JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JB.01265-06v1
188/24/8413    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J. K.
Right arrow Articles by Hausinger, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J. K.
Right arrow Articles by Hausinger, R. P.

 Previous Article  |  Next Article 

Journal of Bacteriology, December 2006, p. 8413-8420, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01265-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein{triangledown} ,{dagger}

Jong Kyong Kim,1 Scott B. Mulrooney,2 and Robert P. Hausinger1,2,3*

Cell and Molecular Biology Program,1 Department of Microbiology and Molecular Genetics,2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-43203

Received 10 August 2006/ Accepted 3 October 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Urease is a nickel-containing enzyme that catalyzes the hydrolysis of urea in plants, selected fungi, and many bacteria (10). The bacterial enzyme is especially well studied because it functions as a virulence factor and is implicated in urinary stone formation and gastric ulceration (20). Crystallographic analyses reveal that the three structural subunits (encoded by ureA, ureB, and ureC) of most bacterial ureases associate into a trimer of trimers [({alpha}ß{gamma})3], with each UreC subunit containing a dinuclear nickel active site bridged by a carbamylated lysine (1, 12, 30). Variations on this theme are found in strains of Helicobacter in which the two shorter structural genes are fused and the resulting gene products assemble into a larger macromolecular complex {[({alpha}ß)3]4} (9) and in fungi and plants in which all three genes are fused to yield a single gene product that associates into a homotrimeric or homohexameric enzyme (34). The nickel ions are essential for the urease catalytic mechanism and function to bind and activate both substrate and the hydrolytic water molecule (5, 10).

In addition to the extensive literature related to the enzyme itself, much effort has focused on the steps of urease metallocenter assembly (23). This multistep process is guided by the action of several accessory proteins that, in bacteria, are typically encoded by genes located in the same cluster as the structural genes. Klebsiella aerogenes possesses the best-studied urease activation system involving the ureDABCEFG urease gene cluster, in which UreD, UreF, and UreG form a GTP-dependent molecular chaperone (37) and UreE functions as a metallochaperone by delivering nickel to the urease apoprotein (UreABC) (8, 25, 36). Among these accessory proteins, only UreE and UreG are highly soluble, resulting in the publication of many studies to characterize these proteins (primarily using the recombinant K. aerogenes proteins produced in Escherichia coli or the native proteins from Bacillus pasteurii) at a biochemical and structural level (7, 8, 21, 26, 32, 35, 39). Unlike UreE and UreG, very little is known about UreD and UreF as individual proteins because they are insoluble when overexpressed in E. coli.

Sequence analysis of the urease gene cluster in Bordetella bronchiseptica, a common ureolytic pathogen, suggested that ureE and ureF are fused to form a single gene in this organism (18); however, no protein biochemical studies were carried out to confirm this finding. That study suggested the intriguing idea that the UreEF fusion protein may result in tighter coordination of the functions of the two proteins, ensuring productive incorporation of nickel by preventing premature nickel binding before the correct formation of the active site. We hypothesized that the fusion of K. aerogenes ureE and ureF genes may encode a functional protein and the highly soluble nature of UreE may assist in rendering UreF more soluble as a fusion protein. Here, we characterize the soluble UreEF fusion protein and show that it is functional based on its interactions with other urease components and its participation in urease activation. These studies present the first biochemical analyses of soluble UreF.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction. All restriction digestions and DNA manipulations were performed by following standard procedures (33). Primers were synthesized at Integrated DNA Technologies, Inc. (Coralville, Iowa) for construction of plasmids used in this study (Table 1). Site-directed mutagenesis was performed to introduce two nucleotides (GC) before the stop codon of ureE, using pTBEF (3) as a template and the QuickChange mutagenesis kit (Stratagene). This procedure removed the stop codon of ureE by frameshift, created a new NheI site for easy screening of the mutant plasmid, and inserted two extra amino acids (Ala and Ser) between UreE and UreF. The resulting plasmid (pTBEF-GCins), containing a translational fusion of the ureE and ureF genes, was digested with BstXI and AatII and was cloned into similarly digested pKK17 (7) to yield pKK-EF (containing the entire K. aerogenes urease operon, but with ureE and ureF fused). pKK-{Delta}E and pKK-{Delta}F (containing the complete urease operon with ureE or ureF deleted) were generated by the subcloning procedures described in Table 1, using pACT-KK{Delta}2-136f (25) and pKAU17{Delta}ureF L2 (16), respectively. pET-EF was constructed by cloning the BamHI-AatII fragment of pTBEF-GCins into similarly digested pETH144*{Delta}G, resulting in the insertion of the ureEF fusion gene into pET21 for purification of the fusion protein. To construct a template plasmid for generation of deletion mutants of the UreEF fusion protein, pKK-EF was digested with BamHI and KpnI, and the resulting fragment (covering the ureEF fusion gene and ureG) was cloned into pUC19 to yield pK-EFG. Mutants with deletions at either the N or C terminus of UreF in the UreEF fusion protein were obtained by PCR-based methods by using the primers indicated (Table 1) and Pfu Turbo DNA polymerase (Stratagene). After sequencing, each deletion mutant plasmid was digested with BstXI and KpnI and the appropriate fragment was subcloned into similarly digested pKK-EF to replace the ureEF fusion fragment.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Plasmids and primers used in this study

 
Bacterial strains and culture conditions. E. coli C41(DE3) cells (19) were used as the host for expression for all plasmids. E. coli transformants were cultured in Terrific Broth (TB; Fisher Biotech) supplemented with appropriate antibiotics at 37°C to an optical density at 600 nm of ~0.4, induced with 0.5 mM isopropyl ß-D-thiogalactopyranoside (IPTG; Roche), and harvested after 14 to 16 h. Cultures for urease activity measurements were grown with supplementary NiCl2 in the medium under the same conditions described above.

Preparation of cell extracts for urease and protein assays. Cell pellets were resuspended in 20 mM Tris buffer (pH 7.4) containing 150 mM NaCl and 1 mM EDTA (STE buffer) and disrupted by sonication (Branson Sonifier). Intact cells and debris were removed by centrifugation at 10,000 x g for 20 min at 4°C.

Purification of UreEF fusion protein and UreEF-UreABC complexes. UreEF fusion protein and UreEF-containing protein complexes were purified by using the methods described previously (3). Cell pellets of E. coli C41(DE3) containing pET-EF (for UreEF fusion protein) or pKK-EF (for UreEF-UreABC complexes) were resuspended in buffer A (20 mM Tris [pH 7.8], 500 mM NaCl, 60 mM imidazole) and disrupted by sonication. The cell extracts were obtained by centrifugation at 100,000 x g for 45 min at 4°C and loaded onto an Ni-nitrilotriacetic acid (NTA) (Novagen) column charged with 50 mM NiCl2 and equilibrated with buffer A. The Ni-NTA resin was washed with buffer A until the A280 reached the baseline. Bound proteins were eluted in 20 mM Tris buffer (pH 7.8) containing 500 mM NaCl and 1 M imidazole, and fractions were analyzed by gel electrophoresis. Samples of interest were dialyzed against 20 mM Tris buffer (pH 7.8) containing 85 mM NaCl, 1 mM EDTA, and 20% glycerol. For equilibrium dialysis and UV-visible spectroscopy, the samples were further dialyzed against the identical buffer without EDTA several times to remove EDTA from the samples.

Ni-NTA pull-down assay. Cell pellets of E. coli C41(DE3)[pET-EF] were resuspended in binding buffer (20 mM Tris (pH 7.8), 300 mM NaCl, 60 mM imidazole) and disrupted by sonication. The resulting cell extracts were incubated for 15 min at room temperature with 200 µl of Ni-NTA slurry (50% suspension) equilibrated with the binding buffer. The UreEF-bound resin was washed with 10 bed volumes of the binding buffer twice and incubated with 600 µg K. aerogenes UreD-UreABC complex (purified by Soledad Quiroz according to reference 29) in 1 ml for either 20 min at room temperature or overnight at 4°C. After incubation, the resin was washed in the same manner as the first wash, followed by elution with 20 mM Tris (pH 7.8) containing 300 mM NaCl and 1 M imidazole. Eluted proteins were analyzed by polyacrylamide gel electrophoresis PAGE).

Polyacrylamide gel electrophoresis and Western blot analysis. Sodium dodecyl sulfate (SDS)-PAGE was carried out for protein analysis with the buffers described by Laemmli (15), and utilized 15%, 13.5%, or 12% polyacrylamide running gels with 4.5% stacking gels. The gels were either stained with Coomassie brilliant blue (Sigma) or electroblotted onto Immobilon-P polyvinylidene difluoride membrane (Millipore), probed with anti-K. aerogenes UreE antibodies (17) or anti-K. aerogenes UreG antibodies (21), and visualized with anti-rabbit immunoglobulin G-alkaline phosphatase conjugates (Sigma).

Urease and protein assays. Urease activity was measured by quantifying the rate of ammonia release from the hydrolysis of urea by formation of indophenol, which was monitored at 625 nm (38). One unit of urease activity was defined as the amount of enzyme required to hydrolyze 1 µmol of urea per min at 37°C. The standard assay buffer contained 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid; pH 7.8) and 50 mM urea. Protein concentrations were determined by using a commercial assay (Bio-Rad) with bovine serum albumin as the standard (2).

Gel filtration chromatography. The native molecular weight of the purified UreEF fusion protein was estimated by gel filtration chromatography using two different columns, a KW-804 (8 by 300 mm; Shodex) and a Protein-Pak 125 column (7.8 by 300 mm; Waters), connected to a Waters Breeze chromatography system. Isocratic elution utilized 20 mM Tris buffer (pH 7.8) containing 200 mM NaCl and 0.1 mM EDTA. Mixtures of protein molecular weight standards (Bio-Rad) were used to standardize the columns.

Equilibrium dialysis. Protein samples (10 µM in 50 mM Tris buffer (pH 7.8) containing 85 mM NaCl) were dialyzed against the identical buffer containing varied concentrations of 63NiCl2 (1,445 mCi/mmol; Du Pont NEN Research Products, Inc., Wilmington, DE), using an equilibrium microvolume dialyzer (Hoefer Scientific Products, San Francisco, CA) equipped with dialysis membranes (molecular weight cutoff of 10,000; Spectra/Por). After overnight equilibration at 4°C, an aliquot from each compartment was measured for radioactivity by using a Beckman LS7000 liquid scintillation system and Bio-Safe II scintillation fluid (Research Products International Corp.). Data were fitted to a two-cooperative-site Adair equation, as described previously (7).

UV-visible spectroscopy. Electronic absorption spectra of wild-type UreE (100 µM monomer) and UreEF fusion proteins (47 µM monomer) in the presence of increasing concentrations of varied divalent metal ions were analyzed on a Shimadzu 2401PC spectrophotometer with a 1-ml cuvette in 50 mM Tris buffer (pH 7.8) containing 85 mM NaCl.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of recombinant K. aerogenes UreEF fusion protein in E. coli. To generate the K. aerogenes UreEF fusion protein and determine whether the fusion protein is functional in terms of urease activation, the ureE and ureF genes were translationally fused by adding two nucleotides (GC) before the stop codon of ureE in pKK17 (containing the entire K. aerogenes urease operon [see Table 1]). This mutation eliminated the stop codon of ureE by frameshift and inserted two extra amino acids (Ala and Ser) between UreE and UreF, as illustrated in Fig. 1A. The resulting plasmid, pKK-EF, was transformed into E. coli C41(DE3) cells for overexpression of UreEF. The cell extracts of cultures containing pKK-EF did not contain the UreE band like that detected in cell extracts of E. coli C41(DE3)[pKK17], but they possessed a new protein band with an apparent molecular mass of ~43 kDa that was the size expected for the product arising from the fusion of ureE and ureF genes (Fig. 1B, left). To confirm that the new protein band is the UreEF fusion protein, a Western blot analysis with anti-UreE antibody was performed. A single immunoreactive band was detected in E. coli C41(DE3)[pKK-EF] cell extracts, which is at the same position as the band visualized by Coomassie blue staining (Fig. 1B, right). As expected, E. coli C41(DE3)[pKK17] cell extracts had a single immunoreactive band running at the position of UreE. These results indicate that soluble K. aerogenes UreEF protein can be produced in E. coli.


Figure 1
View larger version (68K):
[in this window]
[in a new window]

  		FIG. 1. Expression of the K. aerogenes ureEF fusion gene in E. coli. (A) Nucleotide sequence of the junction of the translationally fused ureE and ureF genes in pKK-EF. Addition of the two nucleotides (GC) before the stop codon of ureE is shown in a shaded box. This creates an NheI site (underlined) encoding the two extra amino acids (Ala and Ser). (B) (Left panel) cultures carrying pKK17 or pKK-EF were induced with 0.5 mM IPTG. The cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. Lanes: M, molecular mass markers; 1, cell extracts of E. coli C41(DE3)[pKK17]; 2, cell extracts of E. coli C41(DE3)[pKK-EF]. (Right panel) Western blot with anti-UreE antibodies for samples described in the lanes of the left panel.

 
Copurification of the UreEF fusion protein with other urease components. UreE contains a His-rich sequence (i.e., 10 His residues in its C-terminal 15 residues) that allows for one-step purification by Ni-NTA affinity chromatography. In addition to facilitating purification, this chromatography approach may be employed for detecting protein-protein interactions by examining the proteins that copurify with the target protein. To explore the possible protein-protein interactions that occur among urease components (both structural and accessory proteins) in vivo, cell extracts of E. coli C41(DE3) cultures containing pKK17 (containing the wild-type urease gene cluster) or pKK-EF (possessing the gene cluster with ureE and ureF fused) were subjected to Ni-NTA column chromatography. As shown in Fig. 2, purified protein from E. coli C41(DE3)[pKK17] cell extracts was exclusively UreE, indicating that UreE did not exhibit stable interactions with other urease components. In contrast, when starting with E. coli C41(DE3)[pKK-EF] the UreEF fusion protein copurified with the UreABC structural proteins, two other urease accessory proteins (UreD and UreG), and an unknown protein with a molecular mass of ~20 kDa. This unknown protein band ran at a similar position to UreE on the gel, but it was not a degradation product of the UreEF based on the lack of immunoreactivity using anti-UreE antibody (data not shown). These results suggest that the UreEF fusion protein is capable of establishing stable interactions with other urease components in vivo, forming a UreD(EF)G-UreABC complex.


Figure 2
View larger version (72K):
[in this window]
[in a new window]

  		FIG. 2. Copurification of other urease components with the UreEF fusion protein. E. coli C41(DE3) cultures harboring pKK17 or pKK-EF were induced with 0.5 mM IPTG, and the cell extracts were subjected to Ni-NTA column chromatography. Eluted fractions of the purified proteins were analyzed by SDS-PAGE. Lanes: M, molecular weight markers; 1, cell extracts of E. coli C41(DE3)[pKK17]; 2 to 4, fractions 2 to 4 of eluted proteins from these extracts; 5, cell extracts of E. coli C41(DE3)[pKK-EF]; 6 to 8, fractions 2 to 4 of eluted proteins from these extracts; Std (standard), purified K. aerogenes UreDFG-UreABC complex.

 
In vitro interactions of UreEF with UreD-UreABC complex. To determine whether the UreEF fusion protein can interact with purified K. aerogenes UreD-UreABC complex in vitro, the Ni-NTA pull-down assay was performed using two different incubation times (Fig. 3). For both conditions tested, UreEF bound to UreD-UreABC complex in vitro, but at substoichiometric levels of the complex that did not equate to the input amount; i.e., ~10% of the added UreD-UreABC complex was estimated to bind UreEF. The observed low stoichiometry suggests that additional cellular components such as molecular chaperones might facilitate the interaction of these proteins within the cell. The binding of UreEF to the UreD-UreABC complex was specific as shown by the lack of interaction with contaminant proteins present in the purified UreD-urease apoprotein complex (lane 2).


Figure 3
View larger version (100K):
[in this window]
[in a new window]

  		FIG. 3. In vitro interactions of UreEF with UreD-UreABC complex. E. coli C41(DE3)[pET-EF] cell extracts were incubated with Ni-NTA resin, and the UreEF-bound resin was incubated with purified UreD-urease apoprotein complex at two different conditions. Eluted protein complexes were analyzed by SDS-PAGE. Lanes: M, molecular mass markers; 1, extracts of E. coli C41(DE3)[pET-EF] cells; 2, purified K. aerogenes UreD-UreABC complex; 3, eluted proteins after incubation of UreEF-bound Ni-NTA resin with UreD-UreABC complex for 20 min at room temperature; 4, eluted proteins after incubation of the mixture overnight at 4°C.

 
Effects of the UreEF fusion on urease activity. The observation that the UreEF fusion protein interacts with other urease components indicated that the fusion protein might be functional in urease activation. To assess the effects of the fusion on individual functions of UreE and UreF, urease activities were measured in cell extracts of E. coli C41(DE3) containing pKK17 or pKK-EF and grown under high or limiting nickel conditions. The effect of the fusion on the function of UreF was examined at high nickel concentration (5 mM), where the effect was likely to be most easily visualized, by comparing it to the activity levels found in cells carrying the wild-type urease operon (pKK17). In contrast, the assessment of UreE function in the UreEF fusion protein was examined using a limiting nickel concentration (0.5 mM) in which the metallochaperone role is more pronounced.

At a high nickel concentration, the E. coli C41(DE3)[pKK-EF] cell extracts had a similar level of activity to that found in E. coli C41 (DE3)[pKK17] cell extracts (Fig. 4A), which suggested that the UreF portion of the UreEF fusion protein is fully functional, in good agreement with its ability to form a stable complex with other urease components (Fig. 2 and 3). Deletion of the ureE gene from the urease operon (creating plasmid pKK-{Delta}E) decreased the activity by ~40%, compared to that in E. coli C41(DE3)[pKK17]. As expected, very low activity was observed in the case of deletion of the ureF gene from the cluster (generating plasmid pKK-{Delta}F), which is consistent with our previous studies showing that UreF is essential for urease activation (16). At limiting nickel conditions, however, a significant decrease in urease activity was observed in extracts of E. coli C41(DE3)[pKK-EF] compared to that found in control cell extracts. In fact, the level of activities was comparable to that detected in extracts of E. coli C41(DE3)[pKK-{Delta}E] cells, which indicated that the function of UreE in the UreEF fusion protein was greatly compromised by the fusion (Fig. 4B).


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 4. Urease activity in recombinant E. coli C41(DE3) cell extracts containing the indicated plasmids. The cells were grown with 5 mM Ni2+ (A) or 0.5 mM Ni2+ (B) in TB medium. Error bars represent the standard deviation for three separate determinations.

 
Characterization of the purified recombinant K. aerogenes UreEF fusion protein. Recombinant K. aerogenes UreEF fusion protein was purified by Ni-NTA affinity chromatography from E. coli C41(DE3) cells containing pET-EF, which includes only the ureEF fusion gene in the pET21 expression vector. Typical yields of the protein were 20 to 30 mg per liter of culture. Using the purified UreEF fusion protein, native molecular weight estimations were performed by gel filtration chromatography with two different size exclusion columns, KW-804 and ProteinPak-125, as described in Materials and Methods. Data from the chromatographic analysis with KW-804 allowed estimation of the native size of the UreEF fusion protein to be 43.0 ± 1.6 kDa, consistent with a monomeric structure. Analysis by using the ProteinPak-125 column gave similar results (51.7 ± 0.9 kDa). These findings support the notion that the UreEF fusion protein is monomeric, rather than the dimer that was anticipated by the dimeric structure of wild-type UreE (35). These results indicate that the fusion of UreE and UreF prevented dimerization of UreE, at least partially accounting for the perturbed UreE function of the UreEF fusion protein (Fig. 4B).

The monomeric structure of the UreEF fusion protein led us to examine the metal binding properties of the UreEF fusion protein and to compare them to those of wild-type UreE protein. The number of nickel ions that bound to the UreEF fusion protein was determined for a range of nickel concentrations by equilibrium dialysis, and the data were fitted to a two-cooperative-site Adair equation (7). The UreEF fusion protein binds 3.32 ± 0.17 mol of nickel per mol of monomer with a Kd1 of 64.0 ± 10.4 µM and a Kd2 of 7.0 ± 2.1 µM, which compares to about 6 nickel ions per wild-type UreE homodimer, with an average Kd of 9.6 µM (17). The metal binding properties of the UreEF fusion protein were further characterized by UV-visible spectroscopy and compared to the spectra of metal-bound wild-type UreE. Many studies have focused on spectroscopic metal binding analysis of H144* UreE that lacks the His-rich carboxyl terminus (3, 6, 8, 25), but no UV-visible studies have been reported on wild-type UreE until this analysis. As illustrated by Fig. S1 in the supplemental material, the UreEF fusion protein exhibited distinct UV-visible spectra from those of wild-type UreE for all three metal ions. Binding of Cu2+ to wild-type UreE resulted in a significant increase in absorbance at 366 nm, caused by a thiolate-to-Cu2+ charge-transfer transition involving Cys79 (6), while this feature is completely absent in the corresponding spectrum of the UreEF fusion protein. A plot of the absorbance changes at 366 nm versus the Cu2+ concentration exhibited sigmoidal effects for wild-type UreE, with a midpoint Cu2+ concentration of 2.60 ± 0.04 mol of Cu2+/mol of monomer. In contrast, only a broad feature near 700 nm along with a uniform increase throughout the spectrum was generated when cupric ions were added to the fusion protein. There was no turbidity detected in this sample throughout the titration, ruling out protein aggregation. The addition of Ni induced an absorbance increase at 366 nm as well as an increase throughout the spectrum in wild-type UreE, but neither of these features is as pronounced in the UreEF fusion protein. Similar differences were noted when Co2+ was added to the two proteins. The lack of thiolate-to-metal charge transfer transitions suggests that, unlike the case for wild-type UreE (6), the Cys79 of the UreEF fusion protein is not accessible for metal binding in solution.

Effects of UreEF deletions on interactions with other urease components and function as a molecular chaperone. To gain further insight into the interactions of UreEF fusion protein with other urease components (Fig. 2) in relation to its function, N- or C-terminal deletion mutants of UreEF were generated by in-frame truncation of amino acid residues of UreF, as illustrated in Fig. 5A. As shown in Fig. 5B, deletion of 24 residues at the UreF N terminus did not affect its interactions with other urease components in the complex. The urease subunits and UreD were clearly visible in this sample, while UreG was not distinguishable on the gel because of the lower band intensity and the presence of other nonspecific proteins similar in molecular mass to UreG. However, UreG was detected on a Western blot with anti-UreG antibodies using the same sample, suggesting that UreG is less stably associated with the urease apoprotein complex than UreD and UreEF (data not shown). Compared to the case of the UreEF control, much lower levels of N{Delta}24 mutant protein were purified due to its decreased solubility caused by the truncation. The small amount of truncated UreEF protein led to a near stoichiometric correlation with the other urease components (UreABC and -D), indicating that the deletion mutant protein is primarily in the complex [UreD(N{Delta}24/EF)-UreABC], rather than a mixture of free protein and protein in the complex, as observed in the case of the UreEF control.


Figure 5
View larger version (67K):
[in this window]
[in a new window]

  		FIG. 5. Interactions of the UreEF deletion mutants with other urease components. (A) Schematic diagram of the N- or C-terminal deletion mutants of the UreEF fusion protein. Deletions are denoted by the black rectangles starting and ending at the designated amino acid sequence number of UreF. The linker sequence generated by the fusion of UreE and UreF is indicated by AS (Ala and Ser). (B) Interactions of UreEF deletion mutants with other urease components. E. coli cultures expressing the entire urease gene cluster containing each UreEF deletion mutant were used for monitoring protein-protein interactions, following the same procedures described in the legend to Fig. 2. (C) Expression of UreEF deletion mutants. Deletion mutant proteins in cell extracts were visualized by Western blot analysis with anti-UreE antibodies. The sample sizes were adjusted to provide comparable amounts of the UreEF fusion protein.

 
As opposed to the N-terminal deletion, truncation of the C terminus of UreF totally abolished the interactions with other urease components in the complex. As in the case with N{Delta}24, the C{Delta}49 mutant produced very little soluble protein, which resulted in much smaller protein levels after Ni-NTA column purification, and a major nonspecific band (~60 kDa) was more prominent (Fig. 5B). We also generated the C{Delta}61 mutant, but encountered a similar problem involving decreased solubility of the severely truncated protein (data not shown). Nevertheless, all deletion mutants tested produced some soluble proteins, as detected by Western blot analysis with anti-UreE antibodies (Fig. 5C).

The next question we asked was how the deletions of the UreEF protein would affect its function in urease activation. Urease activities were measured in E. coli cell extracts containing each deletion mutant. As shown in Table 2, the pKK-EF control cell extracts exhibited a high urease activity, consistent with the results in Fig. 4A, but with even higher activity due to slight changes in culture conditions. As expected, both the C{Delta}15 and C{Delta}49 mutants showed almost no activities, in good agreement with their inability to interact with other urease components in the complex. Surprisingly, the N{Delta}24 mutant also failed to activate urease and conferred low activity comparable to that of the C-terminal deletion mutants of UreEF. These results suggest that the N terminus of UreF is not required for protein-protein interactions in the complex, but it is essential for UreF to function as a molecular chaperone in the process of urease activation.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Urease activity in recombinant E. coli C41(DE3) cell extracts containing the indicated UreEF deletion mutants grown with 5 mM NiCl2

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Urease has served as a paradigm for understanding the activation of many metalloenzymes that require accessory proteins for the production of catalytic activity (14). K. aerogenes possesses the best-characterized urease metallocenter assembly system, and several complexes of the UreABC apoenzyme plus accessory proteins have been described (22, 27, 28). The essential urease accessory protein UreF has been an elusive subject for biochemical and structural analyses because it is insoluble when overexpressed in E. coli. A fusion protein with the maltose-binding protein was produced in E. coli (13); however, no further characterization of the fusion protein was described, and, when separated from the maltose-binding domain by digestion with factor Xa, the UreF protein was unstable. To overcome this hurdle, we generated the K. aerogenes UreEF fusion protein by creating a translational fusion of ureE and ureF genes.

The translationally fused UreEF protein yielded a soluble and active version of UreF on the basis of (i) its ability to form a UreD(EF)G-UreABC complex, (ii) its ability to activate urease in vivo, and (iii) its capacity to bind UreD-UreABC to form a UreD(EF)-UreABC complex in vitro. While the UreF portion of the UreEF fusion protein appears to be fully functional, the fusion significantly affected the role of the UreE portion of the UreEF fusion protein. The monomeric UreEF differs from the dimeric UreE, which contains an essential interfacial pair of His96 residues needed for Ni incorporation into the active site of urease (7, 8, 35). UV-visible spectroscopy reveals another aspect of changes in the UreE portion of the UreEF fusion protein: i.e., Cys79 of UreE is not accessible for metal binding. The seclusion of this residue may arise from physical blocking by UreF, by a conformational change within UreE caused by UreF, or by changes in the pKa of the sulfhydryl group of Cys79 in the fusion protein.

Although the fusion of UreE to UreF significantly altered the oligomeric structure and metal binding properties of UreE, it provided a convenient tool to examine the protein-protein interactions between UreEF and other urease components by Ni-NTA affinity chromatography. In particular, we found that UreEF copurified with UreD, UreG, and the UreABC apoprotein, consistent with in vivo formation of a UreD(EF)G-UreABC complex. In addition, UreEF binds to UreD-UreABC to form a UreD(EF)-UreABC complex in vitro. Furthermore, UreEF exhibits only weak or transient interactions with UreG based on analyses using the Ni-NTA pull-down assay with UreEF and UreG as the only urease components (data not shown). These results can be compared with previous efforts to examine interactions among the urease components. A yeast two-hybrid analysis of the Helicobacter pylori system indicated that UreF interacts with UreH (corresponding to UreD in other microorganisms), but not with UreG (31). A similar approach with the Proteus mirabilis urease components also revealed that UreF interacts with UreD (11). The yeast two-hybrid results complement our earlier biochemical and immunological findings that the K. aerogenes UreD-UreABC complex forms independently of the presence of other accessory proteins (27), and the presence of UreF masks the immunoreactivity of UreD bound to UreABC (22). Together, these results suggest that UreD may be crucial for recruitment of the UreF to the apoprotein complexes; i.e., with regard to the present study, the UreD tethers UreEF to UreABC. Interactions also may exist directly between UreF and UreABC. For example, chemical cross-linking/tryptic digestion/mass spectrometry studies showed the existence of a chemical cross-link between the UreF N terminus (residues 1 to 7) and UreB Lys76 of the UreDF-UreABC complex along with evidence for a significant conformational change between the UreD-UreABC and the UreDF-UreABC complexes (4). While we detected weak or transient interactions between UreG and UreEF when studying a mixture of the individual proteins (data not shown), it remains unclear how UreG associates with the UreDF-UreABC complex. For example, we cannot rule out the possibility of additional stabilizing interactions between UreG and the other urease components in the complex.

Although we were unable to identify the exact interaction sites among different urease components in the UreD(EF)- and UreD(EF)G-UreABC complexes, the UreEF deletion mutant study provided evidence for distinct subdomains of UreF with separate functions related to urease activation. We hypothesize that UreF acts in a two-step process in which the C terminus recognizes and binds to UreD-UreABC, perhaps inducing a conformational change in this complex, thus positioning the N terminus of UreF in an orientation that can interact with other urease components to achieve urease activation. This scenario fits well with the results from the UreEF deletion mutant study, in which the N{Delta}24 mutant (with an intact UreF C terminus) interacts with the UreD-UreABC complex, but does not lead to its activation, while the C-terminal deletion mutants fail to form any apoprotein complex. This proposal is consistent with all other available data on protein interactions involving UreF described above.

In summary, we demonstrate that the translational fusion of K. aerogenes ureE to ureF provides a soluble and functional form of UreF that is useful for further biochemical and structural studies.


   ACKNOWLEDGMENTS
 
We thank Soledad Quiroz for providing the purified UreD-urease apoprotein.

This work was supported by National Institutes of Health grant DK45686.


   FOOTNOTES
 
* Corresponding author. Mailing address: 6193 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 48824-4320. Phone: (517) 355-6463, ext. 1610. Fax: (517) 353-8957. E-mail: hausinge{at}msu.edu. Back

{triangledown} Published ahead of print on 13 October 2006. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Benini, S., W. R. Rypniewski, K. S. Wilson, S. Miletti, S. Ciurli, and S. Mangani. 1999. A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels. Structure 7:205-216.[Medline]
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
  3. Brayman, T. G., and R. P. Hausinger. 1996. Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus. J. Bacteriol. 178:5410-5416.[Abstract/Free Full Text]
  4. Chang, Z., J. Kuchar, and R. P. Hausinger. 2004. Chemical crosslinking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease. J. Biol. Chem. 279:15305-15313.[Abstract/Free Full Text]
  5. Ciurli, S., S. Benini, W. R. Rypniewski, K. S. Wilson, S. Miletti, and S. Mangani. 1999. Structural properties of the nickel ions in urease: novel insights into the catalytic and inhibition mechanisms. Coord. Chem. Rev. 190-192:331-355.[CrossRef]
  6. Colpas, G. J., T. G. Brayman, J. McCracken, M. A. Pressler, G. T. Babcock, L.-J. Ming, C. M. Colangelo, R. A. Scott, and R. P. Hausinger. 1998. Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein. J. Biol. Inorg. Chem. 3:150-160.[CrossRef]
  7. Colpas, G. J., T. G. Brayman, L.-J. Ming, and R. P. Hausinger. 1999. Identification of metal-binding residues in the Klebsiella aerogenes urease nickel metallochaperone, UreE. Biochemistry 38:4078-4088.[CrossRef][Medline]
  8. Colpas, G. J., and R. P. Hausinger. 2000. In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE. J. Biol. Chem. 275:10731-10737.[Abstract/Free Full Text]
  9. Ha, N.-C., S.-T. Oh, J. Y. Sung, K.-A. Cha, M. H. Lee, and B.-H. Oh. 2001. Supramolecular assembly and acid resistance of Helicobacter pylori urease. Nature Structure Biol. 8:505-509.
  10. Hausinger, R. P., and P. A. Karplus. 2001. Urease, p. 867-879. In K. Wieghardt, R. Huber, T. L. Poulos, and A. Messerschmidt (ed.), Handbook of metalloproteins. John Wiley & Sons, Ltd., West Sussex, United Kingdom.
  11. Heimer, S. R., and H. L. T. Mobley. 2001. Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology. J. Bacteriol. 183:1423-1433.[Abstract/Free Full Text]
  12. Jabri, E., M. B. Carr, R. P. Hausinger, and P. A. Karplus. 1995. The crystal structure of urease from Klebsiella aerogenes. Science 268:998-1004.[Abstract/Free Full Text]
  13. Kim, K. Y., C. H. Yang, and M. H. Lee. 1999. Expression of the recombinant Klebsiella aerogenes UreF protein as a MalE fusion. Arch. Pharm. Res. 22:274-278.[Medline]
  14. Kuchar, J., and R. P. Hausinger. 2004. Biosynthesis of metal sites. Chem. Rev. 104:509-526.[CrossRef][Medline]
  15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.[CrossRef][Medline]
  16. Lee, M. H., S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger. 1992. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J. Bacteriol. 174:4324-4330.[Abstract/Free Full Text]
  17. Lee, M. H., H. S. Pankratz, S. Wang, R. A. Scott, M. G. Finnegan, M. K. Johnson, J. A. Ippolito, D. W. Christianson, and R. P. Hausinger. 1993. Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly. Protein Sci. 2:1042-1052.[Abstract]
  18. McMillan, D. J., M. Mau, and M. J. Walker. 1998. Characterization of the urease gene cluster in Bordetella brochiseptica. Gene 208:243-251.[CrossRef][Medline]
  19. Miroux, B., and J. E. Walker. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane protein and globular proteins at high levels. J. Mol. Biol. 260:289-298.[CrossRef][Medline]
  20. Mobley, H. L. T., M. D. Island, and R. P. Hausinger. 1995. Molecular biology of microbial ureases. Microbiol. Rev. 59:451-480.[Abstract/Free Full Text]
  21. Moncrief, M. B. C., and R. P. Hausinger. 1997. Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease. J. Bacteriol. 179:4081-4086.[Abstract/Free Full Text]
  22. Moncrief, M. B. C., and R. P. Hausinger. 1996. Purification and activation properties of UreD-UreF-urease apoprotein complexes. J. Bacteriol. 178:5417-5421.[Abstract/Free Full Text]
  23. Mulrooney, S. B., and R. P. Hausinger. 2003. Nickel uptake and utilization by microorganisms. FEMS Microbiol. Rev. 27:239-261.[CrossRef][Medline]
  24. Mulrooney, S. B., H. S. Pankratz, and R. P. Hausinger. 1989. Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease. J. Gen. Microbiol. 135:1769-1776.[Medline]
  25. Mulrooney, S. B., S. K. Ward, and R. P. Hausinger. 2005. Purification and properties of the Klebsiella aerogenes UreE metal-binding domain, a functional metallochaperone of urease. J. Bacteriol. 187:3581-3585.[Abstract/Free Full Text]
  26. Neyroz, P., B. Zambelli, and S. Ciurli. 2006. Intrinsically disordered structure of Bacillus pasteurii UreG as revealed by steady-state and time-resolved fluorescence spectroscopy. Biochemistry 45:8918-8930.[CrossRef][Medline]
  27. Park, I.-S., M. B. Carr, and R. P. Hausinger. 1994. In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly. Proc. Natl. Acad. Sci. USA 91:3233-3237.[Abstract/Free Full Text]
  28. Park, I.-S., and R. P. Hausinger. 1995. Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation. J. Bacteriol. 177:1947-1951.[Abstract/Free Full Text]
  29. Park, I.-S., and R. P. Hausinger. 1996. Metal ion interactions with urease and UreD-urease apoproteins. Biochemistry 35:5345-5352.[CrossRef][Medline]
  30. Pearson, M. A., L. O. Michel, R. P. Hausinger, and P. A. Karplus. 1997. Structure of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease. Biochemistry 36:8164-8172.[CrossRef][Medline]
  31. Rain, J.-C., L. Selig, H. de Reuse, V. Battaglia, C. Reverdy, S. Simon, G. Lenzen, F. Petel, J. Wojcik, V. Schächter, Y. Chemama, A. Labigne, and P. Legrain. 2001. The protein-protein interaction map of Helicobacter pylori. Nature 409:211-215.[CrossRef][Medline]
  32. Remaut, H., N. Safarof, S. Ciurli, and J. Van Beeumen. 2001. Structural basis for Ni2+ transport and assembly of the urease active site by the metallo-chaperone UreE from Bacillus pasteurii. J. Biol. Chem. 276:49365-49370.[Abstract/Free Full Text]
  33. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  34. Sheridan, L., C. M. Wilmont, K. D. Cromie, P. van der Logt, and S. E. V. Phillips. 2002. Crystallization and preliminary X-ray structure determination of jack bean urease with a bound antibody fragment. Acta Crystallogr. D 58:374-376.[CrossRef][Medline]
  35. Song, H. K., S. B. Mulrooney, R. Huber, and R. P. Hausinger. 2001. Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation. J. Biol. Chem. 276:49359-49364.[Abstract/Free Full Text]
  36. Soriano, A., G. J. Colpas, and R. P. Hausinger. 2000. UreE stimulation of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein complex. Biochemistry 39:12435-12440.[CrossRef][Medline]
  37. Soriano, A., and R. P. Hausinger. 1999. GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins. Proc. Natl. Acad. Sci. USA 96:11140-11144.[Abstract/Free Full Text]
  38. Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39:971-974.[CrossRef]
  39. Zambelli, B., M. Stola, F. Musiani, K. De Vriendt, B. Samyn, B. Devreese, J. Van Beeumen, A. Dikiy, D. A. Bryant, and S. Ciurli. 2005. UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+. J. Biol. Chem. 280:4684-4695.[Abstract/Free Full Text]

Journal of Bacteriology, December 2006, p. 8413-8420, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.01265-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Other Versions of this Article:
JB.01265-06v1
188/24/8413    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J. K.
Right arrow Articles by Hausinger, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J. K.
Right arrow Articles by Hausinger, R. P.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS