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Structural basis for Ni transport and assembly of the urease active site by

the metallo-chaperone UreE from Bacillus pasteurii

Han Remaut1, Niyaz Safarof2, Stefano Ciurli2*, Jozef Van Beeumen1*

1 Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat

35, B-9000 Gent, Belgium

2 Department of Agro-Environmental Science and Technology, University of Bologna, Viale Berti

Pichat 10, I-40127 Bologna,, Italy

Running Title: Structure of UreE from B. pasteurii

*Corresponding authors:

S. Ciurli: Phone: +39-051-209-9794; Fax: +39-051-243362; e-mail: sciurli@agrsci.unibo.it

J. Van Beeumen: Phone: +32-92-645109; Fax: +32-92-645338; e-mail: jozef.vanbeeumen@rug.ac.be

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 15, 2001 as Manuscript M108304200 by guest on A

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ABSTRACT

Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni2+ ions in

the active site of urease. The X-ray structure of the protein has been determined for two crystal

forms, at 1.7 and 1.85 Å resolution, using SIRAS phases derived from a Hg-derivative.

BpUreE is composed of distinct N- and C-terminal domains, connected by a short flexible

linker. The structure reveals the topology of an elongated homodimer, formed by interaction

of the two C-terminal domains through hydrophobic interactions. A single Zn2+ ion, bound to

four conserved His100 residues, one from each monomer, connects two dimers resulting in a

tetrameric BpUreE, known to be formed in concentrated solutions. A large hydrophobic patch,

surrounding the metal ion site, is surface-exposed in the biologically relevant dimer. The

BpUreE structure represents the first for this class of proteins, and suggests a possible role for

UreE in the urease nickel-center assembly.

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INTRODUCTION

The molecular details of the mechanism by which specific metal co-factors are transported

in the cell and sorted into the correct metallo-enzyme among the many biological systems that

employ transition metal ions for their function, without being involved in alternative adventitious

and possibly poisonous binding, are still rather unclear. The role of a new class of soluble metal-

binding proteins, known as “metallo-chaperones”, is emerging as being fundamental for the

accomplishment of this process. However, the only case for which structural information is

available concerns the metallo-chaperones involved in copper transport (1). The present article

describes the structure of a novel protein involved in nickel metabolism.

The in vivo assembly of urease (EC 3.5.1.5), a nickel-containing enzyme that catalyzes urea

hydrolysis in the last step of nitrogen mineralization (2) requires at least four accessory proteins

(3,4). These proteins, named UreD (Mr ca. 30 kDa), UreE (Mr ca. 18 kDa), UreF (Mr ca. 25 kDa),

and UreG (Mr ca. 22 kDa), play fundamental roles in the transport and insertion of two Ni2+ ions

into the urease active site (5,6). The specific function of these four accessory proteins is not fully

understood, even though enough knowledge has been gathered on the Klebsiella aerogenes (Ka)

urease assembly system to propose a specific role for each of them (3,5,6). KaUreD was found to

form a complex with inactive apo-urease, suggesting that it is a specific chaperone that facilitates

nickel insertion into apo-urease by stabilizing a proper protein conformation (7). The sequence of

UreG features a nucleotide-binding motif suggesting a possible role in an energy-dependent step

during in vivo urease assembly (8,9). KaUreG and KaUreF form a super-complex with the Ka

UreD-apo-urease aggregate (10), suggesting that such large complexes could be required for in

vivo activation of urease (9). Finally, KaUreE, the only urease accessory protein shown to bind

Ni2+ ions (11-14), is thought to interact with the Ka apo-urease/UreD/UreF/UreG super-complex

and facilitate Ni2+-incorporation in the urease active site (5). KaUreE possesses a His-rich C-

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terminus (10 of the last 15 residues are histidines) and is able to bind ca. 6 Ni2+ per dimer in a

coordination environment of 3-5 histidines, with an average Kd of ca. 10 µM (11). However, not

all UreE proteins feature such a long His-rich C-terminus region (3). A genetically modified and

truncated form of KaUreE, named H144* KaUreE, lacks the last 15 residues, but displays

properties and physiological activity comparable to that of the wild-type KaUreE (12). The

truncated H144* KaUreE is able to bind ca. 2 Ni2+ ions per dimer (13) in a pseudo-octahedral N/O

coordination environment. Site-specific mutagenesis experiments have suggested the role of

His96, His110, His112, Asp111 and Cys79 as Ni2+-binding residues (14). However, only His96

and Asp111 are critically involved in Ni2+-incorporation into the urease active site (14-16).

In a continuing effort to characterize the proteins encoded by the urease operon in Bacillus

pasteurii (Bp) (17-21) we have recently reported the cloning, over-expression, purification and

characterization of BpUreE (Ciurli et al., submitted). In particular, we have shown that

recombinant BpUreE contains a single Zn2+ ion per dimer, while no Ni2+ is present. BpUreE

undergoes dimerization in diluted (≤ ca. 0.2 mM) solutions, while at higher concentrations the

protein is found to aggregate to form a tetramer. The presence of Zn is not required for

dimerization. Paramagnetic NMR spectroscopy on concentrated (2 mM) BpUreE solutions, using

either Zn2+-BpUreE or the corresponding apo-form, revealed a 1 Ni2+/tetramer stoichiometry, with

the Ni2+ ion bound to histidines in an octahedral coordination environment. This observation

suggested that Ni2+ substitutes Zn2+, or that it binds in a different site.

We report here the X-ray structure of BpUreE. The structure is the first for this class of

proteins, and reveals features that suggest a possible molecular role in the urease assembly, a

puzzle process requiring five proteins, for which only two pieces have been structurally

characterized so far. Knowledge of all the molecular details for this biological system could also

provide additional targets for potential drugs able to diminish the negative effects of bacterial

urease activity both in human health and environmental settings (3,22).

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MATERIALS AND METHODS

Crystallization and data collection

Recombinant BpUreE was expressed and purified as described elsewhere (Ciurli et al.,

submitted). The purified protein used for crystallization was 10 mg mL-1 in 50 mM Tris•HCl, pH

7.5, 100 mM NaCl. Crystals were grown in hanging drops containing the protein and reservoir

solution at a 1:1 ratio. Two crystal forms were obtained by equilibration against 0.5 mL of

reservoir solution containing 86-92 % citrate, 100 mM Tris•HCl, pH 7 at 294 K. Both crystal

forms are in the I222 space group, are morphologically similar and grow approximately in a 15:1

ratio. Native crystals of type I diffract to 1.77 Å resolution and have unit cell dimensions a =

50.50 Å, b = 60.72 Å and c = 130.58 Å at 100 K. Native type-II crystals diffract to 1.5 Å and have

cell dimensions a = 44.32 Å , b = 63.51 Å and c = 129.29 Å at 100 K. Both crystal forms contain

one molecule of UreE in the asymmetric unit. An isomorphous Hg-derivative was obtained by

soaking BpUreE crystals of type I for 3 – 12 hours in 5 µL of reservoir solution containing 1 mM

HgCl2. A summary of the collected data is given in Table 1. Data were collected on the EMBL

wiggler line BW7B of the DORIS storage ring at DESY- Hamburg, using a MAR345 image plate

detector. The data sets were recorded in two sweeps, at different exposure times, in order to

accurately record both the strongest, low-resolution, and the weakest, high-resolution diffraction

intensities. The Hg CuKα data (Table 1) were collected on a DIP 2030 image plate using CuKα

radiation generated with a FR591 rotating anode generator (Enraf_Nonius, Delft) equipped with

double-mirror focussing optics. All data were collected at 100 K using crystals soaked for 1-2

minutes in reservoir solution containing 5% v/v glycerol as cryogenic protectant. Data were

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processed and merged with DENZO and SCALEPACK (23). Native data for type-I and type-II

crystals were truncated to 1.85 Å and 1.7 Å respectively.

Structure determination and refinement

The BpUreE structure was determined using SIRAS phases obtained from a single Hg-

derivative. Type-I crystals were used for structure determination. A single Hg2+ position was

identified from isomorphous and anomalous Patterson maps. Refinement of the occupancy and

position of the heavy atom and subsequent phase extraction was performed by MLPHARE (24) to

2.6 Å. Experimental protein phases were solvent flattened and extended to 1.85 Å using DM (24).

ARP/wARP (25) was used to further improve protein phases and 90% of the initial model was

auto-built in 50 cycles of warpNtrace. Phases for the type-II data were derived from a molecular

replacement where the type-I model was used as search model (AMORE (24)). A single solution

for the rotation and translation vectors was found with R-factor 41.2 % and correlation coefficient

of 54.3. The final models contain 143 and 142 residues out of 147 for type-I and type-II crystal

forms, respectively, and were refined to a final R- and free R-factor of 22.8 and 26.4 % for type-I,

and 21.3 and 23.2 % for type-II model (CNS (26)). Side-chain atoms for which no clear electron

density was observed were modeled with B-factors fixed at 100 Å2. Residues 13-17 were mutated

from the expected sequence (YESSD) to a sequence (LSHNI), generated supposedly as a cloning

artifact. Ramachadran plots of the type-I and type-II models show 98% and 99% of the residues to

be located within the allowed regions (PROCHECK (27)).

Coordinates and structure factors for both models of UreE have been deposited in the

Protein Data Bank with the accession codes 1EB0 and 1EAR for the type-I and type-II models,

respectively.

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RESULTS AND DISCUSSION

Overall structure

The structure of BpUreE was determined for two alternative oligomerization states

obtained from separate crystal forms (I and II), by using SIRAS phases derived from a Hg-

derivative of type-I crystals. The structure was determined at 1.85 Å and 1.70 Å resolution for

type-I and type-II crystal forms, respectively. Details of the crystallization and structure solution

and refinement are given in the Methods section and in Table 1.

BpUreE is organized as a (α2)2 tetramer (ca. 40 x 70 x 95 Å3) made of the dimerization of

two elongated dimers, each of approximate dimensions 40 x 35 x 95 Å3. Each monomer is made

up of two distinct domains and has a unique tertiary structure, in agreement with the lack of

significant sequence similarity with other known proteins (Ciurli et al., submitted). The N-

terminal domain encompasses residues 1-73 and is composed of two three-stranded mixed β-sheets

that stack upon each other in a nearly perpendicular orientation (Fig. 1a,b). A DALI search (28)

reveals that a similar topology is reported only for a heat-shock protein domain (Hsp40 (29); Z-

score = 3; Z-values > 4 are considered to indicate significant structural similarity (28)). A short

helical (310) region, residues 27-31, is present between strands 2 and 3. The N-terminal domain is

separated from the C-terminal domain by a short linker (residues 74-75). The C-terminal domain

encompasses residues 76-147 and is composed of a four-stranded anti-parallel β-sheet and two α-

helices organized in a βαββαβ-fold (Fig. 1a,b). Such topology, characterized as the ‘ferredoxin-

like’ fold (SCOP (30)), is found in a broad range of different proteins. The latter include human

procarboxypeptidase A2 (ref. (31)) (Z = 6.1), bacterial elongation factor G (32) (Z = 5.8), the

papillomavirus E2 regulatory protein (33) (Z = 5.4) and archaeal bDNA polymerase (34) (Z = 5.0)

amongst the proteins in which the respective domains most closely resemble the C-terminal

domain of BpUreE. The function of these domains varies, depending on the protein in which they

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are present. In BpUreE, the C-terminal domain is involved in dimerization and metal ion binding.

The C-terminal end of the protein is found as a long coil fragment encompassing residues 135-143,

while the remaining four amino acids (Gly144-His145-Gln146-His147) are not visible in the

electron density map due to disorder.

Oligomerization state and metal binding site

The physiologically significant dimeric form of BpUreE is built by a head-to-head

interaction of two monomers (Fig. 1c,d). The hydrophobic face of an amphiphilic helix of the C-

terminal domain is involved in the dimerization, this region constituting the central part of the

dimer, with the N-terminal domains at the two ends protruding from the core. No S-S bridges are

present. The inter-helix interaction is mainly stabilized by induced dipole interactions between

pairs of Met86, Met89, Glu90, Ala93, and Gly97, one on each monomer, at a distance of ca. 4-5 Å.

These five amino acids are either conserved or conservatively mutated in all UreE proteins (Ciurli

et al., submitted). The dimer interface further encompasses three strictly conserved residues,

Gly97, Asn98, and His100. The latter is involved in the coordination to a Zn2+ ion, located on the

peripheral surface in the central portion of the BpUreE dimer (Fig. 1c,d). Asn98 Oδ forms an H-

bond with His100 Nδ, further stabilizing the His100 Nε – Zn2+ bond through a well-conserved

‘elec-His-Zn’ motif (35). The BpUreE-Zn2+ interaction, although not indispensable for the

formation of the dimer (Ciurli et al., submitted), supposedly further contributes to its stability.

In both crystal forms the protein dimer is found to further dimerize around this single Zn2+

ion (Fig. 2). The metal center is positioned on the origin of the I222 cell so that each monomer is

donating a single His100 to the ion, which represents the only shared atom between the two

dimers. The coordination environment of the Zn2+ ion differs in the two forms characterized

crystallographically (Fig. 2, 3a,b), which are otherwise very similar in overall structure,

superimposing with a root mean square (rms) deviation of 0.89 Å for 142 out of 147 Cα atoms. In

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type-I crystals, Zn2+ is coordinated pseudo-octahedrally (Fig. 2a,b,c, 3a), with the Nε atoms of the

four His100 residues laying in the equatorial plane, and the fifth and sixth axial coordination sites

occupied by water molecules. In type-II crystals, Zn2+ is coordinated only to the four His100 Nε

atoms in a pseudo-tetrahedral coordination (Fig. 2d,e,f, 3b). This difference in coordination results

in a different relative orientation of the two dimers within the tetramer (Fig. 2). In type-I crystals

the two dimers are staggered with a closing angle of ca. 40°, and no dimer-dimer interaction is

present (Fig. 2c). On the other hand, type-II crystals have a smaller absolute closing angle, with

the dimers staggered at ca. -30° (Fig. 2f). As a consequence, in type-II crystals an interaction

patch is formed at the N-terminal domains, involving residues 33-34 and 48-52, causing the

formation of an unfavorable clash between two Arg33 residues on opposing dimers. Furthermore,

the residues in the region 48-52 feature increased temperature factors, reflecting a lower degree of

order likely due to local strain on the peptide chain. A further consequence of these dimer-dimer

contacts is a reorientation of the N-terminal domains relative to the core of the dimers (Fig. 3c).

The N-terminal domains are pivoted around the linker (residues 74-75) separating the N- and C-

terminal domains, suggesting that this region may serve as a hinge within the UreE dimer. Taken

all together, the absence of dimer-dimer interactions in type-I crystals, together with the nature of

these interactions in type-II crystals, explains why the BpUreE tetramer is found only in highly

concentrated protein solutions.

The disordered C-terminal region (residues 142-147), which is absent from both models,

encompasses two additional histidines (His 145 and His 147) and is located in close proximity to

the metal binding site. In diluted solutions, when BpUreE is primarily present as a dimer, one or

both of these histidine residues may provide additional ligands to the metal ion, stabilizing the

protein-metal complex. It should be noted that in all UreE proteins, the C-terminal tail contains at

least one His, most often two in a HXH motif, and for several cases it is constituted naturally by a

long His-rich stretch (Ciurli et al., submitted). In the case of KaUreE, the second His of the HXH

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motif in the wild type protein was artificially removed, producing a truncated form (H144*

KaUreE) that was used for most of the physiological studies (12). The fact that these C-terminal

fragments are disordered in the crystallographically established structure of the tetrameric BpUreE

indicates a substantial flexibility of this protein stretch. In the crystallographic tetramer, the HXH

‘arm’ motifs, supposedly coordinating the metal ion in the dimer, must have been displaced from

their position by the incoming pair of His100 residues from the adjacent BpUreE dimer.

Mechanistic implications

The structure of BpUreE allows a rationalization of the biochemical data available for this

protein (Ciurli et al., submitted). In particular, the molecular determinants for the stabilization of

the dimeric form of BpUreE (and also KaUreE (11)) in diluted solutions in the presence and

absence of a metal ion (Zn2+ or Ni2+), as revealed by mass spectrometry (Ciurli et al., submitted),

are now available. Furthermore, the topology of the tetrameric form of BpUreE, detected at higher

protein concentrations (larger than ca. 0.2 M) (Ciurli et al., submitted) has been elucidated. Most

importantly, the structure clearly indicates a single metal binding site on the protein dimer. This

site is characterized by the presence of only His residues as putative coordinating ligands. This

observation, together with the NMR evidence of a Ni2+-Hisn binding site (Ciurli et al., submitted),

supports the hypothesis that Ni2+, when present, substitutes the Zn2+ ion, ruling out the possibility

that the two metal ions bind at different sites in the protein. The peculiar surface-exposed and

peripheral position of the metal ion binding site, together with the flexibility of the proximal C-

terminal regions on the two adjacent monomers, each containing two His residues, strongly

suggests a mode of action for this metallo-chaperone in the process of Ni-uptake and transport, as

well as release to the apo-urease/UreD/UreF/UreG super-complex, formed prior to the interaction

with UreE (4). The flexible arms at the C-terminus could confer flexibility to the metal-binding

site, adopting a ‘closed’ conformation while binding the metal ion with one or two terminal His

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residues, or an ‘open’ conformation, freeing coordination sites on the metal ion during the process

of release to the target metal site. The latter process must occur upon or after formation of a

protein complex involving Ni2+-UreE. In this respect, the presence of an extended hydrophobic

surface on the same side of the dimer where the metal binding site is located, indicates a region of

the protein most likely involved in the formation of such super-complex between the urease

chaperones and the apo-enzyme itself. This hydrophobic region comprises the entire protein

length, contains some smaller positively charged patches (including the metal ion), and features the

most hydrophobic areas towards its outer ends, positioned in the N-terminal domains (Fig. 4). On

the opposite side of the dimer, the protein instead features an extended negatively charged and

hydrophilic surface. It may be worth noticing that the length of the extended BpUreE dimer (ca.

95 Å) is comparable to the length of one edge of the α3β3γ3 trimer constituting Bp urease (2),

suggesting that three BpUreE dimers, one on each side of the triangular apo-urease, can

independently operate to provide Ni2+ ions to the three active sites of the enzyme. In order to

elucidate the properties and operational mode of this ensemble of accessory proteins in the

assembly of the Ni2+-containing active site of urease, the determination of the structure of the

remaining urease chaperones is necessary. Work is currently in progress in our laboratories

towards this goal.

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FIGURE LEGENDS

Fig. 1 Structure and topology of the BpUreE monomer and dimer. Figures are based on the type

II, 1.7 Å model. a, Topology diagram of the BpUreE monomer. Helices are colored yellow and

red, strands are colored green and blue for the N- and C-terminal domain respectively b, Drawing

of the BpUreE monomer. The N-terminal domain encompasses residues 1-73 and is composed of

two three-stranded mixed β-sheets (1 (1-3), 2 (19-25), 3 (34-38), 4 (44-48), 5 (59-63) and 6 (67-

73)). A short 310 helix (A (27-31)) is present between strands 2 and 3. The C-terminal domain,

residue 76 – 142, is composed of a four-stranded anti-parallel β-sheet (7 (76-82), 8 (103-106), 9

(109-113) and 10 (128-134)) and two α-helices (B (86-98) and C (116-125)) organized as a

‘ferredoxin-like’ βαββαβ-fold. A Zn2+ ion (gray sphere) is positioned near the C-terminal end of

helix B. c, d, Front and top view of the BpUreE dimer along the respective twofold axes of the

I222 cell. A central core is formed by a head-to-head interaction of the two C-terminal domains.

Separated by a short linker, the N-terminal domains protrude from this central core resulting in an

elongated dimer with approximate dimensions of 40 x 35 x 95 Å3. The Zn2+ ion is located at the

intersection of the two-fold axes above the dimerization interface. Figures b, c and d were made

with MOLSCRIPT (36) and Raster3D (37).

Fig. 2 Quaternary assembly in type-I and type-II BpUreE models. a, b, c, Front, side and top

views along the respective twofold axes in the type-I tetramer. The Zn2+ ion (gray sphere),

positioned at the intersection of the axes, is pseudo-octahedrally coordinated by four His100

residues, one of each monomer. All four His ligands lay in the equatorial plane. The axial fifth

and sixth coordination sites are occupied by water molecules (red spheres). d, e, f, Tetrameric

organization in the type-II model. In figure d and e the molecule is oriented such that the upper

dimer superimposes with that in the type-I figures (the respective twofold axes intersect the plane

of the figure at about 45 degrees). Figure f gives a top view of the tetramer along the twofold axis.

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In the type-II tetramer, the Zn2+ ion is pseudo-tetrahedrally coordinated by four His residues,

His100, one from each monomer. For the upper dimer the color scheme used in Fig. 1 was

applied, while for the lower dimer, the colors cyan and violet were used to separate the two

monomers. The figures were made with MOLSCRIPT (36) and Raster3D (37).

Fig. 3 Comparison of the Zn2+ coordination in both BpUreE models. a, b, Ball and stick

representation of the coordination spheres of Zn2+ in type-I and type-II crystals respectively. c,

Superposition of the Cα trace of type-I (orange) and type-II (blue) models. The superposition was

performed only considering the Cα atoms in the C-terminal domain (residues 76 –142), which

superimpose with an rms deviation of 0.46 Å, whereas for the resulting N-terminal domains

(residues 1-73) an average displacement of 2.75 Å is found. When only the residues in the N-

terminal domain are considered, an rms deviation of 0.70 Å is calculated. It can be concluded that

the N-terminal domains pivot around the small linker, residues 74-75, connecting both domains.

All figures were made with MOLSCRIPT (36) and Raster3D (37).

Fig. 4 Solid surface representation of the electrostatic potential of BpUreE. The molecular

surface and electrostatic mapping were generated by the program GRASP (38), using a probe

radius of 1.4 Å. The electrostatic potential was calculated using a simple version of a Poisson-

Boltzmann solver, with the GRASP full charge set, and the Zn2+ ion group included in the

calculations. All histidine residues were considered neutral, and the N- and C-terminal residues

were charged. Dielectric constants of 80 and 2 were used for the solvent and protein interior,

respectively. The surface is colored according to the calculated electrostatic potential contoured

from –15.9 kT/e (intense red) to +15.7 kT/e (intense blue). The protein is shown with the Zn2+-

binding patch towards the viewer (panel a) and rotated by 180° about the long horizontal axis

(panel b).

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Table 1 Data collection and refinement statistics

Data collection Native cell 1 Hg BW7B Hg CuKα Native cell 2

Wavelength (Å) 0.8453 0.8453 1.5418 0.8453Resolution (Å) 20.0 - 1.85 2.05 2.80 1.7Reflections, total 311356 699986 23822 292340Reflections, unique 17790 12925 5255 20619Av. Redundancy1 10.2 (5.1) 15.8 (14.0) 1.9 (2.1) 6.2 (3.2)Completeness1 (%) 99.7 (99.9) 99.3 (99.9) 79.4 (84.6) 99.0 (96.8)Rsym

1,2 (%) 4.8 (3.37) 10.1 (39.3) 7.4 (27.5) 5.8 (28.1)Av. I/σ(I)1 43.80 (3.96) 29.90 (4.41) 18.10 (3.07) 32.60 (3.63)I>2 σ(I) last shell (%) 64.6 70.3 59.0 63.9

Phasing

Resolution (Å) 15.0 – 2.6Phasing power3 (acentric/centric) 1.61 / 1.09 2.27 / 1.48Rcull

3 (%) 0.73 / 0.71 0.62 / 0.63Rcull anomalous3 (%) (15.0 – 3.5 Å) 0.83Mean figure of merit

Acentric1,3 0.47 (0.38)Centric1,3 0.61 (0.53)All1,3 0.49 (0.40)

Mean figure of merit, after density modification1,4 (15 – 1.85 Å) 0.753 (0.49)

RefinementModel Type I Type IIR-factor5 (%) 22.8 21.3Rfree

5 (%) 26.4 23.2Average B-factorsDomain N-term C-term Overall N-term C-term OverallMain chain atoms 27.4 32.4 30.2 17.5 41.8 28.8Side chain atoms 31.9 37.0 34.4 23.0 46.5 33.0

1highest resolution bin in parenthesis2Rsym = Σhkl_Σj Ij − <I> / Σhkl Σj Ij where I is the intensity of a reflection, and <I> is the mean intensity of all symmetryrelated reflections j.3taken from MLPHARE32

4taken from DM32

5R-factor = Σhkl Fobs− Fcalc/ Σhkl Fobs. For Rfree, Fobs are (5%) test set amplitudes not used in refinement.

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Acknowledgements. This work was supported by the Italian Ministero dell’Università e della

Ricerca Scientifica e Tecnologica (MURST), PRIN title: "The role of metallic cofactor in

inorganic structural biology", awarded to S.C. H.R. is a research fellow of the ‘Vlaams Instituut

voor de bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie’ (IWT).

The authors acknowledge support of the European Community - Access to Research Infrastructure

Action of the Improving Human Potential Program to the EMBL Hamburg Outstation. We wish to

thank W.R. Rypniewski for his assistance with data collection at the DESY-Hamburg outstation.

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Han Remaut, Niyaz Safarof, Stefano Ciurli and Jozef J. Van Beeumenmetallo-chaperone UreE from Bacillus pasteurii

Structural basis for Ni transport and assembly of the urease active site by the

published online October 15, 2001J. Biol. Chem. 

  10.1074/jbc.M108304200Access the most updated version of this article at doi:

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