The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2+ ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2+ insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni2+- and Zn2+-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 Å (apo), 1.59 Å (Ni2+) and 2.52 Å (Zn2+) resolution, show the conserved proximal and solvent-exposed His102 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His152. The analysis of X-ray absorption spectral data obtained using solutions of Ni2+- and Zn2+-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.

INTRODUCTION

Urease [1,2] is a Ni2+-dependent enzyme that plays a crucial role in the nitrogen cycle by catalysing the hydrolysis of urea to ammonia and carbamate with a 3×1015-fold rate enhancement with respect to the uncatalysed reaction [3] (1).

Mechanisms of urea decomposition in water

The active site contains two Ni2+ ions that are bridged by a post-translationally carbamylated lysine residue and a hydroxide ion, and are bound to the protein framework by four histidine imidazole nitrogen atoms and one aspartate residue carboxylate oxygen atom [47]. The co-ordination geometry of the Ni2+ ions is completed by labile water molecules, yielding one penta-co-ordinated Ni2+ ion with a distorted square-pyramidal geometry, and one hexa-co-ordinated Ni2+ ion with a distorted octahedral geometry (2).

Activation of urease and building of the nickel-containing active site

Urease is initially produced in the apo form, devoid of Ni2+ ions and enzymatic activity. The apo-enzyme is modified in several successive steps that require a dedicated set of accessory proteins, usually comprising UreD, UreF, UreG and UreE. [8] This process leads to carbamylation of the active-site lysine and incorporation of the binuclear metallic active site, with consequent enzyme activation (2). The activity of urease is strictly required for the survival and growth of bacterial pathogens that colonize human and animal gastric mucosa as well as intestinal and urinary tracts, and therefore both the enzyme and the accessory proteins represent targets for drug development [9,10]

The urease activation process entails the formation of a multimeric complex between the apoenzyme and UreD, UreF and UreG, with the latter protein probably responsible for lysine carbamylation following GTP hydrolysis. UreE appears to act as a metallo-chaperone by delivering Ni2+ to the UreDFG complex [1113]. This role for UreE is supported by the evidence that the concentration of Ni2+ required for proper assembly of the urease active site is considerably reduced, and a larger amount of enzyme is activated, in the presence of UreE [14]. Another crucial role for UreE is the enhancement of the GTPase activity of UreG [13], which relies on the direct UreE–UreG interaction shown to occur in vivo and in vitro [15,16].

Recombinant UreE proteins from different sources, including KaUreE (Klebsiella aerogenes UreE) [17], BpUreE (Bacillus pasteurii UreE) [18] and HpUreE (Helicobacter pylori UreE) [19], have been structurally characterized. These orthologues consistently exhibit a homodimeric architecture composed of an N-terminal domain and a C-terminal domain, the latter mediating head-to-head dimerization. A conserved metal-binding site involves a pair of closely spaced histidine residues, one per monomer, located on the protein surface at the homodimer interface (His96 in KaUreE, His100 in BpUreE and His102 in HpUreE).

Despite sharing common structural features, UreE proteins have different metal-binding capabilities. In particular, they exhibit a variable stoichiometry for Ni2+-binding that ranges from one metal ion bound per dimer for HpUreE [15,20] to six ions for KaUreE [21], with two Ni2+ ions bound in the case of BpUreE [22]. Interestingly, this peculiarity is reflected by the nature of their C-terminal regions. KaUreE, possessing the highest Ni2+-sequestering activity, features a histidine-rich tail containing ten histidine residues among the last fifteen amino acids. BpUreE displays two C-terminal histidine residues at the end of its sequence, in the context of a His-Gln-His motif, whereas in this region HpUreE contains a single histidine residue (His152) [23].

All UreE proteins contain at least one histidine residue in the C-terminal portion, an observation that suggested a role for the C-terminus in modulating the metal-trafficking activity of UreE proteins, in terms of both selectivity and stoichiometry of metal binding [15,22,23]. However, the structural and functional details of these regions are not well established. The crystal structure of KaUreE (PDB codes 1GMW, 1GMU and 1GMV) was determined on a truncated form of this protein (H144*KaUreE) that lacks the last 15 residues [17], whereas the crystal structure of BpUreE (PDB codes 1EAR and 1EBO) showed a solid-state disorder that prevented the observation of the Gly-His-Gln-His motif at the C-terminus [18]. The recently described structures of HpUreE (PDB codes 3L9Z, 3NXZ, 3NYO and 3LA0) in the apo form or bound to Cu2+, Ni2+ or an unidentified metal ion cover only residues 1/2–148/149 and therefore do not include His152 [19]. The only exception is the tetrameric (dimer of dimers) form of Ni-HpUreE, which features a square pyramidal Ni2+ site composed of four His102 residues (one from each protomer) in the basal plane and a single His152 in the axial position [19]. However, this type of tetrameric arrangement, even though observed commonly in the solid state probably because of the elevated concentrations utilized for protein crystallization, does not correspond to the simple dimeric form of the protein present in solution, as established using static and dynamic light scattering [15,22].

The present study describes the crystal structures of recombinant HpUreE in the apo, Ni2+-bound and Zn2+-bound forms. These structures reveal the architecture of the C-terminal arm and the metal-binding mode of the His152 residue located in this region. The structures, determined in the solid state, were corroborated by X-ray absorption spectroscopy in frozen solutions of the wild-type and the H152A HpUreE mutant proteins in the presence of bound Ni2+ and Zn2+, providing accurate metric parameters in the vicinity of the metal ions. The results of the present study clarify the role of the protein framework for Ni2+ and Zn2+ trafficking effected by UreE within the urease activation process.

EXPERIMENTAL

Protein crystallization

Recombinant apo-HpUreE was purified as described previously [15]. Crystallization trials were carried out using the hanging-drop vapour-diffusion method. Crystals of the apoprotein were obtained by mixing 2 μl of an 8 mg/ml protein solution in Tris/HCl buffer, pH 7, with a reservoir solution containing 0.1 M sodium citrate, pH 5.6, 0.5 M ammonium sulfate and 1.0 M lithium sulfate. Cuboidal crystals appeared within 2 weeks. Crystals of Zn-HpUreE were obtained by mixing 2 μl of an 8 mg/ml protein solution in Tris/HCl buffer, pH 7, containing an equimolar amount of Zn2+ (ZnSO4), with 2 μl of a reservoir solution made of 4.0 M sodium formate and 0.1 M sodium cacodylate, pH 6.5. Crystals with a hexagonal cross-section formed within 2 weeks. Crystals of Ni-HpUreE were obtained by mixing 2 μl of an 8 mg/ml protein solution in Tris/HCl buffer, pH 7, containing an equimolar amount of Ni2+ (NiSO4), with 2 μl of a reservoir solution made of 4.0 M sodium formate and 0.1 M sodium acetate, pH 5.5. One block-shaped crystal appeared after approximately 30 days.

X-ray diffraction data collection, structure determination and refinement

X-ray diffraction data were recorded on the BL14.1 and BL14.2 beam lines at BESSY (Berlin Electron Storage Ring Company for Synchrotron Radiation) (Berlin, Germany) at 100 K in the presence of 20% (v/v) glycerol or 4.0 M sodium formate as a cryo-protectant. Data collection and refinement statistics are summarized in Table 1.

Table 1
X-ray diffraction data collection and refinement statistics

Values in parentheses are for the highest-resolution shell.

 Zn-HpUreE   Ni-HpUreE Apo-HpUreE 
Data collection      
 Beamline BESSY BL14.2   BESSY BL14.2 BESSY BL14.1 
 Temperature (K) 100   100 100 
 Peak Inflection Remote   
 Wavelength (Å) 1.28183 1.28316 1.27655 0.91885 0.91841 
 Space group P6522   C2221 P212121 
 Cell constants (Å)      
  a 109.34   67.55 69.00 
  b 109.34   117.14 70.47 
  c 280.34   98.66 123.34 
 Resolution (Å) 50.00–2.52 (2.56–2.52) 50.00–2.59 (2.63–2.59) 50.00–2.77 (2.82–2.77) 50.00–1.59 (1.62–1.59) 20.00–2.00 (2.03–2.00) 
Rsym 0.092 (0.434) 0.095 (0.586) 0.100 (0.558) 0.049 (0.448) 0.060 (0.593) 
I/σ(I22.4 (3.4) 21.5 (2.3) 20.8 (2.3) 24.9 (2.6) 21.6 (2.0) 
 Completeness (%) 99.9 (98.2) 99.7 (95.9) 99.5 (92.9) 97.9 (87.7) 99.7 (96.6) 
 Redundancy 9.4 (6.9) 9.1 (5.2) 9.0 (4.9) 8.4 (3.2) 5.5 (4.9) 
Refinement      
 Resolution (Å) 2.52   1.59 2.00 
 No. of reflections 33219   48955 38745 
Rwork/Rfree 0.203/0.245   0.185/0.210 0.208/0.267 
 Number of protein atoms 4697   2402 4734 
 Number of metal atoms   
 Number of water molecules 186   154 100 
 Average B-factor for protein atoms (Å243.815   33.694 44.327 
 Average B-factor for metal atoms (Å237.275   28.650 − 
 Rmsd of bond lengths (Å) 0.021   0.031 0.022 
 Rmsd of bond angles (°) 1.909   2.578 1.947 
 PDB code 3TJ9   3TJ8 3TJA 
 Zn-HpUreE   Ni-HpUreE Apo-HpUreE 
Data collection      
 Beamline BESSY BL14.2   BESSY BL14.2 BESSY BL14.1 
 Temperature (K) 100   100 100 
 Peak Inflection Remote   
 Wavelength (Å) 1.28183 1.28316 1.27655 0.91885 0.91841 
 Space group P6522   C2221 P212121 
 Cell constants (Å)      
  a 109.34   67.55 69.00 
  b 109.34   117.14 70.47 
  c 280.34   98.66 123.34 
 Resolution (Å) 50.00–2.52 (2.56–2.52) 50.00–2.59 (2.63–2.59) 50.00–2.77 (2.82–2.77) 50.00–1.59 (1.62–1.59) 20.00–2.00 (2.03–2.00) 
Rsym 0.092 (0.434) 0.095 (0.586) 0.100 (0.558) 0.049 (0.448) 0.060 (0.593) 
I/σ(I22.4 (3.4) 21.5 (2.3) 20.8 (2.3) 24.9 (2.6) 21.6 (2.0) 
 Completeness (%) 99.9 (98.2) 99.7 (95.9) 99.5 (92.9) 97.9 (87.7) 99.7 (96.6) 
 Redundancy 9.4 (6.9) 9.1 (5.2) 9.0 (4.9) 8.4 (3.2) 5.5 (4.9) 
Refinement      
 Resolution (Å) 2.52   1.59 2.00 
 No. of reflections 33219   48955 38745 
Rwork/Rfree 0.203/0.245   0.185/0.210 0.208/0.267 
 Number of protein atoms 4697   2402 4734 
 Number of metal atoms   
 Number of water molecules 186   154 100 
 Average B-factor for protein atoms (Å243.815   33.694 44.327 
 Average B-factor for metal atoms (Å237.275   28.650 − 
 Rmsd of bond lengths (Å) 0.021   0.031 0.022 
 Rmsd of bond angles (°) 1.909   2.578 1.947 
 PDB code 3TJ9   3TJ8 3TJA 

Three datasets for the Zn-HpUreE crystal were collected in a MAD (multiwavelength anomalous dispersion) experiment in the 2.52–2.77 Å resolution range (1 Å=0.1 nm). The images were processed with the HKL package [24] using an ‘anomalous’ option during scaling. The Zn-HpUreE structure was solved with a three-wavelength MAD protocol in Auto-Rickshaw [25]. Heavy-atom searching was performed with SHELXD [26], and the resulting positions were refined with phase calculation using SHARP [27]. The density modification and phase extension were performed using DM [28] and RESOLVE [29]. An initial model was built using ARP/wARP [30,31]. Heavy-atom analysis based on the initial model, together with phasing and building cycles, were performed using PHASER [32], MLPHARE [33], SHELXE [34], RESOLVE [29] and BUCCANEER [33]. Three heavy-atom sites were detected, which included two Zn2+ ions and the sulfur atom of Cys95. Initially, 598 residues from the four protomers in the asymmetric unit were found by automatic modelling, followed by manual model building and refinement using REFMAC [35].

One dataset at 1.59 Å resolution was recorded for the Ni-HpUreE crystal. The data were processed with the HKL package [24]. The structure was solved by molecular replacement using PHASER [32] with one chain of Zn-HpUreE as the search model. Solutions were found that corresponded to two HpUreE protomers in the asymmetric unit. The atomic model was refined using REFMAC [35].

One dataset at 2.00 Å resolution was recorded for the apo-HpUreE crystal. The data were processed with the HKL package [24]. The structure was solved by molecular replacement using PHASER [32], with one chain of Ni-HpUreE as the search model. Four apo-HpUreE protomers were found in the asymmetric unit. The atomic model was refined using REFMAC [35].

X-ray absorption spectroscopy sample preparation

Metal ions (0.9 equivalents) as sulfate salts were added to stock solutions of wild-type HpUreE dimer (0.60 mM) and H152A-HpUreE dimer (0.25 mM for Zn2+ and 0.87 mM for Ni2+) to prepare Zn2+- and Ni2+-bound protein samples. Final sample concentrations are listed in Table 2. Samples were prepared in 20 mM Tris/HBr buffer at pH 7, containing 150 mM NaBr. The protein samples were incubated for 5 min upon metal addition, loaded into sample cells consisting of a polycarbonate sample holder with a kapton window, and frozen in liquid N2. The samples were stored at −80°C and transported in liquid N2 before being used at the synchrotron beam line. On the basis of established Kd values and protein concentrations [15], less than 2% of the Ni2+ and Zn2+ added should be dissociated under these conditions.

Table 2
Final sample concentrations used for EXAFS analysis of HpUreE wild-type and H152A mutant
Sample Protein (mM) Ni2+ (mM) Zn2+ (mM) 
Ni-HpUreE 0.53 0.48  
Zn-HpUreE 0.53  0.48 
H152A Ni-HpUreE 0.77 0.69  
H152A Zn-HpUreE 0.22  0.19 
Sample Protein (mM) Ni2+ (mM) Zn2+ (mM) 
Ni-HpUreE 0.53 0.48  
Zn-HpUreE 0.53  0.48 
H152A Ni-HpUreE 0.77 0.69  
H152A Zn-HpUreE 0.22  0.19 

X-ray absorption spectroscopy data collection and analysis

Datasets were collected at SSRL (Stanford Synchrotron Radiation Lightsource; 3 GeV ring) beam line 9-3 equipped with a 100-element Ge X-ray fluorescence detector array (Canberra). The only exception is the H152A HpUreE Ni2+ sample, which was run at beam line 7-3 using a 30-element Ge detector. Both stations consisted of a Si(220) ϕ=0° double-crystal monochromator, and a liquid helium cryostat for the sample chamber. Söller slits were used to reduce scattering and 3 μm Z-1 element filters were placed between the sample and the detector. Internal energy calibration was performed by collecting spectra simultaneously in transition mode on the relevant metal foil (Zn or Ni).

Data averaging and energy calibration was performed using SixPack [36]. The first inflection points from the XANES (X-ray absorption near-edge spectroscopy) spectral regions were set to 9660.7 eV for Zn foil (Zn samples) and to 8331.6 eV for Ni foil (Ni samples). The AUTOBK algorithm available in the Athena software package was employed for data reduction and normalization [37]. A linear pre-edge function followed by a quadratic polynomial for the post-edge was used for background subtraction followed by normalization of the edge-jump.

Data limits were chosen to maximize resolution and signal-to-noise ratio. The Zn-HpUreE EXAFS (extended X-ray absorption fine structure) data were extracted using an Rbkg of 0.9 and a spline with a range for k of 2–13.5 Å−1, with a rigid spline clamp at higher k. The k3-weighted data were fitted in r-space over the k range 2–13.5 Å−1 using an E0 of 9670 eV. The Ni-HpUreE EXAFS data were extracted using an Rbkg of 1, and a spline from k=2 Å−1 to k=13.5 Å−1 with a strong clamp at high k values for wild-type HpUreE, and a spline from k=2 Å−1 to k=12.5 Å−1 with a rigid clamp at higher k values for H152A HpUreE. The k3-weighted data were fitted in r-space over the k=2–13.5 Å−1 region for wild-type HpUreE and k=2–12.5 Å−1 for the mutant, with E0 for Ni2+ set to 8340 eV in both cases. All datasets were processed using a Kaiser–Bessel window with a dk of 2 (window sill).

Artemis software employing the FEFF6 and IFEFFIT algorithms was used to generate and fit scattering paths to the data [3739]. Single-scattering and multiple-scattering fits were performed as described in subsequent sections. Single-scattering fits were generally carried out over an r-space of 1–2.0 (and up to 2.5) Å, whereas multiple-scattering fits were generated over the 1–4.0 (and up to 4.2) Å range of r-space, as specified in Supplementary Tables S1–S8 at http://www.BiochemJ.org/bj/441/bj4411017add.htm. Average values and bond lengths obtained from crystallographic data were used to construct rigid imidazole rings to fit histidine residues [40]. The position of the imidazole ring with respect to the metal centre was fitted in terms of the metal–ligand bond distance (Reff) and the rotation angle α (Figure 1) [41,42].

Definition of the α angle involving histidine imidazole and bound metal ion M

To assess the goodness-of-fit from different fitting models, the R-factor, χ2 and reduced χ2ν2) were minimized. Increasing the number of adjustable parameters is generally expected to improve the R-factor; however, χν2 may go through a minimum and then increase, indicating that the model is over-fitting the data. These parameters are defined as follows:

 
formula

and:

 
formula

where Nidp is the number of independent data points defined as:

 
formula

where Δr is the fitting range in r-space, Δk is the fitting range in k-space, Npts is the number of points in the fitting range, Nvar is the number of variables floating during the fit, ϵ is the measurement uncertainty, Re() is the real part of the EXAFS Fourier-transformed data and theory functions, Im() is the imaginary part of the EXAFS Fourier-transformed data and theory functions, χ(Ri) is the Fourier-transformed data or theory function and:

 
formula

RESULTS

X-ray crystallography on HpUreE in the apo and holo forms bound to Ni2+ and Zn2+

Three crystal structures have been analysed: HpUreE in the apo form at 2.00 Å resolution (apo-HpUreE), the protein co-crystallized with Ni2+ at 1.59 Å resolution (Ni-HpUreE), and the protein co-crystallized with Zn2+, solved at 2.52 Å resolution (Zn-HpUreE). Each structure is in a different crystal form.

The crystals of the apoprotein are orthorhombic, P212121, with four polypeptide chains assembled into two dimers in the asymmetric unit (Figure 2). Each chain was modelled starting from the N-terminal methionine residue, but at the C-termini the last 20–22 residues are disordered and therefore invisible. Unambiguous electron density extends to Ser149 in chain A, to Met150 in chains B and D, and to Val148 in chain C.

Crystal structure of apo-HpUreE

Figure 2
Crystal structure of apo-HpUreE

Ribbon schemes of the crystallographic structural model of the apo-HpUreE dimer of dimers in the asymmetric unit. (B) Representation of the same structure in (A) rotated by 90° about the horizontal axis. Each of the protomers forming the dimer on the left is coloured from blue in the proximity of the N-terminus to red at the C-terminus in order to highlight the secondary structure elements along the protein sequence. The dimer on the right shows the two protomers in different colours (purple and pink). The side chains of the conserved His102 are represented as ball-and-stick models coloured according to the CPK (Corey–Panling–Koltun) colour code.

Figure 2
Crystal structure of apo-HpUreE

Ribbon schemes of the crystallographic structural model of the apo-HpUreE dimer of dimers in the asymmetric unit. (B) Representation of the same structure in (A) rotated by 90° about the horizontal axis. Each of the protomers forming the dimer on the left is coloured from blue in the proximity of the N-terminus to red at the C-terminus in order to highlight the secondary structure elements along the protein sequence. The dimer on the right shows the two protomers in different colours (purple and pink). The side chains of the conserved His102 are represented as ball-and-stick models coloured according to the CPK (Corey–Panling–Koltun) colour code.

The Ni-HpUreE crystals are orthorhombic, C2221, with one protein dimer and one Ni2+ ion in the asymmetric unit (Figure 3). Amino acid residues were modelled from Met1 to His149 for chain A and from Met1 to His152 for chain B.

Crystal structure of Ni-HpUreE

Figure 3
Crystal structure of Ni-HpUreE

(A) Ribbon scheme of the crystallographic structural model of the Ni-HpUreE dimer in the asymmetric unit, with each protomer coloured from blue in the proximity of the N-terminus to red at the C-terminus in order to highlight the secondary structure elements along the protein sequence. The symmetry-related dimer that carries the Glu4B' residue bound to Ni2+ (represented as a black sphere) is shown as transparent gold ribbon. The side chains of the Ni-bound ligands His102A, His102B, His152B and Glu4B', as well as the solvent molecule, are represented as ball-and-stick models coloured according to the CPK (Corey–Pauling–Koltun) colour code. (B) Close-up view of the co-ordination environment of the Ni2+ ion together with the 2FoFc electron density map contoured at 1.5 σ (light blue) and the FoFc electron density map contoured at 3.0 σ (magenta).

Figure 3
Crystal structure of Ni-HpUreE

(A) Ribbon scheme of the crystallographic structural model of the Ni-HpUreE dimer in the asymmetric unit, with each protomer coloured from blue in the proximity of the N-terminus to red at the C-terminus in order to highlight the secondary structure elements along the protein sequence. The symmetry-related dimer that carries the Glu4B' residue bound to Ni2+ (represented as a black sphere) is shown as transparent gold ribbon. The side chains of the Ni-bound ligands His102A, His102B, His152B and Glu4B', as well as the solvent molecule, are represented as ball-and-stick models coloured according to the CPK (Corey–Pauling–Koltun) colour code. (B) Close-up view of the co-ordination environment of the Ni2+ ion together with the 2FoFc electron density map contoured at 1.5 σ (light blue) and the FoFc electron density map contoured at 3.0 σ (magenta).

The Zn-HpUreE crystals are hexagonal, P6522, and contain two protein dimers and two Zn2+ ions in the asymmetric unit (Figure 4). The polypeptide chains were modelled from the N-terminal Met1. Chains B and C could be traced to residue Ser153 and Glu154 respectively. Chains A and D were modelled to Ser149, whereas the electron density from residues 150 to154 is unclear and has been interpreted as statically disordered residues (see below). In addition, the electron density in chain D is disordered from Leu13 to Ser19 and from Lys65 to Ile71.

Crystal structure of Zn-HpUreE

Figure 4
Crystal structure of Zn-HpUreE

Ribbon scheme of the crystallographic structural model of the Zn-HpUreE dimer of dimers in the asymmetric unit. Each protomer of the dimer on the top is coloured from blue in the proximity of the N-terminus to red at the C-terminus in order to highlight the secondary structure elements along the protein sequence. The dimer on the bottom shows the two protomers in different colours (purples and pink). The side chains of the histidine residues binding the Zn2+ ions (shown as black spheres) are represented as ball-and-stick models coloured according to their position in the sequence and in the protomer. (B) Close-up view of the co-ordination environment of the Zn2+ ions together with the 2FoFc electron density map contoured at 1.0 σ (light blue).

Figure 4
Crystal structure of Zn-HpUreE

Ribbon scheme of the crystallographic structural model of the Zn-HpUreE dimer of dimers in the asymmetric unit. Each protomer of the dimer on the top is coloured from blue in the proximity of the N-terminus to red at the C-terminus in order to highlight the secondary structure elements along the protein sequence. The dimer on the bottom shows the two protomers in different colours (purples and pink). The side chains of the histidine residues binding the Zn2+ ions (shown as black spheres) are represented as ball-and-stick models coloured according to their position in the sequence and in the protomer. (B) Close-up view of the co-ordination environment of the Zn2+ ions together with the 2FoFc electron density map contoured at 1.0 σ (light blue).

The polypeptide fold is similar to the previously reported crystal structures of KaUreE [17] (PDB codes 1GMW, 1GMU and 1GMV), BpUreE [18] (PDB codes 1EAR and 1EB0) and HpUreE [19] (PDB codes 3L9Z, 3NXZ, 3NY0 and 3LA0). Each protein subunit contains two domains (Figures 2–4). The N-terminal domain includes residues from Met1 to Asp77 and consists of two three-stranded mixed β-sheets with two extended loops connecting strands 1 and 2, and strand 2 with strand 3. Each of the two loops contains a β-turn. The C-terminal domain includes residues from Ser78 and has a ferredoxin-like βαββαβ fold. Residues Glu144–Leu146 form a short fifth β-strand and the last ordered residues stretch along the two α-helices of the other subunit of the dimer. The segment from Ser149 to Glu154 is poorly ordered but interpretable in Zn-HpUreE, whereas the remaining residues, up to the C-terminal Lys170, are not visible in the electron density.

The proteins in the three crystal structures form dimers. The core of their interface is formed by two α-helices (residues 88–102) running in parallel. Each helix is braced on the other side by a segment of residues 146–150 from the other subunit. Both hydrophobic and hydrogen-bonded interactions occur between the two helices, and between the helices and neighbouring residues and the poorly ordered C-terminal stretch, with a notable hydrophobic cluster formed by pairs of Val88 and Val91 from the two subunits. Symmetric inter-subunit hydrogen bonds are found between Tyr96 and Ala103, Asn100 and His102, Ala89 and Gln111, and Glu97 and Ser149.

The Ni2+ ion in Ni-HpUreE co-ordinates six ligands arranged in a pseudo-octahedral co-ordination geometry (Figure 3). It interacts with His102A, His102B, His152B, Glu4B' (a residue located on chain B of a symmetry-related molecule), one water molecule and another unidentified ligand (Figure 5A). The electron density of this moiety is elongated, and therefore it is unlikely to be a water molecule (Figure 3B). It could be His152A, but there is no continuity in the electron density between this ligand density and the nearby Ser149A, which is the last visible residue of chain A. This implies a disordered chain comprising residues 150 and 151. The role of Glu4B' in forming a dimer of dimers is probably a solid-state effect since only dimers (not tetramers) are observed in solution using multi-angle scattering [15,22], and this dangling Ni2+ ligand could easily be replaced by a water molecule in solution.

Schematic depiction of the ligand environments around the metal ions in Ni-HpUreE and Zn-HpUreE

Figure 5
Schematic depiction of the ligand environments around the metal ions in Ni-HpUreE and Zn-HpUreE

(A) Interaction of the Ni2+ with six ligands in the Ni-HpUreE structure. The A/B dimer is shown in grey, and the symmetry-related dimer A'/B' is shown by broken lines. The ambiguous interaction with His152A is indicated with a grey line. (B) Interactions of the two Zn2+ ions with four ligands each in the Zn-HpUreE structure. Dimers A/B and C/D are shown in grey. The alternative interactions with His152A and Glu154A are shown using a grey line.

Figure 5
Schematic depiction of the ligand environments around the metal ions in Ni-HpUreE and Zn-HpUreE

(A) Interaction of the Ni2+ with six ligands in the Ni-HpUreE structure. The A/B dimer is shown in grey, and the symmetry-related dimer A'/B' is shown by broken lines. The ambiguous interaction with His152A is indicated with a grey line. (B) Interactions of the two Zn2+ ions with four ligands each in the Zn-HpUreE structure. Dimers A/B and C/D are shown in grey. The alternative interactions with His152A and Glu154A are shown using a grey line.

In Zn-HpUreE, each of the two Zn2+ ions have four ligands arranged in a pseudo-tetrahedral co-ordination geometry (Figure 4B). Zn(1) interacts with His102C, His102D, His152C and a partially disordered Glu154D, whereas Zn(2) is bound by His102A, His102B, His152B and the partially disordered His152D from the neighbouring C/D dimer (Figure 5B). The electron density corresponding to the segment His152D–Glu154D suggests an alternative interpretation, with the side chain of His152A taking the place of Glu154D, and Glu154A replacing His152D. The first alternative seems to have a higher occupancy factor, but some residual density indicates that the second alternative also occurs in the crystal.

All of the subunits of the apo and metal-bound HpUreE models were superposed using the Cα atoms. The rmsd (root-mean-square deviation) values ranged from 0.6 to 1.1 Å. The number of outliers, pairs of atoms deviating more than 3 rmsd, ranged from zero to nine. A comparison with the previously determined structures of UreE from H. pylori (PDB codes 3MY0 and 3L9Z) gave similar statistics. Comparing the two apo-HpUreE dimers or the two Zn2+ dimers present in the asymmetric unit gave a similarly good fit (0.8–0.9 Å, with zero and three outliers respectively). Significantly larger differences were observed only when apo-HpUreE dimers were compared with metal-loaded dimers (rmsd 1.3–1.4 Å with 30–66 outliers) and when Zn2+-bound dimers were compared with Ni2+-bound dimers (rmsd 0.9 Å with 48 outliers), with the largest differences observed in the outer (C-terminal) domains of the dimer.

X-ray absorption spectroscopy on the Ni2+-binding site in wild-type and H152A HpUreE

The Ni K-edge XANES spectra of both the wild-type and H152A mutant Ni-HpUreE samples exhibit a single intense white line at ~8347 eV, and a small pre-edge peak that is associated with a 1s→3d transition at 8331.6 eV, consistent with a six-co-ordinate Ni2+-binding site and octahedral geometry (Figure 6A) [43]. The difference between the spectra obtained for wild-type and the mutant HpUreE samples arises from the nature of the ligands involved, as revealed by the analysis of the EXAFS spectra (see Supplementary Tables S1–S4 at http://www.BiochemJ.org/bj/441/bj4411017add.htm).

X-ray absorption spectroscopic analysis of HpUreE at the Ni K-edge

Figure 6
X-ray absorption spectroscopic analysis of HpUreE at the Ni K-edge

(A) Ni K-edge XANES spectra of wild-type (WT) and H152A Ni-HpUreE. (B) Fourier-transformed Ni K-edge EXAFS spectra of wild-type Ni-HpUreE [no phase correction, Fourier transform (FT) window=2–13.5 Å−1]. The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles). (C) Fourier-transformed Ni K-edge EXAFS spectra of H152A Ni-HpUreE (no phase correction, FT window=2–12.5 Å−1). The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles).

Figure 6
X-ray absorption spectroscopic analysis of HpUreE at the Ni K-edge

(A) Ni K-edge XANES spectra of wild-type (WT) and H152A Ni-HpUreE. (B) Fourier-transformed Ni K-edge EXAFS spectra of wild-type Ni-HpUreE [no phase correction, Fourier transform (FT) window=2–13.5 Å−1]. The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles). (C) Fourier-transformed Ni K-edge EXAFS spectra of H152A Ni-HpUreE (no phase correction, FT window=2–12.5 Å−1). The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles).

The best multiple-scattering fits of the wild-type Ni-HpUreE (Supplementary Tables S1 and S2) are consistent with the presence of four histidine residues around the Ni2+ centre, spread over two shells of N/O-donor ligands (Figure 6B). The features between 2 and 4 Å are best described using a combination of histidine ligands with an angle α of 5° and 10°, separated in two scattering shells. At 2.06(2) Å, a shell is formed by a pair of histidine residues with an angle α of 10° and two N/O ligands. The second shell consists of an additional two histidine residues (α=5°) at 2.15(1) Å (Table 3, Figure 6B and Supplementary Table S2). The separation between the two shells of ~0.09(3) Å is at the limit of the resolution (~0.1 Å) for the dataset. Splitting the histidine residues into two shells significantly improves the goodness-of-fit, although the reduced χ2 only drops by a factor of 1.3 (compared with an optimal factor of 1.7). Such a two-shell model is consistent with the wild-type HpUreE Ni2+-binding site described by the crystallographic data in the present study, as well as theoretical models [15]. Both studies show the presence of two distinct sets of histidine residues at the Ni2+ site in the wild-type HpUreE dimer, the His102 pair and the His152 pair, where the His102 pair is essential for metal binding.

Table 3
Best fit EXAFS models

WT, wild-type.

Sample Ligand* r (Å) σ2 (×103 Å2%R χν2 
WT Ni-HpUreE 2 NHis10 2.15(2) 3(1) 3.8 36.6 
 2 NHis5 2.06(1) 2.5(7)   
 2 N/O 2.06(1) 2.5(7)   
H152A Ni-HpUreE 2 NHis10 2.09(1) 3.7(5) 5.2 32.4 
 4 N/O 2.09(1) 3.7(5)   
WT Zn-HpUreE 2 NHis5 1.99(2) 5(1) 3.78 17.3 
 2 N/O 2.07(3) 12(5)   
 1 Br 2.38(1) 4.3(4)   
 2 NHis5 2.00(1) 4(1) 2.61 12.8 
 1 NHis5 2.16(1) 1(1)   
 1 N/O 2.00(1) 3(2)   
 1 Br 2.39(1) 4.8(5)   
H152A Zn-HpUreE 2 NHis5 2.01(1) 6.7(5) 1.85 3.96 
 2 N/O 2.01(1) 6.7(5)   
 1 Br 2.39(1) 3.4(2)   
Sample Ligand* r (Å) σ2 (×103 Å2%R χν2 
WT Ni-HpUreE 2 NHis10 2.15(2) 3(1) 3.8 36.6 
 2 NHis5 2.06(1) 2.5(7)   
 2 N/O 2.06(1) 2.5(7)   
H152A Ni-HpUreE 2 NHis10 2.09(1) 3.7(5) 5.2 32.4 
 4 N/O 2.09(1) 3.7(5)   
WT Zn-HpUreE 2 NHis5 1.99(2) 5(1) 3.78 17.3 
 2 N/O 2.07(3) 12(5)   
 1 Br 2.38(1) 4.3(4)   
 2 NHis5 2.00(1) 4(1) 2.61 12.8 
 1 NHis5 2.16(1) 1(1)   
 1 N/O 2.00(1) 3(2)   
 1 Br 2.39(1) 4.8(5)   
H152A Zn-HpUreE 2 NHis5 2.01(1) 6.7(5) 1.85 3.96 
 2 N/O 2.01(1) 6.7(5)   
 1 Br 2.39(1) 3.4(2)   
*

The superscripted number is the angle α for the histidine ligands.

A weakening of the Ni2+ complex formation was observed upon mutagenesis of His152, with the dissociation constant increasing from 0.15 μM in the wild-type protein to 0.89 μM in the H152A mutant [15]. EXAFS analysis of the H152A Ni2+ site indeed reveals a significant change in the co-ordination environment, which could explain the change in the binding constants. The best fit of the EXAFS spectra for the H152A HpUreE mutant suggests the presence of only two co-ordinating histidine residues around Ni2+, together with four other N/O-donor ligands (Figure 6C and Table 3). Although still six-co-ordinate, the H152A HpUreE Ni2+-binding site is best fitted using a single shell of ligands, as evident from single-scattering fits (Supplementary Table S3). The multiple-scattering analysis (Supplementary Table S4) shows that this shell contains only two histidine residues, presumably His102, with an angle α of 10°. This shell is complemented by four additional N/O-donor ligands at 2.09(1) Å. Therefore it is plausible that upon mutating the more weakly bound histidine residues [His152 most probably occurring at 2.15(1) Å in the wild-type HpUreE] there is a rearrangement in the Ni2+-binding site of HpUreE resulting in changes in the orientation of His102 to facilitate Ni2+ co-ordination by an additional pair of N/O-donor ligands that compensate for the removal of His152.

X-ray absorption spectroscopy on the Zn2+-binding site in wild-type and H152A mutant HpUreE

The differences in the Zn K-edge XANES spectra of wild-type and H152A Zn-HpUreE (Figure 7A) bound to one Zn2+ equivalent are not significant and are consistent with a four- or five-co-ordinate Zn2+ site. The normalized fluorescence intensity approaches 1.5 at its maximum, which favours a five-co-ordinate over a four-co-ordinate binding site [44]. Furthermore, the lack of resolution among the post-edge XANES features suggests that Zn2+ co-ordination is dominated by N/O donors [44].

X-ray absorption spectroscopic analysis at HpUreE at the Zn K-edge

Figure 7
X-ray absorption spectroscopic analysis at HpUreE at the Zn K-edge

(A) Zn K-edge XANES spectra of wild-type (WT) and H152A Zn-HpUreE. (B) Fourier-transformed EXAFS spectra of wild-type Zn-HpUreE [no phase correction, Fourier transform (FT) window=2–13.5 Å−1]. The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles). (C) Fourier-transformed Zn K-edge EXAFS spectra of H152A Zn-HpUreE (no phase correction, FT window=2–13.5 Å−1). The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles).

Figure 7
X-ray absorption spectroscopic analysis at HpUreE at the Zn K-edge

(A) Zn K-edge XANES spectra of wild-type (WT) and H152A Zn-HpUreE. (B) Fourier-transformed EXAFS spectra of wild-type Zn-HpUreE [no phase correction, Fourier transform (FT) window=2–13.5 Å−1]. The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles). (C) Fourier-transformed Zn K-edge EXAFS spectra of H152A Zn-HpUreE (no phase correction, FT window=2–13.5 Å−1). The inset shows the k3-weighted unfiltered EXAFS spectra: data (black line), best fit (white circles).

The EXAFS spectrum of wild-type Zn-HpUreE is distinct from the Ni2+ complex and shows two features in the Fourier-transformed spectrum that indicate the presence of two scattering shells. For biological samples, this is consistent with a shell of N/O-donor ligands at 1.5 Å (in r-space, uncorrected for phase shifts), and a second shell of sulfur/halogens ligands at 2.0 Å [45]. Single-scattering fits suggest the presence of a bromide ligand in addition to five N/O donors (see Supplementary Table S5 at http://www.BiochemJ.org/bj/441/bj4411017add.htm). Features between 2.5 and 4 Å in r-space are generally attributed to histidine residues. Two models for wild-type Zn-HpUreE emerge from the multiple-scattering analysis. The best-fit model consists of three histidine residues arranged in two shells (Figure 7B). The first shell at 2.00(1) Å consists of an N/O-donor ligand in addition to two histidine ligands with α=5°. The second shell at 2.16(1) Å consists of a single histidine residue and an N/O-donor ligand, whereas a third shell contains a bromide ion at 2.39(1) Å. This model is in agreement with the crystallographically determined dimer-of-dimer wild-type Zn-HpUreE crystal structure, which indicates that at least three histidine residues play a role in Zn2+ co-ordination. Modelling the EXAFS with four histidine ligands did not improve the fit (see Supplementary Table S6 at http://www.BiochemJ.org/bj/441/bj4411017add.htm). Although the model with three histidine ligands described above gives the best fit in terms of both goodness-of-fit (R-factor) and reduced χ2, it is not statistically distinct from a second model with only two histidine ligands (Table 3). The difference in reduced χ2 for the two models differ by only a factor of 1.4, and not the optimal 1.8 that would allow the two-histidine model to be ruled out.

Removal of the His152 pair in the H152A mutant HpUreE results in a Zn2+-binding site that is also five-co-ordinate and has a bound bromide at 2.39(1) Å. However, only two histidine residues are readily simulated in the EXAFS analysis, which together with two other N/O-donor ligands, form a scattering shell at 2.01(1) Å (Table 3, Figure 7C and Supplementary Tables S7 and S8 at http://www.BiochemJ.org/bj/441/bj4411017add.htm).

In summary, the best descriptions of the metal-binding sites from XAS analysis indicate that in wild-type HpUreE the Ni2+ site is six-co-ordinate with six N/O-donors, of which four are histidine ligands, and the Zn2+ site is five-co-ordinate with two or three histidine ligands and a bromide ion. In H152A HpUreE, a six-co-ordinate (N/O)6Ni site is retained, but includes only two HisN-donor ligands. Similarly, the corresponding Zn2+ site in H152A HpUreE retains a five-co-ordinate (N/O)4Br site that is similar to the wild-type Zn2+ site, but contains only two HisN-donor ligands.

DISCUSSION

The present study represents an attempt to clarify the role of the disordered C-terminal portions of the two protomers in homodimeric UreE proteins in the metal binding and release steps that occur when UreE acts as a metallo-chaperone in the process of Ni2+ insertion in the urease active site. A role for the C-terminal protein region was initially suggested in the case of BpUreE by metal-binding experiments coupled with X-ray absorption spectroscopy, which indicated the presence of a binuclear Ni2+-binding site involving the fully conserved His100 as well as the C-terminal histidine residues [22]. Subsequently, ITC (isothermal titration calorimetry) coupled with site-directed mutagenesis indicated that binding of a single Zn2+ or Ni2+ ion to the homodimeric HpUreE involves His102 on the protein surface, and that mutation of His152 on the disordered C-terminal arm alters the metal-binding properties of the protein [15]. Although Ni2+ is essential for enzymatic activity, being present in the active site of the functional urease enzyme, Zn2+ has been found to mediate and stabilize the interaction between H. pylori UreE and UreG in vitro [15]. UreG is another accessory protein with a GTPase role in the metallocentre assembly, suggesting a possible functional role for Zn2+, in addition to Ni2+, in this process [15]. So far, structural information on the Ni2+ and Zn2+ metal-binding environment of the homodimeric functional form of UreE has been hindered by protein oligomerization, occurring in the solid state, coupled with the molecular disorder of the HpUreE C-terminal region, which is known to be involved in metal binding on the basis of solution studies. In particular, although the crystal structure of apo-UreE from H. pylori was determined to be a dimer, the metal-bound form was described as a tetramer, or a dimer of dimers, with one metal ion bound between the four protein subunits [19]. A similar tetrameric arrangement was observed around a single Zn2+ ion in a structural study of BpUreE [18]. These observations prompted us to investigate further the Ni2+- and Zn2+-binding properties of HpUreE both in the solid state, using X-ray crystallography, and in solution, using X-ray absorption spectroscopy. This multifaceted approach yields consistent results that are in agreement with previous calorimetric studies, and reveals the structural details of the co-ordination environment of a single Ni2+ or Zn2+ ion bound to a single protein homodimer. The key role played by the conserved histidine ligands in the C-terminal arm of each protomer is thus demonstrated, and the protein motif is observed to gain significant structural ordering upon metal binding.

A comparison of all the HpUreE models (two apo dimers, two Zn2+-bound dimers and one Ni2+-bound dimer from the present study, as well as the previously reported apo and Ni2+-bound structures) indicates some flexibility of the protein through the linker chain connecting the central domain. The central domain consists of two C-terminal halves of the protein, dimerizing head-to-head, and two peripheral N-terminal domains. On the basis of temperature factors and rmsd values, the most mobile parts of the models are the loops between strands 1 and 2 and between strands 5 and 6 on the surface of the N-terminal domain. The most disordered region is found towards the end of the C-terminal region, which, in the best case, becomes untraceable in electron density maps after residue Gly154.

In the crystal structures presented herein, a single metal ion (Ni2+ or Zn2+) is found per protein dimer, in agreement with the stoichiometry previously obtained using ITC [15]. This metal ion is co-ordinated by two His102 residues, one from each UreE monomer subunit. This arrangement, suggestive of a pair of tweezers, holds the metal cation on the surface of the protein dimer. The rest of the co-ordination environment involves the C-terminal segment, which is in contact with the metal ions through His152. This protein region is not visible in the apo-protein, but becomes significantly more ordered upon metal binding. Nevertheless, some disorder in the electron density was still observed. The data suggest that the HpUreE binding arrangement, stable on one side and transient on the other, can be easily disengaged, and thus represents a fine balance between binding the metal ion and releasing it to its partners, a balance that is necessary for the chaperone function of UreE in Ni2+ trafficking.

In the crystal structure, the co-ordination of the Zn2+ site is tetrahedral, whereas the Ni2+ ion adopts an axially elongated distorted octahedral site that is approximately square bipyramidal. This reflects the intrinsic co-ordination preferences of the two metal ions, with Zn2+ being d10 and closed-shell, having no stereo-electronic preferences and leading to a co-ordination number and geometry imposed only by steric constraints, whereas Ni2+, being d8 and open-shell, has stereo-electronic preferences towards a tetragonal 4+2 co-ordination geometry due to the ligand field stabilization energy. The ability of the different cations to achieve their preferred co-ordination number in the complex with UreE indicates a significant flexibility of the protein. Indeed, the angle His102 Nϵ2–Ni2+–His102 Nϵ2 is close to 90° and it becomes 106–109° with Zn2+. The other ligands also appear in appropriate positions and numbers. The arrangement of the protein around the Ni2+ ion is more open. In addition to the pair of His102 residues and a pair of His152, the Ni2+ ion takes two additional ligands (a water molecule and Glu4' from a neighbouring protein molecule in the crystal lattice). The Zn2+ is surrounded by the two pairs of histidine residues, His102 and His152 (with one of the latter histidine ligands possibly displaced by Glu154D).

Conclusions

The structural features of HpUreE established in the present study allow us to propose a role for the C-terminal portions of the UreE dimer in molecular recognition and metal-ion delivery. In particular, the observations strongly support the idea that UreE could exist in two different conformations. In a ‘closed’ state, the delivered metal ion would be bound to the protein through the two conserved His102 residues (H. pylori numbering) and to histidine residues invariably found in the C-terminal region of this class of proteins (His152 in the case of HpUreE). The C-terminal region, disordered in the absence of the metal, but gaining order upon metal binding, would change into an ‘open’ state when protein–protein interactions between UreE and a partner protein, or protein complex, prone to receive the metal ion are present. In this form, His152 and analogous residues would be replaced with amino acid residues located on the surface of the protein partner receiving the metal ion from UreE.

Different roles of HpUreE bound to Ni2+ and Zn2+ are suggested by the observation that Zn2+, but not Ni2+, stabilizes the interaction between HpUreE and its cognate GTPase HpUreG [15]. The cross-talk between UreE, Ni2+ and Zn2+ suggests a specific functional role for different metal complexes of this urease accessory protein in regulating the formation of protein–protein complexes involved in enzyme maturation. The present study has shown that the metal-ion selectivity of UreE is based on the different metal-ion co-ordination environments that are dictated by the electronic properties of the metal ion in a mechanism that is facilitated by the flexibility of the C-terminal protein region.

Abbreviations

     
  • BpUreE

    Bacillus pasteurii UreE

  •  
  • EXAFS

    extended X-ray absorption fine structure

  •  
  • HpUreE

    Helicobacter pylori UreE

  •  
  • ITC

    isothermal titration calorimetry

  •  
  • KaUreE

    Klebsiella aerogenes UreE

  •  
  • MAD

    multiwavelength anomalous dispersion

  •  
  • rmsd

    root mean square deviation

  •  
  • XANES:

    X-ray absorption near-edge spectroscopy

AUTHOR CONTRIBUTION

Matteo Bellucci and Barbara Zambelli expressed and purified HpUreE and its mutants. Katarzyna Banaszak carried out protein crystallization, X-ray diffraction data collection and processing, protein structure solution, refinement and analysis. Vlad Martin-Diaconescu carried out X-ray absorption data collection and analysis. Wojciech Rypniewski, Michael Maroney and Stefano Ciurli designed the study and interpreted the data. All authors contributed to writing and editing the paper, and approved the final version of the paper.

We acknowledge the Helmholtz-Zentrum Berlin – Electron storage ring BESSY II for provision of synchrotron radiation at beamlines 14.1 and 14.2. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL), a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences.

FUNDING

This work was supported by the European Community's Seventh Framework Programme (FP7/2007-2013) [grant number 226716 (to W.R.)]; the National Institutes of Health [grant number R01-GM-69696 (to M.M.)]; and the Italian Ministero dell'Istruzione, dell'Università e della Ricerca PRIN2007 (to S.C.). The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health National Center for Research Resources Biomedical Technology Program.

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Author notes

1

These authors contributed equally to this work.

The structural co-ordinates reported will appear in the PDB under accession codes 3TJA, 3TJB and 3TJ9.

Supplementary data