline ID23-1 at ESRF, Grenoble. The data were processed with MOSFLM (Leslie, 2006) and scaled with SCALA (Winn et al., 2011). The data-collection statistics are summarized in Table 1.
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2.4. Structure solution and refinement
The crystal belonged to I2 and contained six molecules in the The structure was determined by the molecular-replacement method with MOLREP (Vagin & Teplyakov, 2010) using the coordinates of the WT Mt-NDPK structure as the search model (PDB entry 1k44 ; Chen et al., 2002). was performed using phenix.refine in PHENIX (Adams et al., 2010) alternated with manual model building in Coot (Emsley & Cowtan, 2004). (NCS) restraints were implemented during excluding residues 44–64, with positional weights of 0.1 and thermal weights of 10.0. TLS groups were designated using the TLSMD server (Painter & Merritt, 2006). In the final model, a few residues belonging to the short loop connecting helices αA and α2 (residues 56–58, 55–58 and 54–61 in chains B, D and F, respectively) were absent owing to poor electron density in these regions. The Fo − Fc maps revealed electron density for citrate in the active site. The occupancy factor of citrate was fixed at 1. Atomic coordinates and experimental structure factors for R80N mutant Mt-NDPK at 1.9 Å resolution have been deposited in the PDB as entry 4ane . The figures were produced using PyMOL (DeLano, 2002). The stereochemistry of the R80N mutant Mt-NDPK structure was good as assessed by MolProbity (Chen et al., 2010). The are summarized in Table 2.
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3. Results and discussion
The thermal Mt-NDPK and its R80N and D93N mutants was followed by The Tm was 76.0°C and 68°C for Mt-NDPK and the R80N mutant, respectively, while it drastically decreased to 48.4°C for the D93N mutant (Supporting Fig. S11). Similar Tm values were obtained on measuring the enzyme inactivation for each protein, suggesting simultaneous dissociation/unfolding (data not shown). Since Arg80 and Asp93 interact via an intersubunit salt bridge, a neutral molecule (urea) or a salt (guanidinium hydrochloride) was used as a chemical denaturant. For the WT protein and the R80N mutant, the unfolding and the loss of occurred simultaneously (Supporting Fig. S2), while the D93N mutation lowered the stability of the hexamer to urea since the hexamer first dissociates into monomers before unfolding (Georgescauld et al., 2013). The salt bridge is clearly involved in the stability of the hexamer. However, such large differences in melting temperature and in behaviour towards chemical for the R80N and D93N mutants constituted a puzzling result. To gain insights into the structural basis of these differences, determinations were carried out.
of WTThe Mt-NDPK R80N mutant crystallized in a monoclinic with 47% solvent content, while the WT and the D93N mutant crystallized in orthorhombic space groups with 56–68% solvent content. Thus, the R80N mutant crystals diffracted to significantly higher resolution (1.8 Å) than the WT crystals (2.6 Å). The overall structures of the monomer (r.m.s.d. of 0.6 Å for Cα atoms) and the hexamer (r.m.s.d. of 0.9 Å for Cα atoms) are essentially unaltered by the R80N mutation (Fig. 1). Compared with the WT structure, the αA–α2 helical loop of every chain is 10° more tilted towards the central β-sheet, closing the catalytic pocket, independent of the presence of citrate and of crystal-packing interactions. At the trimeric interface, relative to the WT, the inter-subunit salt bridge Arg80–Asp93 was obviously lost. However, similarly to Arg80, Asn80 is hydrogen-bonded to the main-chain carbonyl of Leu109 of the neighbouring subunit and moreover to the Asp93 carboxyl group via a water molecule (Fig. 2). The other intersubunit hydrogen bonds present in the WT (Lys29 to the carbonyl of Pro94, Gly105 and Gly108; Arg16 to the carbonyl of Arg28) are conserved in the R80N mutant. This explains why the stability of the R80N hexamer is less disturbed compared with that of the D93N hexamer.
Among the six subunits in the A, D and F) have a bound citrate in the active site. The citrate anion is docked via electrostatic and hydrogen-bonding interactions with six conserved residues: a lysine (Lys10), two arginines (Arg86 and Arg104), a threonine (Thr92), an asparagine (Asn114) and the catalytic histidine (His117) (Fig. 3). It is also hydrogen-bonded to the main-chain N atom of Gly118 and to the His49, His50, Tyr50 and Thr92 side chains through a water molecule. All of the O atoms of the three carboxyl groups and the hydroxyl group are involved in binding. The citrate binding does not appears to be linked to the R80N mutation. Although citrate fits well in the active site and occupies the same location as the nucleotide phosphate groups, a of WT or mutant NDPK in complex with citrate has never been reported.
three subunits (Supporting information
3D view: 4ane
PDB reference: nucleoside diphosphate kinase, R80N mutant, 4ane
Figure S1. Thermal 10.1107/S2053230X13034134/hv5248sup1.jpg
The R80N mutant decreased by 8 degrees Celcius and behaves more similarly to the wild-type protein than to the D93N mutation, for which a dramatic decrease of 28 degrees Celcius was observed. The temperature dependence of the excess molar of wild-type Mt-NDPK (full circles), the R80N mutant (open squares) and the D93N mutant (full squares) is shown. Each curve displays a single calorimetric peak. The protein concentration was 0.2-0.3 mg ml-1. DOI:Figure S2. Denaturation/renaturation by urea/GuHCl of wild-type and R80N mutant Mt-NDPK. The unfolding and the loss of 10.1107/S2053230X13034134/hv5248sup2.jpg
are indicated by the excess heat and the enzymatic activity, respectively. Unfolding (full squares) and subsequent refolding (open squares) were monitored by following the intrinsic fluorescence of Mt-NDPK (A in urea, B in GuHCl) and R80N mutant (C in urea, D in GuHCl). The residual enzymatic activity for the unfolding is shown by open circles. The protein concentration was 10 mg ml-1. The measurements are normalized to the maxima; fn is the fraction of native protein. DOI:Figure S3. Binding scheme of citrate in chain A of D80N mutant Mt-NDPK. The figure was drawn with LigPlot+ (Laskowski and Swindells, 2011). DOI: 10.1107/S2053230X13034134/hv5248sup3.pdf
Acknowledgements
Pr Ioan Lascu and Pr Anna Giartosio are acknowledged for their support and helpful discussions. The authors wish to thank the beamline staff at the ESRF, Grenoble, France for their help in performing X-ray diffraction. This project was supported by the ESRF.
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