UreG, a chaperone in the urease assembly process, is an … · 4 and CO2, the participation of four...

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UreG, a chaperone in the urease assembly process, is an intrinsically

unstructured GTPase that specifically binds Zn2+

Barbara Zambelli,‡ Massimiliano Stola,‡ Francesco Musiani,‡ Kris De Vriendt,§ Bart Samyn,§

Bart Devreese,§ Jozef Van Beeumen,§ Paola Turano,¥ Alexander Dikiy,‡ Donald A. Bryant,#

Stefano Ciurli‡*

‡ Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and

Technology, University of Bologna, Italy

§ Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry,

Physiology and Microbiology, University of Gent, Belgium

¥ Magnetic Resonance Center and Department of Chemistry, University of Firenze, Italy

# Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University,

University Park, PA, USA

Running Title: UreG is a natively unfolded GTPase that binds Zn2+

* To whom correspondence should be addressed to: Laboratory of Bioinorganic Chemistry,

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

Giuseppe Fanin 40, 40127 Bologna, Italy. Telephone: +39-051-209-6204; FAX: +39-051-

209-6203; e-mail: [email protected]

JBC Papers in Press. Published on November 12, 2004 as Manuscript M408483200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was

over-expressed in E. coli BL21(DE3) and purified to homogeneity. The identity of the

recombinant protein was confirmed by SDS-PAGE, protein sequencing and mass

spectrometry. Combination of size exclusion chromatography, multi-angle and dynamic laser

light scattering established that BpUreG is present in solution as a dimer. Analysis of circular

dichroism spectra indicated that the protein contains large portions of helices (15%) and

strands (29%), while NMR spectroscopy indicated the presence of conformational

fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP

with a kcat = 0.04 min-1, confirming a role of this class of proteins in coupling energy

requirements and nickel incorporation into the urease active site. BpUreG binds 2 Zn2+ ions

per dimer with a KD = 42±3 µM, and has ten-fold lower affinity for Ni2+. A structural model

for BpUreG was calculated using threading algorithms. The protein, in the fully folded state,

features the typical structural architecture of GTPases, with an open β-barrel surrounded by

α-helices and a P-loop at the N-terminus. The protein dynamic behavior observed in solution

is critically discussed relatively to the structural model, using algorithms for disorder

predictions. The results suggest that UreG proteins belong to the class of intrinsically

unstructured proteins (IUP) that need the interaction with co-factors or other protein partners

to perform their function. It is also proposed that metal ions such as Zn2+ could have

important structural roles in the urease activation process.


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INTRODUCTION

Urease is a nickel-containing enzyme found in plants, fungi and bacteria that catalyzes

the hydrolysis of urea in the last step of nitrogen mineralization (1,2) (Scheme 1).

C

O

H2N NH2

+ H2O C

O

H2N O+ NH4

+ H2OC

O

HO O+ NH3

urease

HN C O + NH3ca. 3.6 years

ca. 1 µs

NH

O O

Ni(1) Ni(2)OH

(His)N

(His)N

N(His)

N(His)

H2O H2O O(Asp)

Lys

Over the past few years, intensive studies have been carried out to achieve an

elucidation of its catalytic mechanism. Structures of the native enzyme isolated from

Klebsiella aerogenes (Ka) (3), Bacillus pasteurii (Bp) (4) and Helicobacter pylori (Hp) (5),

revealed a dinuclear metallo-center, with two Ni2+ ions bridged by a carbamylated lysine

residue and a hydroxide ion. The enzyme, consisting of a heterotrimeric α3β3γ3 quaternary

structure with the three different subunits encoded by the ureC, ureB, and ureA genes,

respectively, is synthesized in the apo-form that is devoid of nickel. The incorporation of Ni2+

into the active site, leading to the activation of the enzyme, is still poorly understood, and is

thought to occur in vivo as a step-wise assembly process (6).

The assembly of the active site in vitro can be achieved using high, non-physiological,

concentrations of Ni2+ ions and bicarbonate as the source of CO2, which is needed for the

carbamylation of the active site lysine residue (7). At physiological concentrations of Ni2+


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and CO2, the participation of four accessory proteins is required (8-12). These proteins

(UreD, UreF, UreG, UreE) are encoded by four genes, which are also present in the urease

operon together with ureA, ureB, and ureC (12).

Many functional studies have been carried out on the urease accessory proteins from

K. aerogenes. KaUreD (~ 30 kDa) binds to apo-urease and appears to induce a

conformational change required for the next steps of the activation process (13,14). KaUreF

(~25 kDa) binds the KaUreD/apo-urease complex and seems to facilitate carbamylation of the

Ni-bridging lysine residue and to prevent Ni2+ binding to the non-carbamylated apo-urease

(15). Cross-linking experiments showed an interaction between KaUreD and the α and β

subunits of apo-urease, while KaUreF was shown to interact with the β subunit and to induce

a conformational change capable of increasing the accessibility of the nickel ions and CO2 to

residues in the active site (16). The interaction of UreD with UreF and with the α-subunit of

apo-urease was suggested by immuno-precipitation and two–hybrid studies carried out on

these proteins from Proteus mirabilis (17). In H. pylori a similar experiment indicated the

interaction between UreF and UreH, the latter corresponding to UreD in other bacteria (18).

KaUreG (~22 kDa) can form a quaternary complex with KaUreDF/apo-urease, suggesting

that such large aggregate is the minimum competent species for the in vitro urease activation

(7,19) and could be required for the process occurring in vivo (20). Finally, KaUreE (a

homodimer of ca. 35 kDa) is thought to bind the KaUreDFG/apo-urease complex, acting as a

nickel-transporter that delivers Ni2+ to the active site of the enzyme (21-25).

Among the four accessory proteins, UreG plays an essential role in coupling cellular

metabolism and bioenergetics to the assembly of urease. This protein contains a fully

conserved P-loop motif, which is also present in many nucleotide-binding proteins and which

is probably related to the in vivo GTP requirement for assembly of the urease active site (26).

Proving a direct relationship between UreG and GTP requirement, GTP is needed for


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activation of the KaUreDFG/apo-urease complex, although it has an inhibitory effect on the

Ni-reconstitution of the apo-urease and of the KaUreD/apo-urease and KaUreDF/apo-urease

complexes (26). UreG is also involved in delivering CO2 necessary for the carbamylation of

the Ni-bridging lysine: the curves that correlate the urease activation to different bicarbonate

concentrations indicate a higher rate and level of enzymatic activation in the presence of

UreDFG/apo-urease complex (26). In vitro, optimal levels of apo-urease activation require

0.5 mM GTP. Larger GTP concentrations lead to a decrease of urease activity, probably

caused by the chelation of Ni2+ by the nucleotide, consistently with the fact that elevated

levels of Mg2+ ions partially restore the activation (26). In the presence of UreE, this

inhibitory effect is lost, and the urease activation occurs at significantly lower GTP

concentrations (27). These observations indicate a correlation between GTP hydrolysis by

UreG and Ni2+-transfer to apo-urease by UreE. Additional evidence of the UreE-UreG

interaction in vivo has been obtained using two-hybrid systems and immuno-precipitation

experiments in H. pylori (18). It has been proposed that UreG may induce GTP-dependent

structural changes of the apo-urease, increasing accessibility of both Ni2+ and CO2 to the

developing active site. Alternatively, UreG may use GTP and CO2 to synthesize

carboxyphosphate, which could serve as an excellent CO2 donor to the Lys residue (26).

The thorough functional studies described above have paved the way to an

understanding of the mechanism of the urease accessory proteins, and in particular of UreG,

in Ni2+ trafficking and metabolic regulation. The next echelon in the comprehension of the

urease chemistry implies the study of the interaction mechanisms between the accessory

proteins and the enzyme at the molecular level, in order to understand how the urease active

site is assembled. This goal cannot be achieved without detailed structural information on

each accessory protein and its biochemical properties. The only crystal structures available

for the urease chaperones are those of UreE from K. aerogenes (28) and B. pasteurii (29).


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The high degree of similarity between these two structures and between the structures of Ka-

and Bp-urease are indications of a conserved molecular mechanism of urease activity,

activation and metal-center building in different species (30). No structural detail is available

for the other three chaperones. A study, published in 1997, reported that KaUreG is

monomeric in solution and that it does not, by itself, hydrolyze GTP or ATP (20). Another

report, dated 2003 and concerning HpUreG, essentially confirmed these results (31).

This paper describes a thorough study performed on the recombinant UreG from B.

pasteurii. In particular, the cloning, expression and purification of the protein in its native

and His-tagged forms are described, together with evidences confirming the identity of the

isolated protein. The oligomerization state and the hydrodynamic properties of the protein in

solution were examined using size exclusion chromatography coupled with light scattering

experiments, and the protein folding was checked using circular dichroism, mass

spectrometry and NMR. The metal-binding capability and enzymatic GTPase activity of

BpUreG were established for the first time and discussed. Finally, threading (fold

recognition) algorithms were applied to calculate a model for the protein structure that is

consistent with its solution properties and enzymatic activity. The results represent a

significant contribution to the understanding of the role of this metallo-chaperone in the

urease active site assembly.

EXPERIMENTAL PROCEDURES

BpUreG cloning

In accordance with the DNA sequence of the B. pasteurii urease operon available from

GeneBank (accession number AF361945), two 24- and 26-bp oligonucleotide primers were


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designed and synthesized to amplify the ureG gene by the PCR technique using the pUC19

plasmid containing the B. pasteurii ure operon (32) as template. The forward and reverse

primers shown below introduced NdeI and BamHI restriction enzyme recognition sites

respectively (bold faced):

5’ CTAGGAGATTGTGCATATGAAAAC 3’

5’ CAATATCGAGGGATCCAAACGGTATT 3’

Taq polymerase for the PCR reaction and dNTPs were from Display System Biotech

(USA). The oligonucleotide primers were synthesized in the Nucleic Acid Facility at the

Pennsylvania State University (University Park, PA, USA). The PCR product obtained by

using these primers was digested by a combination of NdeI and BamHI restriction enzymes

(New English BioLabs), purified by electrophoresis on a 1% (w/v) agarose gel, extracted, and

precipitated. Using T4 DNA ligase (Promega) this DNA fragment was ligated at a two-fold

excess of insert to vector, into plasmid pET3a (Novagen), which had been digested with NdeI

and BamHI, treated with alkaline phosphatase, and purified by electrophoresis. For the His-

tagged BpUreG, the plasmid pET15b was used. Plasmid DNA was isolated from

transformants of E. coli strain DH5α (Bethesda Research Laboratories Inc.) by the rapid

alkaline extraction method, as described (33), digested with appropriate restriction enzymes

and analyzed by agarose gel electrophoresis. The resulting pET3a::ureG and pET15b::ureG

plasmids were purified using the StrataPrep™ Plasmid MiniPrep Kit (Stratagene). The

sequence of the cloned BpUreG gene was confirmed by DNA sequencing. The constructs for

both plasmids were inserted by electroporation (BioRad GenePulser II) into the E. coli

BL21(DE3) expression host (Novagen) grown in shaking flasks at 37 °C in a medium with

the Luria-Bertani (LB) composition (Amersham-Pharmacia Biotech) or on agar (1.5 %) plates

with the same composition.

BpUreG expression


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Based on the T7 system (34), large scale expression of BpUreG and His-tagged

BpUreG was achieved in 2.5 L batches of minimum M9 liquid media (1 L contained 6 g of

Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1.25 g of (NH4)2SO4, 0.246 g of MgSO4)

supplemented with 4 g of glucose per liter of culture. The 15N-enriched proteins were

obtained using a medium containing (15NH4)2SO4. Transformed E. coli BL21(DE3) cells were

grown at 37 °C (28 °C for the His-tagged protein) with vigorous stirring, until the OD600

reached 0.5-0.8. Expression was induced by addition of IPTG (isopropyl β -

thiogalactopyranoside) to a final concentration of 0.5 mM. The cells were harvested 4 hours

after induction by centrifugation at 8,000 g for 10 min, at 4 °C. The cells were re-suspended

in 25 mL of 50 mM TrisHCl buffer, pH 8 containing 5 mM EDTA and lysozyme (200

µg/mL). After incubation at 30 °C for 20 min, followed by the addition of DNAse I (20 µg

mL-1) and additional incubation at 37 °C for 20 min, the cells were disrupted by two passages

through a French Pressure cell (SLM-Aminco) at 20,000 psi. The cell pellet was separated

from the supernatant by centrifugation at 15,000 g for 15 min at 4 °C.

BpUreG purification

In the case of native BpUreG, the pellet was washed (resuspended in 25 mL of buffer

using a mixer homogenizer and centrifuged at 15,000 g for 15 min at 4 °C) three times with

50 mM TrisHCl buffer, pH 8 containing 5 mM EDTA, 1 mM DTT and 2 % (w/v) Triton X-

100, and three times with the same buffer without Triton X-100. The pellet was subsequently

resuspended and incubated overnight at 4 °C in 50 mM TrisHCl buffer, pH 8 containing 1

mM DTT and 2 M urea. The soluble fraction, obtained after removal of the precipitated

material by centrifugation (15,000 g, 15 min), was loaded onto a Q-Sepharose XK 26/10

column (Amersham-Pharmacia Biotech) that had been pre-equilibrated with two volumes of

50 mM TrisHCl buffer, pH 8 containing 1 mM DTT and 2 M urea. The column was washed

using a flow rate of 3 mL min-1 with the starting buffer until the baseline was stable. The


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protein was eluted from the column with a 400 mL linear gradient of NaCl (0 to 1 M).

Fractions containing BpUreG were combined, diluted with the elution buffer to a protein

concentration of 0.3 mg/mL, and dialyzed (5 kDa molecular weight cut-off – MWCO –

membrane) overnight at 4 °C against 50 mM TrisHCl buffer, pH 8. The resulting solution of

BpUreG was concentrated using 5 kDa MWCO Amicon and Centricon ultra-filtration units

(Millipore), to a final volume of 5 mL, and centrifuged (15 min at 14,000 g) to remove the

precipitated material. The resulting solution was loaded onto a Superdex 75 XK 26/60

column conditioned with 50 mM TrisHCl buffer, pH 8 containing 0.15 M NaCl and 1 mM

DTT. BpUreG was eluted at a flow rate of 2 mL min-1, and the purified protein, amounting to

ca. 30 mg per liter of culture, was concentrated to 2.5 mg mL-1 and stored at -80 °C.

In the case of His-tagged BpUreG the supernatant after pellet separation was loaded

onto a column containing 8 mL of the Ni-NTA Superflow affinity resin (Qiagen) pre-

equilibrated with 40 mL of 50 mM TrisHCl buffer, pH 8.0, containing 5 mM imidazole,

washed with 30 mL of the same buffer containing 20 mM imidazole, and eluted using the

same buffer containing 100 mM imidazole.

Protein purity, as well as the molecular mass of BpUreG in denaturing conditions, was

estimated by SDS-PAGE according to the method of Laemmli (35), by using a BioRad Mini-

Protean II apparatus. Proteins were separated on 15% (w/v) acrylamide-bisacrylamide

separating gels that were stained using either Coomassie brilliant blue R-250 or silver

staining.

Protein concentration was measured using a JASCO 7800 spectrophotometer and a

value for the extinction coefficient (ε280=10,810 M-1 cm-1) calculated from the amino acid

sequence using the ProtParam web site (http://au.expasy.org/tools/protparam.html). This

value is in good agreement with that obtained by using the BioRad assay that is based on the

Bradford colorimetric method (36).


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Protein sequence determination

All reagents, solvents and instruments were obtained from Applied Biosystems. N-

terminal sequence analysis was performed in the gas-pulsed liquid phase using a model 476A

protein sequencer with a micro-reaction chamber and an on-line HPLC system for

phenylthiohydantoin (PTH) analysis. Absorbance was monitored at 269 nm. C-terminal

sequence analysis was performed on a Procise 494C protein sequencer using C-terminal

sequencing chemistry (37,38). Prior to this analysis, the sample was adsorbed on a Prosorb

sample preparation cartridge and, after subsequent washes with milliQ-filtered water, treated

with 200 mM phenylisocyanate in acetonitrile under basic conditions (124 mM diisopropyl

ethylamine/acetonitrile) in order to modify the ε-amino group of the lysine residues into stable

phenylureas. The alkylated thiohydantoin (ATH)-amino acids were analyzed on-line using a

thermostated (38 °C) C18 reverse-phase column (2.1 x 220 mm, 5 µM). A linear gradient

with a flow rate of 300 µL min-1 was formed using a 140C micro-gradient system with the

following solvents: Solvent A, 35 mM sodium acetate buffer/3.5% tetrahydrofuran/MQ-

water, and Solvent B, 100% acetonitrile. The ATH-amino acid derivatives were monitored

using a 785A absorbance detector set at 254 nm, and quantified relative to a 100 pmol ATH-

amino acid standard. The methyl napthyl thiohydantoin amino acid standards were obtained

from the supplier.

Mass spectrometry

All mass spectrometric analyses were performed on a Q-TOF mass spectrometer

(Micromass), interfaced to a chip-based nanoESI source (NanoMate100, Advion

Biosciences). The mass spectra were processed using MassLynx v3.1 software of Micromass.

Before analysis, the buffer was changed to 50 mM NH4OAc, pH 6.5 (OAc, acetate). The

denatured protein (1 µM) was measured in 50 % acetonitrile/0.1 % formic acid. For native

measurements, the protein (5 µM) was kept in 50 mM NH4OAc, pH 6.5. Acquisition


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conditions were a spraying voltage of 1.5 kV (denatured protein) or 1.7 kV (native protein),

gas pressure of 0.3 psi and an acquisition time of 3 min (denatured protein) or 10 min (native

protein) across a m/z range of 500 – 3000.

Hydrodynamic properties

The molecular mass and hydrodynamic radius of the native protein were estimated by

standard size exclusion chromatography. A small amount (100 µL, 2.5 mg/mL) of the

purified protein solution was applied to a Superdex-75 HR 10/30 FPLC column that had been

equilibrated with 50 mM TrisHCl, pH 8.0, containing 0.15 M NaCl, at a flow rate of 0.5 mL

min-1 in order to estimate the apparent hydrodynamic volume of the purified protein. The

column was calibrated using an Amersham low molecular weight gel filtration calibration kit.

Absolute estimates of molecular mass and hydrodynamic radius of BpUreG were

determined using a combination of size exclusion chromatography, MALS (multiple angle

light scattering) and QELS (quasi-elastic light scattering). BpUreG (100 µL, 2.5 mg/mL) in

TrisHCl 20 mM (pH 7.5), 150 mM NaCl, was loaded onto a S-200 16/60 column (Amersham)

pre-equilibrated with the same buffer, and eluted at room temperature at a flow rate of 1

mL/min. The column was connected downstream to a multi-angle laser light (690.0 nm)

scattering DAWN EOS photometer (Wyatt Technology). Quasi-elastic (dynamic) light

scattering data were collected at 90° angle using a WyattQELS device. The concentration of

the eluted protein was determined using a refractive index detector (Optilab DSP, Wyatt).

Values of 0.185 for the refractive index increment (dn/dc) and 1.330 for the solvent refractive

index were used. Molecular weights were determined from a Zimm plot. Data were analyzed

using the Astra 4.90.07 software (Wyatt Technology), following the manufacturer’s

indications.


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Circular dichroism spectroscopy

The CD spectra of BpUreG and its His-tagged analogue were measured at 20 °C, using

a JASCO 710 spectropolarimeter flushed with N2, and a cuvette with 0.01 cm path-length.

The buffer was 20 mM phosphate, pH 7.5, containing 0.15 M NaCl. The spectra were

registered from 190 to 300 nm at 0.2 nm intervals. Ten spectra were accumulated at room

temperature, and averaged to achieve an appropriate signal-to-noise ratio. The spectrum of

the buffer was subtracted. The secondary structure composition of BpUreG was evaluated

with the tool available on the Dichroweb server of the Centre for Protein and Membrane

Structure and Dynamics, http://www.cryst.bbk.ac.uk/cdweb/html/home.html (39) using the

reference sets 4 and 7.

NMR spectroscopy

NMR spectra of 15N-enriched KaUreG and His-tagged BpUreG were recorded at pH

8.0 and 298 K on a Bruker Avance 800 spectrometer operating at 800.13 MHz. The KaUreG

spectrum was recorded using a 5 mm reverse-detection probe on a 1 mM sample, while the

spectrum of His-tagged BpUreG was obtained using a TXI cryoprobe on a 0.45 mM sample.

The spectrum of BpUreG isolated from inclusion bodies was recorded on a 0.45 mM 15N-

enriched sample at pH 8.0 and 298 K using a Bruker DRX Avance 500 spectrometer

operating at 500.13 MHz and equipped with a TXO cryoprobe. 1H,15N–HSQC spectra were

acquired using sensitivity improvement (40-42) and consisted of 8-48 scans, spectral windows

of 11-16 ppm in the proton dimension and 30-40 ppm in the nitrogen dimension, with the

carrier set at the water frequency and 118 ppm, respectively. Relaxation delays (including

acquisition time) in the range 0.9-1.2 s were employed. Matrices of 1024×256 points or

2048x128 points were acquired and transformed into 1024×512 or 2048x512 points.


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Measurement of GTPase activity

GTP-hydrolyzing activity was measured using a colorimetric method. The reaction

mixture, containing 20 mM TrisHCl, pH 8.0, 0.075 M NaCl, 5 mM MgCl2, 2 mM GTP and 5

µM BpUreG, in the absence or presence of 25 µM ZnSO4, was incubated at 37 °C. Aliquots

(90 µL) were removed at different incubation times and added to 30 µL of a 35% trichloro

acetic acid/water solution. Phosphate concentration was determined by the malachite green

assay (43).

Metal-binding experiments

In all operations, care was taken to avoid exogenous metal contamination. Ni2+ and

Zn2+ nitrate salts solutions were prepared starting from ICP 1000 ppm standard solutions (CPI

International) diluted to 1 mM with buffer A, containing NaCl 0.15 M. Equal volumes of

BpUreG and metal solutions were mixed in 1:1 ratio to yield a constant concentration of

protein (40 µM and 20 µM BpUreG for nickel and zinc, respectively) and an increasing

concentration of metal ion. No precipitation was observed during the titration. The resulting

mixtures were incubated 1 h at 37 °C and overnight at 4 °C, and then filtered by

centrifugation using 0.5 mL Centricon (MWCO 5 kDa). 400 µL of the filtered solution were

diluted to 8 mL with milliQ water. Metal analysis was performed using a Spectro Ciros CCD

ICP optical emission spectrometer (Spectro Analytical Instruments) in combination with a

Lichte nebulizer and a peristaltic pump for sample introduction. The ICP-ES system was

calibrated by serial dilutions of appropriate single and multi-element standards (CPI

International). The standardization curve was made using standard solutions in the range 0-

500 µM of Ni and Zn in TrisHCl 2.5 mM, pH 8, and NaCl 7.5 mM with a linear fitting. An

Rf power of 1400 W, a nebulizer gas flow of 0.8 L min-1 and a plasma gas flow of 14 L min-1

were used. The sample uptake was set at 2 mL min-1 for 24 s, and a wash time of 15 s at 4 mL

min-1 plus 45 s at 2 mL min-1, for each sample. Quality control was established by evaluation


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of buffer containing standards. In order to estimate the total metal added to every protein

sample, 200 µL of every metal solution were mixed in a 1:1 ratio with the blank buffer,

diluted to 8 mL with milliQ water, and measured as the filtered samples. The 221.648 and

231.604 nm lines for Ni and the 202.548, 206.191, and 213.604 nm lines for Zn were used for

analysis. The measured metal ion concentrations were corrected with the value obtained for

the filtered solution of the protein incubated with the buffer blank, without metal ion. The

experimental points were fitted using the MacCurveFit software, and the fit optimized using a

Quasi-Newton algorithm.

Calculation of BpUreG molecular structure

A similarity search of the protein sequences related to BpUreG was carried out using

the program FASTA3 (44,45) applied to the SwissProt database. Multiple alignment of all

sequences was performed using ClustalW (http://www.ebi.ac.uk/clustalw) (46). The

alignment was optimized using information deriving from secondary structure predictions

provided by the program JPRED (http://www.compbio.dundee.ac.uk/~www-jpred) (47). The

PONDR VL-XT algorithm (48,49) for the prediction of disordered regions of the UreG

sequences was accessed through the web site http://www.pondr.com/ (Molecular Kinetics,

Inc.).

The sequence of BpUreG was used to search for templates using the 3D-Jury predictor

meta-server (50,51) available at the address http://bioinfo.pl/Meta/. Only the templates with a

3D-Jury score higher then 80% of the best score were selected. Multiple sequence alignment

of the sequence of B pUreG with the selected templates was performed using ClustalW

(http://www.ebi.ac.uk/clustalw) (46). Alignment optimization was carried out comparing

information deriving from the secondary structure calculated for the templates using DSSP

(52) with BpUreG secondary structure prediction provided by the program JPRED

(http://www.compbio.dundee.ac.uk/~www-jpred) (47).


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Model structures were calculated using the program MODELLER 6.2 (53) with the

model-default options. The program PROSA II (Version 3.0, 1994) (54) was used for

selecting the best models provided by MODELLER and for protein structure analysis to test

the coherency and validity of the model structures. The Z-score reported in this work is

derived through the standard “hide and seek” procedure of the program, by which the score is

correlated to the difference in potential energy, calculated using mean field potentials,

between the input structure and other randomly assigned folds for its amino acid sequence. A

lower Z-score corresponds to a more favorable potential energy associated with the structure

under examination.

Structure validation was performed using PROCHECK (55) and WHATIF (56). The

calculated final structure was deposited in the http://www.postgenomicnmr.net site. The

molecular surface and the electrostatic color-coding was generated by the program GRASP

(57) 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. All the 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 topological diagram was drawn using the program TOPS (Topology of Protein Structure)

(58) available at the address http://www.tops.leeds.ac.uk .

RESULTS

BpUreG cloning, expression and purification. The sequence analysis of a 5.3-kbp

DNA fragment isolated from B. pasteurii indicated the presence of four open reading frames

identified as the genes expressing the urease accessory proteins UreE, UreF, UreG, and UreD


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(32). The ureG gene was cloned from B. pasteurii chromosomal DNA by PCR amplification.

The gene was inserted between the NdeI and BamHI sites of a pET3a plasmid, and this

construct was used to overproduce BpUreG in E. coli BL21(DE3) strain by induction with

IPTG. This protocol produced an abundant polypeptide with an apparent molecular mass of

25 kDa that was absent from non-induced cells (Figure 1A, lanes 1 and 2). Fractionation of

cells into soluble and insoluble extracts showed that the over-produced protein product

accumulated almost exclusively in the insoluble fraction (Figure 1A, lanes 3 and 4). In order

to obtain the protein in a soluble form, the insoluble BpUreG was treated with increasing

amounts of urea from 2 to 8 M. These studies showed that 2 M urea was the optimum

denaturing agent concentration to extract the maximum amount of BpUreG without associated

protein degradation or re-aggregation (Figure 1A, lane 5). The extract was purified by ion

exchange chromatography in the presence of 2 M urea, and the isolated fractions were

dialyzed overnight to remove the denaturing agent and to refold the protein. The soluble

protein obtained was further purified by size-exclusion chromatography (yield 50 mg per liter

of culture) and its purity was checked by SDS-PAGE (Figure 1A, lane 6).

The formation of inclusion bodies during heterologous over-expression of recombinant

proteins is generally attributed to the high-levels of protein production that result from

constructs built using plasmids containing the strong T7 promoter. This was a problem

encountered in our studies using the pET system for the over-expression of BpUreG.

Lowering the growth temperature or decreasing the amount of added inducer failed to reduce

the expression level and to increase the protein solubility. Alternative expression systems

involving GST-tagged protein were unsuccessful, and yielded lower amounts of protein

without solving the solubility problem. Therefore, we relied upon the pET system and a novel

protocol for protein purification, which yielded large amounts of pure protein. It is generally

believed that inclusion bodies contain proteins in a misfolded state, and several protocols are


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available to solubilize and purify proteins from inclusion bodies. Proteins are typically

solubilized by large concentrations of denaturing agents such as urea or guanidinium chloride

and are refolded during an extensive dialysis step. These conditions often cause problems in

achieving an efficient and reliable folding in vitro due to the many re-folding pathways

potentially available for the random-coiled protein and to the re-aggregation processes

occurring at intermediate denaturant concentrations. In the case of BpUreG, the amount of

urea found to solubilize the inclusion bodies was relatively low (2 M) suggesting that the

protein was only partially unfolded in the insoluble aggregate. The low urea concentration

also prevents the complete unfolding of the protein, a state from which a native fold is

difficult to attain.

Another approach to solve the solubility problem of BpUreG was attempted by cloning

and expressing the His-tagged protein using the pET15b plasmid. This method, similarly to

the pET3a system, produced large amounts of protein in the inclusion bodies, but allowed the

purification of the protein from the supernatant, in a single chromatographic step using a Ni2+-

affinity column. The yield of His-tagged BpUreG was significantly lower than in the case of

the protein purified from the insoluble pellet (10 mg per liter), but this amount allowed us to

compare the fold properties of the protein isolated from the soluble and insoluble fraction,

using CD and NMR spectroscopy, as well as activity assay.

ESI Q-TOF mass spectra of BpUreG in denaturing conditions (Figure 1B) confirmed

the high purity of the isolated protein and indicated a mass of 23084.5 Da, in agreement with

the theoretical mass (23084.1 Da).

### Figure 1 here ###


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As expected from the gene sequence, Edman degradation of the N-terminus of

BpUreG yielded the sequence MKTIHL. The protein was also subjected to C-terminal, amino

acid sequence analysis, which provided the expected sequence ESK. In the case of the latter

experiment, the sensitivity dropped rather rapidly, yielding only the last three residues, most

likely because of the presence of consecutive ‘problematic amino acids’ (59). However, this

information, together with the mass of the recombinant protein, is sufficient to demonstrate

that the recombinant purified BpUreG protein is intact and unmodified.

Hydrodynamic properties and oligomerization of BpUreG. Gel filtration experiments

performed on BpUreG revealed the presence of two peaks corresponding, according to their

retention volumes, to hydrodynamic radii (Rh) of 2.8 nm (minor peak) and 3.5 nm

(predominant form), and apparent molecular mass of 39 and 59 kDa. The gel filtration profile

for the soluble His-tagged BpUreG analogue was very similar to that of BpUreG isolated from

inclusion bodies, with a smaller amount of the minor peak. These masses are not easily

related to multimeric forms of the BpUreG monomer (Mw = 23084 Da). These data, based on

the calibration of the column with globular standard proteins, assuming a globular structure

for BpUreG, are strongly dependent on the properties of the protein and the column, as well as

on the possible interactions between the protein and the solid phase. Therefore, a better

estimate of the molecular masses and hydrodynamic radii of the protein eluted in the two

different peaks was obtained using a combination of size exclusion chromatography (SEC),

multi-angle light scattering (MALS) and quasi-elastic (dynamic) light scattering (QELS)

(60,61). The advantage of such a system is that the molecular mass determination is based on

the fundamental light scattering properties of the macromolecule. The results obtained are

shown in Figure 2. The elution profile and the MALS data are consistent with the presence of

a predominant dimeric form of the protein in solution (≥95%, Mw = 40.0±0.5 kDa), while the

monomeric BpUreG represents only a minor fraction (≤5%, Mw = 28.6±4.9 kDa). The MALS


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data confirm the anomalous chromatographic behavior of the protein and suggest either a

non-spherical (prolate or oblate) shape of the protein, or the presence of non-specific

interactions between the protein and the column phase. Because the scattering of the applied

light (wavelength 690 nm) by such small molecules is isotropic, no information on the protein

size can be obtained from the static MALS. However, such information can be derived from

dynamic light scattering (QELS) experiments performed simultaneously with the MALS

measurements. The hydrodynamic radius (Rh) of the dimeric BpUreG, as determined by

QELS, is 2.00±0.02 nm, while the value of Rh for the monomer is larger (2.70±0.20 nm). The

apparent inconsistency between the mass and the volume of the monomer and dimer of

BpUreG can be resolved by considering that the monomer is largely unfolded. Considering

the larger value of Rh for the monomer, the retention volume for the monomeric form in the

size exclusion column is expected to be smaller than that for the dimeric protein: the aberrant

behavior of the monomer in the gel filtration experiment could be explained with unspecific

interactions occurring between the unfolded monomer and the solid phase of the column. The

mass spectrum of BpUreG under non-denaturing conditions reveals the presence of the

monomer only, an indication that the dimerization does not involve the formation of covalent

bonds (not shown). In the ESI-MS experiments, the injected sample must be maintained at

low ionic strength, a condition that could destabilize the dimer, especially if hydrophobic

forces are the main responsible for the dimerization of the protein. Moreover, hydrophobic

interactions are weak in gas phase, further causing the dimer to dissociate in the TOF stage.

The mass spectrum also confirms that the monomer is largely unfolded, with the presence of

populations featuring charge states (from 9+ to 29+) reflecting high solvent accessibility of

protonation sites.



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Circular dichroism and secondary structure of BpUreG. In order to evaluate the

secondary structure composition of BpUreG in solution, the protein was analyzed using

circular dichroism (CD) spectroscopy. The obtained spectrum (Figure 3) shows negative

deflections with a minimum at ca. 208 nm and a pronounced shoulder at ca. 220 nm, as well

as a maximum positive deflection at ca. 193 nm, typical for the presence of both α-helix and

β-strand regions in the protein. The spectrum was quantitatively analyzed in the range 190-

240 nm, on a per amide basis calculated from protein content and sequence, using all the

different fitting programs at the Dichroweb server (39) and all the possible reference sets.

The best fit was selected on the basis of the normalized root mean square deviation (NRMSD

= 0.038) between the experimental and calculated data, obtained using the variable selection

method program CDSSTR (62) and the reference set n. 4. From this analysis, a secondary

structure composition of 15% α-helix, 29% β-strand, 26% turns and 30% random coil was

estimated for BpUreG. The CD spectrum of His-tagged BpUreG confirmed these data,

yielding 18% α-helix, 25% β-strand, 29% turns and 30% random coil.

### Figure 3 here###

NMR spectroscopy of BpUreG. In order to monitor the conformational properties of

the protein in solution, NMR spectroscopy was applied. The 1H,15N-HSQC spectrum of

BpUreG (Figure 4A), His-tagged BpUreG (Figure 4B), and KaUreG (Figure 4C) are very

similar. They are characterized by poorly resolved resonances with little dispersion in the 1H

dimension of the backbone amides, many of them falling in the random coil region (7.6-8.5

ppm chemical shift range). This observation indicates that large portions of the protein

backbone experience exchange between multiple conformations, and lack a well-defined


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secondary structure. The signals of the side chain NH2 groups of the 12 Asn residues present

in the protein are also poorly resolved and give rise to two broad envelopes, centered at 112.5

ppm (15N) and 7.4-6.7 ppm (1H). Lack of differentiation in these resonances suggests lack of

specific interactions for the Asn side chains and therefore confirms the fluxional behavior of

UreG in solution.


Measurement of GTPase activity of BpUreG. UreG is involved in the hydrolysis of GTP

concomitantly with the carbamylation of the lysine residue in the urease active site (26). The

GTPase enzymatic activity of BpUreG was checked and measured using a colorimetric

method that determines the concentration of phosphate released by the hydrolysis of added

GTP at various times (Figure 5). BpUreG and His-tagged BpUreG show a significant and

comparable level of GTPase activity. In the assay mixture, the concentration of substrate

GTP is three orders of magnitude larger than the concentration of enzyme, a condition that

allows the use of the time course data of the reaction for the derivation of the value of kcat =

0.04 min-1 and 0.03 min-1 for wild-type BpUreG and His-tagged BpUreG, respectively.


Ni2+ and Zn2+ binding to BpUreG. The isolated BpUreG did not contain bound metal

ions, as established by ICP-ES metal analysis. Considering that UreG is involved in the

building of the Ni2+-containing active site in the urease activation process (10,20,26) while

UreE, the most known accessory protein in this system, features Ni2+ and Zn2+ binding

properties (21-25), the affinity of BpUreG for Ni2+ and Zn2+ was quantitatively investigated. A


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fixed concentration of BpUreG was titrated with increasing concentrations of Zn2+ and Ni2+

ions, and the concentration of free ions was measured using ICP-ES. The binding curves,

obtained by reporting the amount of total metal bound (Mb) as a function of the total amount

of metal added (Mt), showed saturation with a maximum of four Ni2+ ions and two Zn2+ ions

bound to the BpUreG dimer in the presence of excess metal. Figure 6 reports the amount of

bound metal per dimer (Mb/Ptot) as a function of the free ion per dimer at equilibrium (Mf/Ptot).

Assuming a single-site binding model (that is considering that the metal ions show identical

affinities for the different sites, i.e. homogeneous binding) the experimental points can be

fitted to a curve described by Equation 1:

(1)

where Ptot is the total concentration of BpUreG dimer, KD is the dissociation constant, and n is

the number of binding sites. The best fit was obtained with n = 4 for Ni2+ and n = 2 for Zn2+.

The KD estimated for the binding is 360±30 µM for Ni2+ and 42±3 µM for Zn2+, revealing a

ten-fold higher affinity of BpUreG for Zn2+ than for Ni2+.

###Figure 6 here###

Calculation of a structural model for BpUreG. The deduced amino acid sequence of the

cloned ureG gene confirmed the identity of the encoded protein as belonging to the UreG

family. Multiple alignment of the sequence of BpUreG with related sequences found in a

similarity search highlights the conservation of the predicted secondary structure elements

and the overall sequence profile and motifs (Figure 7). This family of proteins appears to be

characterized by the presence of five long helices and seven short beta-strands. Two

additional helices (H4 and H5) and one strand (S4) are found in some sequences but are not

fully conserved. These structural elements are separated by large portions of turns or coils of


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variable length. The regions predicted to be in a strand conformation are mainly characterized

by the presence of hydrophobic residues, suggesting that they constitute the hydrophobic core

of the protein. On the other hand, the helices are mostly amphipathic, suggesting their

involvement in intermediate regions between the hydrophobic core and the solvent exposed

surface of the protein. The similarity of the secondary structure elements is paralleled by a

high degree of sequence identity among all UreG proteins (between 49% and 62%). Large

portions of the sequences are fully conserved, and these regions mostly occur in the loops and

coils rather than in the helices and strands. This suggests that these less structured regions are

functionally most important, while the helices and strands are only needed to confer the

necessary overall structure to the protein. The P-loop, needed for GTP-binding, is found

between strand S1 and helix H1 in the N-terminal region of the protein, and consists of a

conserved GPVGXGKT motif, where X is usually Ser or, rarely, Ala.

The program PONDR VL-XT (48,49,63), was used to calculate the tendency of each

residue in BpUreG and its homologous proteins to be disordered (PONDR score above 0.5,

residues underlined in Figure 7). The program predicts that large portions of all UreG

proteins (for example 44 residues in BpUreG), found in the central region of the sequence, are

disordered. This type of prediction is consistent with the observation that the monomeric

form of BpUreG is largely unfolded as determined by light scattering, and that the dimeric

form, the predominant oligomer in solution, also undergoes dynamic processes in large

portions of the protein, as detected by NMR spectroscopy.

###Figure 7 here###

In order to calculate a model structure for the fully folded BpUreG, the first choice

involved the use of protein templates having a known structure and high homology with the


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target protein (homology building). However, a database search for homologous proteins of

known structure using FASTA3 (44,45) and PSI-BLAST (64,65) did not result in any hit with

a sequence identity higher than 30%, preventing the use of standard homology modeling

protocols for structure prediction (66). Therefore, algorithms based on fold recognition were

attempted. The 3D Jury approach was singled out for its performance in structure prediction

in the most recent CASP5 experiment (67), and the use of this protocol resulted in five

template structures with a score higher than 81.3 (i.e. higher than 80% of the best), as reported

in Table 1. The 1FFH structure has been refined (68) to a higher resolution (1.10 Å) and the

PDB code 1LS1 was used in its place. All the found templates fall into the c.37.1 SCOP class

(69), indicating that BpUreG is similar to proteins that contain a P-loop and are involved in

GTP-based metabolic processes.

--- Table 1 ---

The sequence alignment of these proteins with the sequence of BpUreG, optimized using

information derived from secondary structure predictions, was used as input to obtain a first

set of model structures, which were then analyzed to identify local fold problems using

PROSA. Whenever possible, these problems were corrected by modifying the alignment in

the interested regions and building new models, following the same procedure used recently

by us to model a set of UreE proteins (30). During the optimization of the structural model,

misfolding problems were encountered in the region between residues 40 and 80, with the

formation of a knotted loop. This is in line with the PONDR prediction that this region is

disordered. In order to avoid this artifact, two template structures (1EGA and 1J8Y) had to be

removed from the templates ensemble. 1J8Y is a GTPase domain found in a signal

recognition particle, and its structure is very similar to 1LS1 (sequence identity: 43%,


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backbone RMSD: 1.26 Å). The use of this structure as template introduces redundancy in the

modeling, somehow hampering the calculation of a good model in the cited region of

BpUreG. 1EGA was excluded because of its low resolution (2.40 Å) and for the absence of

the helix predicted between residues 45 and 51 of BpUreG. The final multiple sequence

alignment of BpUreG and the used structural templates is reported in Figure 8.

The BpUreG model structure obtained from this procedure features a high percentage

of residues in the core and allowed region of the Ramachandran plot (88.8 and 8.7%

respectively), with only few residues localized in the generously allowed region (2.5%), and

no residue found in the disallowed region of the diagram. The low PROSA Z-score (-6.56)

also confirms the good quality of the model.

The fold of the BpUreG monomer is characterized by a central open β-barrel, formed

by seven parallel and two anti-parallel strands, surrounded by six α-helices connected with

loops (Figure 8). The 36% of the modeled residues of BpUreG are involved in α-helices, the

22% are part of the central β-strands, while the remaining 42% comprises turns and coils.

The P-loop is located between strand S1 and helix H1, and is found on one side of a deep

pocket defined, on the other side, by the loop located between strand S5 and helix H4. Figure

8 also report the positions of the so-called Switch I (between strand S2 and helix H2) and

Switch II (between strand S5 and helix H4) regions, found to be important for the binding of

the GTP-Mg2+ adduct in G-proteins (70). The binding of Mg2+ is generally needed for

GTPase function, as it is involved in the GTP phosphate chain binding to the protein at typical

consensus sequence ([DE]XXG) (70). The topological diagram for the model of BpUreG is

also shown in Figure 8. The C-terminal portion of BpUreG, comprising residues 189-211,

was not modeled due to the absence of a structural template for this region. This peptide

sequence is predicted to exist in a α-helical conformation (H7 in Figure 7), whose topology

and orientation with respect to the rest of the protein could not be determined. The presence


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in the model of strand S9 at the C-terminus, in contrast to the predicted presence of an helix

(H7) may be explained by an artifact due to a termination effect of the modeling process.


In order to gain more information on BpUreG model structure characteristics, the solid

surface representation of the electrostatic potential was calculated (Figure 9). The molecular

surface of the model structure is characterized by large patches of negative charge, with the

exception of a large neutral zone situated in the region of helixes H4, H5, and H6. The large

pocket observed in the C-terminal region is probably due to the absence, in the model, of the

C-terminal helix, which comprises the last 24 residues. This suggests that such helix could

fill this pocket in a folded form of the protein.


The deep pocket found near the P-loop, and identified as the putative GTP binding

site, features a negatively charged surface. Consistently with other GTPases co-crystallized

with GTP analogues (see references (71-75) for some recent studies), the pocket also contains

the Mg2+ ion binding site, localized in the proximity of the Thr17 and Thr42 residues,

conserved among UreGs (Figure 7) and template structures (Figure 8).


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DISCUSSION

The thorough functional information collected for UreG in the past few years and

establishing this protein as an essential chaperone for the Ni2+ active site assembly of urease

have been complemented by the present study on the biochemical and structural properties of

this protein in solution, using the recombinant UreG from B. pasteurii. The large amount of

protein required for this type of characterization was obtained by cloning and expressing the

protein, and establishing a protocol for protein purification.

Using a combination of size exclusion chromatography (SEC) and multi-angle laser

light scattering (MALS) (60,61), BpUreG was shown to exist in solution as a dimer. In the

past, hydrodynamic studies on KaUreG reported the presence of only the monomer in solution

(20), while for HpUreG the molecular mass under native conditions was not determined (31).

In the case of KaUreG only chromatographic experiments were used to establish its molecular

aggregation form in solution. This approach is partially uncorrected and subject to errors,

because it is strictly dependent on the shape of the protein and on its interaction properties

with the solid chromatographic phase. This possibility was proven for BpUreG, shown to

produce an aberrant chromatographic profile that could have lead to wrong conclusions were

it not for the use of light scattering techniques, which yielded an incontrovertible value for the

molecular mass of the protein in solution. The aggregation state of BpUreG is similar to that

found for H. pylory HypB, a GTPase involved in the activation of Ni2+-dependent

hydrogenase and reported to exist as a mixture of the monomeric and dimeric form (76). The

hydrodynamic radii measured by dynamic light scattering (QELS) for the dimer and the

monomer BpUreG (2.00±0.02 nm and 2.70±0.20 nm respectively) suggest that the latter is

largely unfolded while the dimer is present with a compact behavior, therefore representing

the actual functional form of the protein.


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In solution, BpUreG showed a well-defined secondary structure, with both α-helices

(15%) and β-strand regions (29%), as determined using CD spectroscopy (Figure 3). These

data do not contain any information regarding the tertiary structure of BpUreG, which has

therefore been evaluated using NMR spectroscopy. 1H,15N-HSQC NMR spectra revealed that

BpUreG does not posses a rigid tertiary structure, but exists in solution in fast equilibrium

among different conformations (Figure 4) and therefore contains large portions of unfolded

backbone. The intrinsically unfolded state of BpUreG in solution is not an artifact due to the

purification method, which involved the use of a small amount of urea to solubilize the

protein from inclusion bodies. This is proven by the essentially identical spectroscopic and

hydrodynamic properties of the His-tagged form of BpUreG, purified in a single step from the

soluble cellular extract.

The large similarity of the NMR spectral properties of KaUreG and BpUreG suggests

that the presence of large portions of the protein backbone undergoing conformational

changes, and therefore causing a substantial intrinsic protein unfolding, is a general feature

for all UreG chaperones. This evidence indicates that UreG belongs to the ever-growing class

of intrinsically unstructured proteins (IUP). These are natively unfolded polypeptides that

undergo disorder-order transitions among the random coil, pre-molten globule, molten

globule or fully folded states during or prior to their biological function (63,77,78). The

observed conformational plasticity of UreG proteins is consistent with their role as

chaperones with GTPase activity, assuming a fully active conformation only in the presence

of a preformed complex with other urease accessory proteins, as experimentally proven (20).

This characteristic could be related with the functional role of UreG in vivo, because it could

permit to minimize the unwanted hydrolysis of GTP unless the protein is ready to perform its

role together with its partner chaperones in the preformed UreDFG-apo urease complex. The

high homology found in the predicted unstructured parts of the UreG sequences could well be


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related to this need, indicating the evolutionary functional importance of these loop regions,

while the α and β elements perform a structural role. Large unstructured portions were

predicted to be present in the central part of all UreG sequences found in the database search

(Figure 7). This characteristic could explain the fluxional behavior detected by NMR

spectroscopy for both BpUreG and KaUreG, confirming that this characteristic is a general

feature of this class of proteins.

The structure of BpUreG in the fully folded state has been modeled, using a fold

recognition procedure. Considering that BpUreG is an IUP we can presume that this model

predicts the structure of the protein when other co-factors or protein partners force it to

assume its functional conformation. The structural prediction shows a fold typical for a

GTPase protein (Figure 8,9) with an identifiable negatively charged P-loop region, likely

capable to contain a GTP molecule, and the Switch I and Switch II regions, putatively

involved in the Mg2+ binding and generally required for the GTPase function. The overall

fold comprises an internal open β-barrel surrounded by α-helices. The hydrophobic and

amphipatic composition of the sequences, predicted respectively as β-strands and α-helices in

the multiple sequence alignment (Figure 7), confirms this architecture. The secondary

structure composition derived from the model is calculated as 36 % for the helix and 22 % for

β-strands, while the remaining 42% is in coils or turns. The partial contrast between this

prediction and the calculation of the secondary structure from the CD spectrum (15% of α-

helices, 29% of β-strands) can be explained considering that the contradiction involves mostly

the more solvent-exposed α-helices, likely more subject to conformational fluctuations, rather

than the β-strands, situated in the internal and protected hydrophobic protein core.

The observation of the presence, in the UreG sequences, of a fully conserved P-loop

motif, has led to consider the possible GTPase activity for this protein. A study, published in

1997, reported that KaUreG does not, by itself, hydrolyze GTP or ATP. Indeed, no


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nucleotide was found to be associated with isolated KaUreG, nor this protein could bind

added GTP or ATP, and no interaction of KaUreG with ATP- or GTP-linked resins was

observed (20). Similar results were obtained for HpUreG, which showed negligible GTPase

activity for the isolated protein (31). The present study demonstrates that BpUreG (and its

His-tagged analogue) features a clear GTPase activity, even if low (kcat= 0.04 min-1) compared

to other GTPases (79) (Figure 5). This activity is lower than (but comparable to) the one

showed by HypB from B. japonicum (kcat= 0.18 min-1) (80) and from E. coli (kcat= 0.17 min-1)

(81). These results indicate that BpUreG, although present in solution as a fluxional, partially

unfolded molecule, displays a level of enzymatic activity indicating that a significant fraction

of UreG molecules is in the correct fold for catalysis. Alternatively, the fold around the

catalytic site could well be correct and the registered activity intrinsic for this protein, with the

unfolded conformation possibly involving a different protein region, as the one indicated by

PONDR prediction.

BpUreG has been demonstrated to bind two Zn2+ ions for dimer, while the affinity is

ten fold lower for Ni2+ ions (Figure 6). The analysis of the structural model of BpUreG

reveals the presence of a putative metal binding site, rich in residues commonly found in zinc-

binding protein sites, and could reasonably be proposed as implicated in the binding of one

Zn2+ per monomer. The fully conserved residues likely involved in the metal binding (Glu64,

Cys68, His70) would fall well within the predicted disordered region of UreG (Figure 7). In

this regard, it is interesting to evidence that this putative metal binding site is in a region

(residues 40 - 80 in BpUreG numeration) that has been very difficult to model, as described

above, probably for its natively unfolded trait. Therefore, we could speculate that the binding

of the Zn2+ ion could induce a conformational change and contribute to the stabilization of the

protein backbone in this region. In this case, the metal ion would assume a structural rather


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than a role in the catalytic activity of the protein. Indeed, no change in GTPase activity was

observed in the presence of a five-fold excess Zn2+ ion in the assay mixture.

The specific metal-binding capability of UreG has never been observed before, and

could be related to its direct role in the assembly of the urease active site (10,20,26). HypB,

the counterpart of UreG in the [Ni,Fe]-hydrogenase system, has often shown a Ni-

sequestering ability, probably due to the presence of an N-terminal His-tag, as in the case of

Rhizobium leguminosarum (82) and Bradyrhizobium japonicum (80), while HpHypB and the

HypB from E. coli, lacking the N-terminal His-tag, does not bind nickel (76,81). Our study

revealed that BpUreG is able to bind zinc even in the absence of a His-tag. In this regard, it is

important to consider that UreE, the best characterized accessory protein in this system, is

able to bind both Ni2+ and Zn2+ with comparable affinity (22,24). The in vivo effect of zinc on

the urease activation is unknown. However, zinc cannot substitute for nickel during urease

biosynthesis in the absence of nickel ions (6). A role for HybF, a Zn-binding protein

analogue to HypA, in the insertion of Ni in [Ni,Fe]-hydrogenase was recently established

(83). The results presented here strongly suggest that metal ions other than nickel play an

important role in the activation of urease, in some sort of co-metabolic metal trafficking

crossroad.

The high sequence homology among UreG proteins is paralleled by a functional

conservation through different species, that was demonstrated in vivo by complementing a

Ka-urease defective mutant with UreG from potatoes, expressed in E. coli (84). UreG has

counterparts in the activation of urease from different organisms as well as in the assembly of

other Ni-containing enzymes. Activation of CO dehydrogenase in Rhodospirillum rubrum

requires the activity of CooC, a nucleotide binding protein related in sequence to UreG

(85,86). UreG is also homologous to HypB, a GTP-binding accessory protein involved in the

GTP-dependent activation of Ni-containing hydrogenase and urease in many organisms; in H.


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pylori inactivation of hypB leads to a phenotype that is both hydrogenase- and urease-negative

(31,87). Eu3, the UreG analogue in Arabidopsis thaliana and soybean (88), has a high degree

of sequence similarity to both UreG and HypB, and also exhibits a putative nucleotide-

binding site. Given the observed sequence similarities among all UreG proteins from

different organisms, as well as the functional parallelism between UreG and the related

proteins in other nickel-containing systems, it is reasonable to presume that the structural and

functional insights obtained in the present study on BpUreG can be extended to different

systems and/or different organisms, and that the results described here contribute to a general

model for Ni2+ incorporation into metallo-proteins.

ACKNOWLEDGEMENTS

Silvia Miletti is acknowledged for performing initial screenings of the BpUreG

cloning. We thank Niyaz Safarov, Suzanne K. Christensen, Katja Kortnetsky, and Ann Brige

for technical assistance in molecular cloning and nucleotide sequencing, and Dr. Andreas

Thiesen (Wyatt Instruments) and Dr. Matthew R. Groves (EMBL Hamburg) for the use of the

Dawn EOS as well as useful advice in data processing. BZ is a recipient of a Ph.D.

fellowship provided by the University of Bologna. MS, FM, and AD are recipients of

fellowships provided by CIRMMP (Consorzio Interuniversitario per le Risonanze Magnetiche

di Metalloproteine Paramagnetiche). The research was supported by grants from the

Ministero Italiano dell’Università e della Ricerca (PRIN-2001 and PRIN-2003). JVB and BD

are supported by the Funds for Scientific Research-Flanders (Grant G.0190.04). Work in the

laboratory of DAB was supported by NIH grant GM-31625. 15N-enriched K. aerogenes UreG

was kindly provided by Gerry Colpas and Robert P. Hausinger (RPH is supported by the NIH

Grant DK45686).


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FIGURE LEGENDS

Figure 1. Expression and purification of BpUreG. A. SDS-PAGE of cell extracts of

BL21(DE3) E. coli cells harboring pET3a::ureG prior to induction (lane 1), after 16 h of

induction with IPTG (lane 2), soluble cell extract (lane 3), insoluble cell extract (lane 4),

soluble fraction after 16 h incubation in 2 M urea (lane 5), purified BpUreG (lane 6),

molecular mass marker (lane 7). B. Electrospray mass spectrum of denatured BpUreG in 50%

acetonitrile/0.1% formic acid. The insert shows the maximum entropy deconvoluted mass

spectrum showing the molecular mass of the protein.

Figure 2. Molar mass distribution plot for BpUreG. The solid line indicates the trace

from the refractive index detector, and the dots are the weight-average molecular weights for

each slice (i.e. measured every second). The experimental conditions are described in the

text.

Figure 3. Circular dichroism spectrum of BpUreG. The experimental points are shown

as hollow circles and the solid line represents the best fit calculated using the Dichroweb

server. The experimental conditions are described in the text.

Figure 4. NMR spectroscopic properties of UreG. 1H-15N HSQC spectra of (A) BpUreG,

(B) His-tagged BpUreG, (C) KaUreG. The experimental conditions are described in the text.

Figure 5. Enzymatic activity of BpUreG. Time course of GTPase activity of BpUreG

(hollow circles) and His-tagged BpUreG (full circles), fitted using a linear regression.

Figure 6. Metal binding properties of BpUreG. Titration curves for the interaction

between BpUreG and Zn2+ (full circles/squares/triangles) or Ni2+ (hollow circles/squares).

The circles, squares and triangles represent different emission wavelengths, as indicated in the

text. The lines represent non-linear curve fits using Equation 1.


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Figure 7. Multiple sequence alignment of UreG proteins. The alignment was obtained

with ClustalW (46), optimized by considering the prediction of secondary structure performed

using JPRED (47). The predicted secondary structural elements are highlighted in yellow

(helix) and turquoise (strand). The P-loop motif is colored red, while residues putatively

involved in Zn2+ binding are in green. Fully conserved residues are indicated by a star (*),

while conservative substitution are marked with a colon mark (:). Residues predicted to be

disordered by the program PONDR VL-XT (48,49,63) are underlined. The sequences

correspond to UreG from: B. pasteurii (1), Helicobacter pylori J99 (2, 62% identity respect to

B. pasteurii), Helicobacter pylori (3, 61%), Staphylococcus xylosus (4, 61%), Bacillus sp.

TB-90 (5, 60%), E. coli (6, 59%), Streptococcus salivarius (7, 58%), K. aerogenes (8, 58%),

Synechocystis sp. (9, 57%), Proteus mirabilis (10, 57%), Haemophilus influenzae (11, 56%),

Ureaplasma parvum (12, 55%), Actinobacillus pleuropneumoniae (13, 55%), Bordetella

bronchiseptica (14, 54%), Yersinia enterocolitica (15, 49%), Yersinia pestis (16, 49%).

Figure 8. Structural modeling of BpUreG. Multiple sequence alignment (top panel) of

BpUreG (BPUG) with G domain of the signal recognition protein Ffh from T. aquaticus

(1LS1), signal recognition particle receptor from E. coli (1FTS) and hypothetical protein Yjia

from E. coli (1NIJ) as obtained from ClustalW and optimized using JPRED. The predicted

secondary structure elements are highlighted in yellow (helices) and turquoise (sheets). The

residues involved in the P-loop are shown in red bold. The residues in italics were not

modeled because of the absence of structural data for the homologous proteins. The fully

conserved residues are evidenced with a star (*). Bottom panels report BpUreG model

structure shown as “cartoon” (left panel) and as topological diagram (right panel). The

secondary structure elements range from deep blue in the proximity of N-terminal to red at

the C-terminal. Bottom left panel was made with MOLSCRIPT and RASTER3D (94,95),

while bottom right panel was made with TOPS (58).


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Figure 9. Surface electrostatic properties of BpUreG. Cartoon (left panels) and solid

surface representations of the electrostatic potential of BpUreG model structure (right panels).

Cartoons are colored as in Figure 8 (bottom panels), while surfaces are colored according to

the calculated electrostatic potential contoured from –10.0 kT/e (intense red) to +10.0 (where

k = Boltzman constant, T = absolute temperature, and e = electron charge) (intense blue). The

protein is shown with the P-loop on the top of the panel (A), toward the viewer (B) and on the

bottom of the panel (C).


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Table 1. 3D-Jury meta-predictions for folding templates of BpUreG

PDB Code Description Source Resolution(Å)

3D-JuryScore

BpUreGidentity

1FTS (89) GTP-dependent signal recognitionparticle receptor (FtsY)

E. coli 2.20 101.60 12.5 %

1FFH (90) GTPase domain of the signalsequence recognition protein (Ffh)

T. aquaticus 2.05 100.00 19,5 %

1EGA (91) GTPase-dependent cell cycleregulator

E. coli 2.40 89.00 13.5 %

1J8Y (92) GTPase domain of the signalsequence recognition protein (Ffh)

A. ambivalens 2.00 87.00 14.6 %

1NIJ (93) Hypothetical protein Yjia E. coli 2.00 84.40 17.3 %


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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Figure 7

S1 H1 S2 H2 S3 S4 H3 S5 | | | | | | | | 1 10 20 30 40 50 60 70 80 90 1 ---------------MKTIHLGIGGPVGSGKTTLVKTLSEALK-EEYSIAVITNDIYTREDANFLINE--NILEKDRIIGVETGGCPHTAIREDASMNFEAIEELKNRF-DDLEIILLE 2 -----------------MVKIGVCGPVGSGKTALIEALTRHMS-KDYDMAVITNDIYTKEDAEFMCKN--SVMPRDRIIGVETGGCPHTAIREDASMNLEAVEEMHGRF-PNLELLLIE 3 -----------------MVKIGVCGPVGSGKTALIEALTRHMS-KDYDMAVITNDIYTKEDAEFMCKN--SVMPRERIIGVETGGCPHTAIREDASMNLEAVEEMHGRF-PNLELLLIE 4 --------------MTDTIKIGVGGPVGAGKHELIEKIVKRLA-KDMSIGVITNDIYTKEDEKILVNS--GVLPEDRIIGVETGGCPHTAIREDASMNFAAIDELKERN-DDIELIFIE 5 ---------------MEPIRIGIGGPVGAGKTMLVEKLTRAMH-KELSIAVVTNDIYTKEDAQFLLKH--GVLPADRVIGVETGGCPHTAIREDASMNFPAIDELKERH-PDLELIFIE 6 -----------MQEYNQPLRIGVGGPVGSGKTALLEVLCKAMR-DTYQIAVVTNDIYTQEDAKILTRA--EALDADRIIGVETGGCPHTAIREDASMNLAAVEELAIRH-KNLDIVFVE 7 -------------MTKRTVIIGVGGPVGSGKTLLLERLTRRMS--DLNLAVITNDIYTKEDALFLAKN--SSLDEDRIIGVETGGCPHTAIREDASMNFEAIETLQERFNHDLDVIFLE 8 -----------MNSYKHPLRVGVGGPVGSGKTALLEALCKAMR-DTWQLAVVTNDIYTKEDQRILTEA--GALAPERIVGVETGGCPHTAIREDASMNLAAVEALSEKF-GNLDLIFVE 9 -------------MAQTPLRIGIAGPVGSGKTALLEALCKALR-QKYQLAVVTNDIYTQEDAQFLVRA--EALTPDRILGVETGGCPHTAIREDASLNLAAIADLEARF-MPLDMVFLE10 -----------MQEYNQPLRIGVGGPVGSGKTALLEVLCKAMR-DSYQIAVVTNDIYTQEDAKILTRA--QALDADRIIGVETGGCPHTAIREDASMNLAAVEELAMRH-KNLDIVFVE11 MSNTVATMINKRNIMRNYIKIGVAGPVGAGKTALIEKLTREIA-SKYSVAVITNDIYTQEDAEFLTKN--SLLPPERIMGVETGGCPHTAIREDASMNLEAVDEMVTRF-PDVEIVFIE12 --------------MKRPLIIGVGGPVGAGKTMLIERLTRYLSTKGYSMAAITNDIYTKEDARILLNT--SVLPADRIAGVETGGCPHTAIREDASMNFAAIEEMCDKH-PDLQLLFLE13 --------------MRKYIKIGVAGPVGAGKTALIERLTREIA-SKYSVAVITNDIYTQEDAEFLTKN--SLLPPERIMGVETGGCPHTAIREDASMNLEAVDEMVARF-PEVELIFIE14 --MHDISSLTTRTKTLPPLRVGVGGPVGSGKTTLLEMVCKAMY-PQFDLIAITNDIYTKEDQRLLTLS--GALPPERILGVETGGCPHTAIREDASINLIAIDQMLEQF-PDADIVFVE15 ------MNSHSTDKRKKITRIGIGGPVGSGKTAIIEVITPILIKRGIKPLIITNDIVTTEDAKQVKRTLKGILDEEKILGVETGACPHTAVREDPSMNIAAVEEMEERF-PDSNLIMIE16 ------MTDKST-ARKKITRIGIGGPVGSGKTAIIEVITPILIKRGIKPLIITNDIVTTEDAKQVKRTLKGILDEEKILGVETGACPHTAVREDPSMNIAAVEEMEERF-PESDLIMIE :*: ****:** :: : : :**** * ** : : ::: ***** *****:*** * *: *: : :::::*

H4 S6 H5 S7 H6 S8 H7 | | | | | | | 101 110 120 130 140 150 160 170 180 190 200 1 SGGDNLSATFSPELVDAFIYVIDVSEGGDIPRKGGPGVTRSDFLMVNKTELAPYVGVDLDTMKNDTIKARNGRPFTFANIKTKKGLDEIIAWIKSDLLLEGKTNESASESK 211 2 SGGDNLSATFNPELADFTIFVIDVAEGDKIPRKGGPGITRSDLLVINKIDLAPYVGADLKVMERDSKKMRGEKPFIFTNIRAKEGLDDVIAWIKRNALLED---------- 199 3 SGGDNLSATFNPELADFTIFVIDVAEGDKIPRKGGPGITRSDLLVINKIDLAPYVGADLKVMERDSKKMRGEKPFIFTNIRAKEGLDDVIAWIKRNALLED---------- 199 4 SGGDNLAATFSPELVDFSIYIIDVAQGEKIPRKGGQGMIKSDFFVINKTDLAPYVGASLERMAEDTKVFRGNRPFTFTNLKTDEGLDEVIEWIEQYVFLKGLA-------- 204 5 SGGDNLAATFSPELVDFSIYIIDVAQGEKIPRKGGQGMIKSVLFIINKIDLAPYVGASLEVMERDTLAARGDKPYIFTNLKDEIGLAEVLEWIKTNALLYGLES------- 204 6 SGGDNLSATFSPELADLTIYVIDVAEGEKIPRKGGPGITHSDLLVINKIDLAPYVGASLEVMEADTARMRPVKPYVFTNLKKKVGLETIIEFIIDKGMLGR---------- 205 7 SGGDNLAATFSPDLVDFTIYIIDVAQGEKIPRKAGQGMIKSDLFLINKTDLAPYVGANLDRMREDTLHFRNEDSFIFTNLNNDDNVKEVEEWIRKNFLLEDL--------- 204 8 SGGDNLSATFSPELADLTIYVIDVAEGEKIPRKGGPGITKSDFLVINKTDLAPYVGASLEVMASDTQRMRGDRPWTFTNLKQGDGLSTIIAFLEDKGMLGK---------- 205 9 SGGDNLAATFSPELVDLTLYVIDVAAGDKIPRKGGPGITKSDLLVINKIDLAPMVGADLGIMDRDAKKMRGEKPFVFTNLKTATGLSTVVDFVEHYLPTKVLAS------- 20610 SGGDNLSATFSPELADLLFMLIDVAEGEKIPRKGGPGITHPDMMVINKIDLAPYVGASLEVMEADTAKMRPVKPYVFTNLKEKVGLETIIDFIIDKGMLRR---------- 20511 SGGDNLSATFSPDLADVTIFVIDVAQGEKIPRKGGPGITRSDLLVINKTDLAPFVGADLSVMERDARRMRNGQPFIFTNLMKKENLDGVIGWIEKYALLKNVEEPASLVR- 22512 SGGDNLSATFSPDLVDFSIYIIDVAQGEKIPRKGGQGMIKSDLFIINKVDLAPYVGANVEVMKADTLKSRGNKDFFVTNLKTDEGLKSVADWIEKRLQLALLEE------- 20613 SGGDNLSATFSPDLADVTIFVIDVAQGEKIPRKGGPGISRSDLLVINKTDLAPFVGADLSVMERDARRMRNGQPFIFTNLMKNENLDGVIGWIEKYALLKNIEDPASLVR- 21114 SGGDNLAATFSPELSDLTLYIIDVASGEKIPRKGGPGITKSDLFIINKTDLAPYVGADLAVMEADTRRMRGDKPFVMCNLKTGDGLDQVIAFLKTEGLFRG---------- 21415 SGGDNLTLTFSPALADFYIYVIDVAEGEKIPRKNGPGLVQADILVINKIDLAPYVGASLDVMESDTKVVRGERPYILTNCKTGQGIEELVDMIMRDFLFTHVQPQGEHA-- 22116 SGGDNLTLTFSPALADFYIYVIDVAEGEKIPRKNGPGLVQADILVINKIDLAPYVGASLDVMESDTKVVRGNRPYILTNCKTGQGIEELVDMIMRDFLFTHVQPEGEQA-- 220 ******: ** * * * : :***: * **** * *: ::::** :*** ** * * *: * * : :


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Figure 8


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Figure 9


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Bryant and Stefano CiurliSamyn, Bart Devreese, Jozef Van Beeumen, Paola Turano, Alexander Dikiy, Donald A.

Barbara Zambelli, Massimiliano Stola, Francesco Musiani, Kris De Vriendt, BartGTPase that specifically binds Zn2+

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured

published online November 12, 2004J. Biol. Chem.

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