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Colonization of Helicobacter pylori in acidic stomach requires the activity of urease, which hydrolyzes urea into ammonia and carbon dioxide. To become enzymatically active, the apo-urease undergoes a post-translation carbamylation of an active-site lysine residue, followed by insertion of two nickel ions essential for metal catalysis to the active site. In H. pylori, the urease consists of two chains, UreA and UreB, and the maturation process requires the participation of four urease accessory proteins - UreE, UreF, UreG, and UreH (UreH is an ortholog of UreD found in other bacterial species). It is postulated that the apo-urease either bind a pre-formed UreG/UreF/UreH complex, or the individual urease accessory proteins sequentially to form a pre-activation complex. The Ni-binding protein UreE then interacts with the UreG of the complex, and triggers the GTP-dependent activation of urease.
How these urease accessory proteins interact with each other and with the urease to form the activation complex is poorly understood, partly because of the lack of high-resolution structures available for these urease accessory proteins. Until recently, the only urease accessory protein whose structure is available is UreE. We have recently determined the crystal structure of UreF, and have obtained a preliminary structure of the UreF/UreH complex. The novel structural information allows us to use the protein engineering approach to address the following questions: (1) Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease? (2) Is the interaction between UreF and UreH essential to the maturation of urease? (3) Where is the interacting surface on the UreF/UreH complex that are responsible for binding UreG and the urease? As we have already obtained crystals of the UreG/UreF/UreH complex that diffract to good resolution, we propose to determine the crystal structure of this ternary complex. Through this work, the structures of all urease accessory proteins involved in urease maturation will be available, and together with the mutagenesis data, we will have a better understanding of how the urease accessory proteins associate with the urease to form the pre-activation complex for the maturation of urease.
1. The project objectives
The activation of urease requires the concerted participation of four accessory proteins UreE, UreF, UreG, and UreH (UreH is the H. pylori ortholog of UreD found in other species). How UreF, UreH, UreG, and the urease interact with each other to form the activation complex is poorly understood.
Here is a summary of the main questions we asked:
(1) Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?
We have determined the crystal structure of H. pylori UreF and UreF/UreH complex. Both of them exist as dimers in the crystal structure. Moreover, we showed that H. pylori UreF/UreH complex exists as a 2:2 dimer in solution. Based on the crystal structures, we will introduce mutations in the dimeric interface to break the dimerization of H. pylori UreF/UreH complex, and test if these mutations affect the in vivo maturation of urease.
(2) Is the interaction between UreF and UreH essential to the maturation of urease?
Our preliminary data showed that the truncation of the C-terminal residues of UreF breaks the UreF/UreH complex, and abolishes the activation of urease. To further investigate the functional importance of UreF/UreH interaction in urease maturation, we will introduce mutations in the interface of the UreF/UreH complex, and test if these mutations affect the formation of UreF/UreH complex, and in vivo maturation of urease.
(3) How does the UreF/UreH complex interact with UreG and the urease?
The UreF/UreH is also known to form bigger complexes with UreG, and with the urease. How UreF/UreH complex associates with UreG and the urease to form the activation complex for the maturation of urease is poorly understood. Based on the crystal structure of the UreF/UreH complex we solved, we propose to perform scanning mutagenesis to map the surface on UreF and UreH for interaction with UreG and with the urease. The mutants' ability to form complex with UreG and the urease will be correlated with their ability to activate urease in vivo.
(4) What is the structure of UreG/UreF/UreH complex?
We have already obtained crystals of UreG/UreF/UreH complex. We propose to solve the high-resolution structure of the complex by X-ray crystallography. The structure will provide the first high-resolution structure of how these urease accessory proteins interact with each other.
Through this work, we will determine the crystal structure of UreF/UreH/UreG complex. Together with the mutagenesis data and the in vivo urease activation assay, our proposed work will contribute a significant step towards a better understanding on the structure-function relationship of these urease accessory proteins.
To test whether the dimerization of the H. pylori UreF/UreH complex is essential to the maturation of urease
To investigate the functional interaction between UreF and UreH by mutagenesis.
To map the interaction surface on the UreF/UreH complex for binding UreG and the urease by scanning mutagenesis.
To determine the structure of UreG/UreF/UreH complex by X-ray crystallography
2. Background of research
Urease accessory proteins are required for in vivo maturation of urease
Urease is a nickel-containing enzyme that hydrolyzes urea into ammonia and carbamic acid, which decomposes spontaneously into carbonic acid and ammonia .
NH2-CO-NH2 + H2O ® NH3 + NH2-COOH
NH2-COOH + H2O ® NH3 + H2CO3
Colonization of Helicobacter pylori, a gram-negative pathogenic bacterium, in the human stomach requires the activity of urease. The ammonia release due to the hydrolysis of urea neutralizes the gastric acid, and allows the pathogen to survive in the acidic environment of the stomach.
The structure of urease has been determined, and it consists of ¡, ¢ and subunits [2-5]. In H. pylori, the ureA gene encodes the b and g subunits as a fusion protein, and the ureB gene encodes the a-subunit. In order for urease to become enzymatically active, the apo-enzyme must undergo post-translational carbamylation of an active site lysine residue, and the insertion of two nickel ions. The in vivo maturation of urease requires the participation of four urease accessory proteins, UreE, UreF, UreG and UreH(UreD). (UreH is the H. pylori ortholog of UreD found in other species)  (Fig. 1).
Crystal structure of UreE and mutagenesis studies suggest that UreE is involved in binding a nickel ion at the dimeric interface, and in delivery of nickel to the urease [6-9]. Sequence analysis indicated that UreG is a SIMIBI class GTPase. Urease activation was inhibited by addition of non-hydrolysable GTP analog, suggesting that GTPase activity of UreG is essential for urease activation . The structure of UreG is not known, but it is predicted to be similar to that of HypB, a hydrogenase maturation factor responsible for nickel delivery . UreE stimulates GTP-dependent activation of urease [7, 10]. UreE and UreG were reported to form complex in a zinc dependent manner .
UreF and UreH(UreD) play pivotal roles in the formation of activation complex for the maturation of urease. UreF was reported to form complex with UreH(UreD) [13-16], and the two proteins interact with UreG to form heterotrimeric complex UreG/UreF/UreH(UreD) [16, 17]. It has been shown that apo-urease forms complex with UreH(UreD), UreF/UreH(UreD), and UreG/UreF/UreH(UreD) [16-19]. It has been proposed that the apo-urease may undergo conformational changes upon binding of UreF/UreH to facilitate the diffusion of nickel ion and carbon dioxide into the active site [20, 21]. By addition of excess carbon dioxide and nickel ion, apo-urease can also be activated in vitro without urease accessory proteins . On the other hand, addition of nickel ion can partially activate apo-urease in the absence of either UreE, UreF or UreG but not in the absence of UreH(UreD) . The formation of complex with UreF/UreH(UreD) and UreG/UreF/UreH(UreD) can increase the rate and the level of in vitro activation of urease [10, 19].
The formation of activation complex for the maturation of urease involves protein-protein interaction among the urease accessory proteins and the urease. However, structure-function studies of how these urease accessory proteins interact with each other was only poorly understood. One obstacle was that expression of UreH(UreD) alone in E. coli resulted in the formation of inclusion bodies. Recently, Hausinger's group has successfully expressed soluble K. aerogenes UreD in fusion with the maltose binding protein (MBP-UreD), which allows for the first time in vitro characterization of UreD(UreH). They showed that UreD can interact with UreF in ~ 1:1 binding ratio, but only weakly with UreG .
Work done by us - preliminary data support the proposed structure-function studies of UreF and UreH
1. We have determined the crystal structure of H. pylori UreF. Our group has determined the crystal structure of H. pylori UreF . We noticed that during purification, the C-terminal residues of UreF were prone to degradation, and a truncated UreF was purified and crystallized. We phased the crystal structure of UreF using the MAD method with Se-Met labeled protein. Diffraction data from the native UreF crystals were collected, and the structure was refined to 1.85Å resolution. The structure of native UreF determined by us is similar to the structure of Se-Met derivative reported independently by Lam et al. . UreF is an all-alpha protein consisting of 10 helices. It forms dimers in the crystal structure (Fig. 2). The dimeric interface is formed by docking of helix-1 to the helix-8 and helix-9 of the opposite UreF molecule.
2. We have established an efficient protocol to express and purify UreF/UreH complex. As mentioned above, one obstacle for the structure-function studies of the UreF/UreH complex was that expression of UreH alone resulted in insoluble inclusion bodies (Fig. 3). We have successfully solved this problem by co-expressing UreH with GST-UreF in E. coli (Fig. 3). After affinity chromatography purification and removal of the GST-fusion tag, the UreF/UreH complex can be purified in large quantity (~10 mg per liter of bacterial culture).
3. We have established assays to correlate in vitro protein-protein interactions with in vivo maturation of urease. We showed that when co-expressed together, UreF and UreH form a soluble complex that can be pull-down by GST affinity column (Fig. 4A, lane 2). We noticed that the C-terminal residues of UreF were protected from degradation upon complex formation with UreH (Fig. 4A, lane 1 & 2). We showed that truncation of the C-terminal residues of UreF (UreF-DC20) disrupted the formation of a soluble UreF/UreH complex (Fig. 4A, lane 3). We have also established an assay to test the in vivo maturation of urease (Fig. 5), and showed that the mutation (UreF-DC20) that disrupted the interaction between UreF and UreH also abolished in vivo maturation of urease. By GST pull-down, we demonstrated that the UreF/UreH complex interacts with UreG (Fig. 4B, lane 4), and with the urease (Fig. 4B, lane 4). These preliminary data demonstrated that feasibility of the proposed structure-function studies.
4. We have obtained the preliminary crystal structure of H. pylori UreF/UreH complex. With the purified UreF/UreH complex, we were lucky to obtain crystals of the complex that diffract to high resolution (Fig. 6A). Diffraction data was collected to 2.5Å resolution. We phased the structure by molecular replacement using the structure of UreF as a search template. Our preliminary structure of UreF/UreH complex showed that the UreF/UreH complex forms a 2:2 dimer in the crystal structure (Fig. 6B). We anticipate that the refinement of the UreF/UreH complex structure will be finished very shortly, and the structure will provide a rational based for the mutagenesis studies proposed in this study.
5. We have showed that the UreF/UreH complex form dimers in solution. To test if the UreF and UreF/UreH form dimers in solution, we have loaded purified samples of UreF and UreF/UreH to an analytical size-exclusion-chromatography column coupled to a static light scattering detector (Fig. 7). The apparent M.W. for UreF was 43 kDa, which is in between the theoretical M.W. of a monomeric (28 kDa) and a dimeric (56 kDa) form of UreF. The results suggest that UreF alone does have a tendency to form dimers, and the dimeric form of UreF is in exchange with the monomeric form in solution. On the other hand, the formation of dimer is more-or-less complete in the UreF/UreH complex. The apparent M.W. measured for UreF/UreH complex was 116 kDa, which is consistent with the theoretical M.W. of 116 kDa for a 2:2 dimer of UreF/UreH complex.
6. We have established an efficient protocol to express and purify UreG/UreF/UreH complex, and obtained crystals of the complex. We have found that the most efficient way to obtain the H. pylori UreG/UreF/UreH complex is to co-express UreG, GST-UreF and UreH together in E. coli. The ternary complex can be easily purified by affinity chromatography followed by removal of GST-fusion tag by protease digestion. In our hand, the yield of UreG/UreF/UreH complex is ~5mg per liter of bacterial culture. More encouraging is that we have successfully obtained crystals of UreG/UreF/UreH that diffracted to a reasonable resolution of ~3Å (Fig. 8). These preliminary data strongly suggest that the proposed structure determination of the ternary complex of UreG/UreF/UreH by X-ray crystallography is highly feasible.
Track Record of PI
The PI has extensive experience on structure determination by both NMR and X-ray crystallography, and using protein engineering to probe the structure-function of proteins. In addition to the structure determination of UreF and UreF/UreH complex discussed above, he has solved the solution structure of barstar, an inhibitor of barnase, and studied its dynamics behavior by NMR spectroscopy [25, 26]. He also studied the effect of mutations on the stability and structural perturbation on the DNA-binding domain of the tumor suppressor p53 by NMR spectroscopy [27, 28]. He has used an approach that combines evidence from NMR experiments and molecular dynamics simulation to study the folding pathway and the denatured states of barnase and chymotrypsin inhibitor-2 [29-31]. Supported by previous GRF grants, he solved the solution  and crystal  structure of ribosomal protein L30e from Thermococcus celer, the crystal structures of a thermophilic acylphosphatase from Pyrococcus horikoshii to 1.5Å , and human acylphosphatase to 1.45Å , an orange fluorescent protein from Cnidaria tube anemone to 2.0Å , seabream antiquitin to 2.8Å , the crystal structure of trichosanthin in complex with the C-terminal residues of ribosomal stalk protein P2 to 2.2Å , and the solution structure of the N-terminal dimerization of P2 . We believe that, with our strong background in structural biology and the solid preliminary data, we are in a leading position to determine the structure of the UreG/UreF/UreH ternary complex, and to study the how the urease accessory proteins interact with each other for the maturation of urease.
3(a). Research plan and methodology
3.1 Objective 1. Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?
Our preliminary data suggest that the H. pylori UreF/UreH complex forms a 2:2 dimer in solution. Both the crystal structure of H. pylori UreF, and the preliminary structure of UreF/UreH complex suggest that the dimerization is likely to be mediated by UreF. It is presently not known whether the dimerization is a unique property of H. pylori UreF - for example H. pylori and K. aerogenes UreF only share 19% sequence identity. Interestingly, the quaternary structure of H. pylori urease is different from ureases from other bacterial species. Unlike the urease (UreABC) from K. aerogenes that forms a trimeric complex (UreABC)3, the H. pylori urease (UreAB) forms a tetramer of trimers ((UreAB)3)4. Nevertheless, that H. pylori UreF/UreH complex exists as a dimer in solution and in crystal structure raises an interesting question - is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?
To address this question, we will introduce mutations that are designed to break the dimerization of UreF and UreF/UreH complex. As shown in Fig. 2, the dimeric interface is formed by docking of helix-1 to the helix-8 and helix-9 of the opposite UreF molecule. A closer look at the dimeric interface of the crystal structure of UreF reveals a number of interactions that may be importance to the dimerization of UreF (Fig. 9). For example, to break the hydrogen bonding network among Q37, Q205 and Q212, we will replace the Gln residue with either alanine or asparagine to create triple mutants of Q37A/Q205A/Q212A and Q37N/Q205N/Q212N. We anticipate that both truncation of and shortening of the amide chain should break the hydrogen bond network. To disrupt the hydrophobic interaction around F33, we will substitute the Phe residue with alanine (F33A) or with a polar residue (e.g. F33R). Substitution of polar residue like arginine at Phe-33 should highly disfavor dimerization because the high desolvation penalty will prevent the polar residue to be buried upon dimerization. If necessary, we will create quadruple mutants (e.g. Q37A/Q205A/Q212A/F33A) to ensure disruption of UreF dimerization.
3.1.1 GST pull-down assay for UreF/UreH interaction. First, we test if these mutants will affect the formation of soluble UreF/UreH complex by GST pull-down assay (Fig. 4A). UreH will be co-expressed with mutants of UreF fused with GST-tag, and the bacterial lysate will be loaded to a GSTrap column (GE Healthcare). After extensive washing with binding buffer (20 mM Tris pH7.5, 0.2M NaCl, 5mM DTT), the proteins will be eluted with 10mM glutathione.
As these mutations are located at the dimerization interface, which are far away from the UreF/UreH interface, we anticipate that they will not affect UreF/UreH interaction.
3.1.2 Size-exclusion-chromatography/static-light-scattering (SEC/LS). We will test if these mutants affect dimerization of UreF by SEC/LS. Purified samples of UreF mutants and its complex with UreH complex will be loaded to an analytical Superdex 200 column connected to an online miniDawn light scattering detector and an Optilab DSP refractometer (Wyatt Technologies). The light scattering data will be analyzed using the ASTRA software provided by the manufacturer to obtain the molecular weight of the protein samples.
If the mutations break the dimerization, we anticipate that the measured molecular weight will be 28 kDa for UreF, and 58 kDa for UreF/UreH complex.
3.1.3 In vivo maturation of urease. We will test if test if these mutants affect in vivo maturation of urease. We have established an assay for in vivo maturation of urease (Fig. 5). We have cloned the H. pylori urease operon, ureABIEFGH, into the pRSETA vector to create the pHpA2H vector. We will introduce the mutations into the ureF gene in the pHpA2H vector. E. coli will be transformed with wild-type and mutant pHpA2H vectors, or the negative control plasmids (pHpAB and the empty vectors). The bacterial cells will be cultured in Luria Broth supplemented with 0.5 mM nickel sulfate, and were induced overnight with 0.4 mM IPTG when OD600 reached 0.5. After cell lysate by sonication, urease activity of the bacterial lysate will be assay in 50 mM HEPES buffer at pH 7.5 with 50 mM urea substrate. Urease activity was measured by the amount of ammonia released, which was quantified using a phenol-hypochlorite reaction as described in . Specific activity of the measured sample will be reported as amount of ammonia released per milligram of total protein per minute.
If the dimerization of UreF and UreF/UreH is essential to the maturation of urease, the mutations that break the dimerization will also abolish the maturation of urease. On the other hand, if the maturation of urease is not affected by these mutations, it is likely that the dimerization is not essential to the urease maturation.
3.2 Objective 2. Is the interaction between UreF and UreH essential to the maturation of urease?
Our preliminary data showed that the truncation of the C-terminal residues of UreF breaks the UreF/UreH complex, and abolishes the maturation of urease. The availability of a preliminary structure of UreF/UreH complex allows us to introduce site-directed mutations that are designed to break the UreF/UreH interaction, and to further investigate the functional importance of UreF/UreH interaction. Our structure showed that upon complex formation, the C-terminal residues of UreF become structured and form an extra helix (helix-11) that dock to a binding cavity of UreH. Three hydrophobic residues V235, I239, and M242 on helix-11 are buried to a hydrophobic pocket of UreH.
To further investigate the functional importance of UreF/UreH interaction in urease maturation, we will create alanine and hydrophobic-to-polar (e.g. V®N) substitutions at V235, I239 and M242, which are designed to break the UreF/UreH complex formation. We will test if these mutations affect the formation of the UreF/UreH complex by the GST pull-down assay described in 3.1.1, and if they affect maturation of urease as described in 3.1.3. If the interaction between UreF and UreH is essential to the maturation of urease, we anticipate the mutations that break the UreF/UreH interaction will also abolish the maturation of urease.
3.3 Objective 3. How does the UreF/UreH complex interact with UreG and the urease?
The UreF/UreH is also known to form bigger complexes with UreG, and with the urease (UreA/UreB). How UreF/UreH complex associates with UreG, and the urease to form the pre-activation complex (UreA/UreB-UreG/UreF/UreH) for the maturation of urease is poorly understood. It has been reported that UreG does not interact directly to the urease, suggesting the UreF/UreH complex serves as a bridge that recruits UreG to the activation complex.
Our group has recently collected 2.5Å diffraction data for the H. pylori UreF/UreH, and has obtained a preliminary structure of the complex, which allows us to identify surface residues of UreF and UreH. To map the interacting surface of UreF/UreH complex for binding of UreG and the urease (UreA/UreB), we propose to perform alanine-scanning mutagenesis of surface residues on UreF and UreH. We will first focus on relatively more conserved surface residues (For UreF: P44, I45, Y48, S51, E55, Y72, E119, R121, Y183, K195, Q201, Q205, H244, E245, R250, L251, S254. For UreH: D60, G61, T78, K84, P111, I115, F177, E140, R146, E151, R213). We will also introduce multiple substitutions at these residue positions, if they are close in space according to the preliminary structure of UreF/UreH complex. We will first test if these mutations affect UreF/UreH interaction as described in 3.1.1. If so, we will exclude those mutants from the library.
After we have created the mutant library of UreF/UreH complex, we will test the mutants' ability to form complex with UreG, and with the urease (UreA/UreB). In brief, mutants of the GST-UreF/UreH complex will be co-expressed in E. coli. Our preliminary data suggest that the GST fusion tag will not interfere with binding of UreG or UreA/UreB (Fig. 4B). The bacterial lysate of GST-UreF/UreH (or its mutants) will be mixed with bacterial lysate expressing UreG or UreA/UreB, and then loaded to a GSTrap column for the pull-down assay. For those mutations that break the interaction, we will also perform the reciprocal pull-down in which the GST-tag is fused to the UreH, UreG, UreA or UreB. This is to confirm that the breakage of interaction is due to the mutations, but not due to a nearby GST-tag.
To address the question if the interaction between UreF/UreH and UreF (or UreA/UreB) is essential to maturation of urease, we will test the ability of the UreF/UreH mutants to activation urease in vivo as described in 3.1.3. If the interaction is essential to urease maturation, we anticipate the mutations that break the interaction will also abolish the urease maturation.
3.4 Objective 4. Structure determination of H. pylori UreG/UreF/UreH complex.
3.4.1 Expression and purification of UreG/UreF/UreH complex - We have established an efficient expression purification protocols for the ternary UreG/UreF/UreH complex. His-GST-tagged UreF, UreG and UreH will be co-expressed together in E. coli BL21(DE3) strain using the expression plasmids pET-Duet-HisGST-UreF/UreG and pRSF-UreH. After affinity chromatography purification, the His-GST fusion tag will be removed by the PreScission Protease (GE Healthcare). The protein complex will be further purified by gel filtration. Typical yield of the UreG/UreF/UreH complex is ~ 5mg per liter of bacterial culture.
3.4.2 Optimization of crystallization conditions - Preliminary screening of crystallization conditions was performed. We have already obtained crystals of the UreG/UreF/UreH that diffract to ~3Å (Fig. 8). We will further optimize the crystallization condition by grid-searching the pH and precipitant concentrations, and addition of additives or detergents. Quality of the diffraction data will be used to guide optimization of the crystallization conditions. We will also optimize the cryo-protection procedures (e.g. the choice of cryo-protectants and their concentration) to improve the quality of diffraction data collected. When necessary, we have access to synchrontron beam line at Diamond Light Source, Oxford, through collaboration with Dr. Yu-Wai Chen (King's College London).
3.4.3 Phase determination - We will first attempt to phase the structure by molecular replacement. At the time of writing this proposal, we are refining the structure of H. pylori UreF/UreH complex. We will use the UreF/UreH complex structure as a search template to solve the phase of the UreG/UreF/UreH complex by molecular replacement. In parallel, we will also prepare selenium-methionine labeled sample of UreG/UreF/UreH for multi-wavelength anomalous diffraction (MAD) phasing by expressing the protein complex in minimal medium containing Se-Met as described in Doublie . The H. pylori UreG, UreF, and UreH proteins contain 9, 10, and 8 methionine out of 199, 254, and 265 residues, which should provide enough phasing power for MAD phasing. The PI's group has previously established the expression protocols for Se-Met labeling for H. pylori UreF, and determined its structure by MAD phasing. We have access to synchrontron beam line at Diamond Light Source for collection of MAD data.
3.4.4 Model building and refinement - Models will be built interactively by the program COOT , and refined using PHENIX . The progress of refinement will be monitored by Rfree- and R-factors. Quality of the crystal structure will be validated by the program MOLPROBITY .