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Journal of Bacteriology, February 2007, p. 706-716, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01334-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Inner Cavity of Escherichia coli DegP Protein Is Not Essential for Molecular Chaperone and Proteolytic Activity{triangledown}

Ahmad Jomaa,1 Daniela Damjanovic,1 Vivian Leong,1 Rodolfo Ghirlando,2 Jack Iwanczyk,1 and Joaquin Ortega1*

Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada,1 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-05402

Received 22 August 2006/ Accepted 13 November 2006


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ABSTRACT
 
The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28°C, but efficiently degrading unfolded proteins above 28°C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.


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INTRODUCTION
 
Heat and chemical stress produce damaged and unfolded proteins that are highly toxic to cells (27). The periplasm of Escherichia coli is more susceptible to stress factors because it is separated from the environment only by a porous outer membrane (17). DegP (also called HtrA or protease Do) (14, 24) is an essential protein for E. coli (13), as it eliminates unfolded and damaged proteins that would otherwise form aggregates and thus compromise the survival of the cell (3).

Chaperones and proteases perform antagonistic functions, but also often work together, as demonstrated by the Clp protein family in which the peptidase ClpP acts together with the chaperone ClpA or ClpX (5, 9, 16). DegP has the unusual property of working as a chaperone below 28°C but efficiently degrading unfolded proteins above 28°C. Thus, DegP is an example of both chaperone and protease functions combined in the same protein (23). Also, unlike other chaperones and proteases whose activities usually require expenditure of ATP, these activities are carried out in the absence of metabolic energy in DegP (10, 23).

The mature DegP monomer (48 kDa) contains a trypsin-like protease domain at the N terminus, followed by two PDZ domains (11). PDZ domains are modular interaction domains involved in protein targeting and protein complex assembly that bind preferentially to the C-terminal three to four residues of their target protein. The structure of DegP determined by X-ray crystallography (Protein Data Bank [PDB] identification no. 1KY9) (11) shows how the DegP monomers associate into a hexameric "cage," best described as a staggered dimer of trimers (15). Each trimer is stabilized by residues of the protease domain and has a funnel-like shape such that the protease domains form the top of the funnel and the PDZ domains extend away much like a "molecular jellyfish" (3). In the hexamer, the six protease domains form the top and bottom of the cage with the LA loops (11) of opposing trimers wound around each other, forming three spacing pillars that delimitate the side entrances to the chamber. In the "closed" conformation observed in the crystal structure, the PDZ domains cover these entrances, but they protrude away from the protease domain in the "open" conformation, leaving the gates open. The classical Asp-His-Ser catalytic triad of trypsin-like proteases is contained in each monomer within the DegP cage. The LA loop from each DegP monomer protrudes into the active site of the opposite monomer, where it interacts with the L1 and L2 loops (18), blocking access to the catalytic site. Moreover, the L2 loop is twisted in a particular conformation such that it prevents the formation of the oxyanion hole and blocks the S1 specificity pocket, thus rendering the protease inactive (11). The nature of the conformational switch that transforms DegP into a proteolytically active enzyme still remains to be elucidated.

In the cytoplasm, the active sites of major proteases such as the proteasome or the Clp proteins are also sequestered inside self-compartmentalized structures, providing a common mechanism to prevent indiscriminate proteolysis (1, 6). In these cytoplasmic proteases, the mechanism of translocation and proteolysis of substrates inside the digestion chamber is relatively well understood. Since DegP is similar to other self-compartmentalized proteases, it has been postulated that unfolded substrates are delivered through the side entrances into the interior of the cage, where the catalytic sites are located. However, in DegP this process is poorly characterized and experimental evidence showing that substrates are channeled into the chamber for either proteolysis or refolding have yet to become available.

To study whether an enclosed cavity is required in DegP for protease and molecular chaperone activities, we engineered several deletion mutants of DegP to disrupt the DegP hexamer into two trimers. An enclosed cavity no longer exists in these mutants. Additionally, we looked into the consequences of significantly reducing the dimensions of the DegP cage. We found that neither a cage of specific dimensions nor the presence of an enclosed cavity is essential for the chaperone or protease activities in DegP. Furthermore, we determined that residues 39 to 78 within the LA loops, as well as the PDZ2 domains, are essential to maintain the integrity of the DegP hexamer. The implications of these results towards a model of DegP acting as a self-compartmentalized protease are discussed.


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MATERIALS AND METHODS
 
Cloning of wild-type DegP and DegP mutants into expression vectors. The DegP gene was amplified from E. coli K-12 (strain S 4362) chromosomal DNA by PCR and was subcloned into the expression vector pET21b (Stratagene) using NdeI-XhoI sites. Reduced, collapsed, and broken cage DegP mutants were obtained from the pET21b-DegP plasmid using the QuikChange site-directed mutagenesis method (Stratagene). The {Delta}PDZ2 DegP mutant was generated by PCR using pET21b-DegP plasmid as the template, and it was subcloned into the expression vector pET21b. To obtain the {Delta}PDZ1 DegP mutant, NheI and SpeI restriction sites were engineered between residues 262 and 263 and 366 and 367, respectively, by QuikChange. The plasmid was digested with NheI and SpeI and purified from the PDZ1 NheI and SpeI fragment by agarose gel electrophoresis and extraction using the QIAEX II kit (QIAGEN). The plasmid was then ligated with T4 DNA ligase (Invitrogen), and the six remaining nucleotides were removed by QuikChange. Proteolytically inactive S210A variants of the wild type and all DegP mutants were constructed by QuikChange.

Protein expression and purification. To express wild-type DegP, the cage, and the {Delta}PDZ domain mutants as C-terminal His tag proteins, the expression vector pET21b containing the wild-type or mutant DegP was transformed into E. coli BL21 (DE3) competent cells. The cells were grown in LB medium at 37°C to an optical density at 600 nm (OD600) of 0.7, and expression was induced with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). Cells were induced for 30 min at 37°C or 3 h at 37°C (S210A DegP mutants). Lysis was performed in 20 ml lysis buffer (50 mM HEPES, pH 7.3, 10% [wt/vol] sucrose, 0.1 M NaCl, 0.149 M ammonium sulfate) by addition of 256 µl of lysozyme (50 mg/ml) and incubation for 3 min at 37°C, followed by sonication on ice. Lysate was cleared by centrifugation at 39,000 x g for 40 min. NaCl was added to bring the concentration to 1 M, and the lysate was filtered with a 0.45-µm filter and added to a HiTrap metal chelating column (GE Healthcare Life Sciences) equilibrated with 50 mM HEPES, pH 7.3, 1 M NaCl, 5% (vol/vol) glycerol. Unspecifically bound proteins were washed with increasing concentrations of imidazole up to 90 mM. DegP and its mutants were eluted with 240 mM imidazole. Purity of the fractions was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions containing pure protein were dialyzed against 50 mM HEPES, pH 7.3, in three steps and stored at 4°C.

Size exclusion chromatography. Gel filtration chromatography was performed on a Superdex 200 10/300 GL column (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES, pH 7.3, containing 100 mM NaCl at 4°C. Protein samples (100 µl) were preincubated at either 4°C, 37°C, or 43°C and applied to the column at concentrations ranging between 0.2 and 3.2 mg/ml at a flow rate of 0.5 ml/min. A gel filtration calibration kit (HMW; GE Healthcare Life Sciences) was used for column calibration.

Analytical centrifugation. Sedimentation equilibrium experiments were conducted at 4°C on a Beckman Optima XL-A analytical ultracentrifuge. Samples (loading volume of 130 µl) were studied at three loading concentrations, corresponding to A280 of approximately 0.3, 0.6, and 0.9 (Table 1), and rotor speeds of 4, 6, 8, and 10 krpm. Data were acquired as an average of four absorbance measurements at a wavelength of 280 nm using a radial spacing of 0.001 cm. Sedimentation equilibrium was achieved within 48 h. Data collected at different speeds and different loading concentrations were analyzed globally in terms of various species analysis models using SEDPHAT 4.0 (P. S. Schuck; http://www.analyticalultracentrifugation.com/sedphat/sedphat.htm) to obtain sample molecular masses. Solution densities, {rho}, were measured at 20°C on a Mettler-Toledo DE51 density meter and corrected to values for {rho} at 4°C. Values of the partial specific volume, v, were calculated based on the amino acid composition using SEDNTERP (J. Philo; http://www.jphilo.mailway.com/).


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  		TABLE 1. Properties of DegP mutants studied by analytical ultracentrifugationa

Sedimentation velocity experiments were conducted in duplicate at 4°C on a Beckman Optima XL-A analytical ultracentrifuge. Samples (loading volume of 350 µl) were studied at a loading absorbance ({lambda} = 280 nm) of about 1.0. One hundred to 125 scans were acquired at the appropriate rotor speed (35 to 45 krpm) as a single absorbance measurement at a radial spacing of 0.003 cm using the shortest time delay possible by setting the time interval to zero. Data were analyzed in SEDFIT (21) (P. S. Schuck; http://www.analyticalultracentrifugation.com/default.htm) to obtain a sedimentation coefficient distribution in the form of c(s). All sedimentation coefficients represent those determined at 4°C in 50 mM HEPES (pH 7.3).

Chaperone activity assay. An ammonium sulfate suspension of citrate synthase (CS) (Roche) was dialyzed at 4°C overnight into TE buffer (50 mM Tris-HCl, pH 8, 2 mM EDTA) and concentrated up to 17 mg/ml using nanosep 30K Omega microconcentrators (Pall Corporation). Preparation of chemically denatured CS was done by dilution in a stock of 6 M guanidinium chloride, 50 mM Tris-HCl, pH 8, and 20 mM dithioerythritol for 2 h at room temperature as previously described (2). DegPS210A protein or the mutant DegP proteins were mixed with a 0.15 µM solution of denatured CS in refolding buffer (40 mM HEPES, pH 7.5) at a molar ratio of 4:1 (DegP monomer to CS monomer) and incubated at room temperature with constant stirring. The volume of the refolding reaction was 200 µl. At specific time points, 20-µl samples were extracted from the refolding reaction and the specific activity of CS was measured using an assay based on the first step of the citric acid cycle. The highest specific activity value obtained for DegPS210A was considered as 100% specific activity, and the calculated specific activity of the mutants was expressed as a percentage of this value. The calculation of the specific activity of CS at a given time point of the reaction was done as previously described (2). In brief, the coenzyme A formed in this assay stoichiometrically reduces the Ellman's reagent dithio-1,4-nitrobenzoic acid (DTNB), resulting in an increased absorbance at 412 nm.

Protease activity assays. Reaction mixtures were assembled in 250 µl containing 240 µg of malate dehydrogenase (MDH) from porcine heart (Roche and Sigma), 480 µg of bovine milk ß-casein (Sigma) or 16 µg of egg white lysozyme (Bioshop) in 50 mM HEPES, pH 7.3. Wild-type DegP or the indicated DegP mutant was added to obtain a molar ratio of 1:16 (DegP monomer to substrate). Reaction mixtures were incubated at 37°C for the ß-casein (10) and lysozyme assays and at 43°C for the MDH experiments (10). Dithiothreitol was added to the lysozyme reaction mixtures up to a final concentration of 1.5 mM (22).

At the indicated times, 20-µl samples were taken and mixed with 2x concentrated SDS-PAGE loading buffer. We resolved 14.5 µl, 12.5 µl, and 12.4 µl of each sample of the MDH, ß-casein, and lysozyme reactions, respectively, by SDS-PAGE. Gels were stained according to the manufacturer's protocols with either Coomassie brilliant blue (GE Healthcare Life Sciences) (MDH and ß-casein) or Deep Purple total protein stain (GE Healthcare Life Sciences) (lysozyme). Proteolysis reactions for each specific mutant and substrate were run in the same gel together with an analogous reaction performed with the wild-type DegP. Only the time points of samples that contained an amount of substrate within the linear range for the stain were taken into consideration for the plots.

Coomassie brilliant blue gels were scanned in a flatbed scanner (HP Scanjet 5400c) and Deep Purple-stained gels were scanned in a Typhoon 9200 scanner (GE Healthcare Life Sciences). Data were analyzed with ImageQuant TL software according to manufacturer's protocols, and a proteolysis curve was calculated for each mutant and wild-type DegP protein. The rate of substrate proteolysis observed for each specific mutant was measured by obtaining the ratio between the slope of the proteolysis curve for the mutant and the wild-type DegP protein. Each proteolysis assay was repeated three times, and an average and standard deviation of the ratios were calculated.


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RESULTS
 
DegP mutants with a reduced cavity are hexamers. The crystal structure of DegP suggests that substrates have to be delivered into the inner cavity of the protein for proteolysis or refolding. To test whether the size of the DegP cavity influences the protease or chaperone activity of this enzyme, we constructed two mutants in which the size of the cavity was reduced. The spacing between the two opposite trimers forming the DegP hexamer is determined by the LA loops. These loops, which protrude from the protease domains, are wound around each other and build the corner pillars of the DegP cage (Fig. 1A). The first mutant was constructed by deleting the two-stranded ß-sheet forming part of the LA loop ({Delta}34-37 plus {Delta}80-83) (Fig. 1A, residues in dark blue). In this reduced cage mutant, the pillar length is partially reduced and thus is the size of the cavity. A second mutant was constructed by reducing the length of the pillars even further ({Delta}34-39 plus {Delta}60-83) (Fig. 1A, residues in dark blue and red), thus causing the two trimers to collapse together, maximally reducing the size of the DegP cavity. This mutant was named the collapsed cage mutant.


Figure 1
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  		FIG. 1. Deletions in the reduced, collapsed, and broken cage DegP mutants. Shown is a ribbon representation of the open conformation (side view) of the DegP hexamer (PDB identification no. 1KY9) with opposite subunits of the hexamer displayed in the same color (green, pink, and orange). (A) Residues from the LA loop removed in the reduced cage mutant (34 to 37 plus 80 to 83) are shown in dark blue. In the collapsed cage mutant, the residues displayed in dark blue and red (34 to 39 plus 60 to 83) were deleted. (B) LA loops after winding around each other reach the opposite ceiling in the cage and interact with the DegP subunit at the other end. Residues from this area of the LA loops shown in dark blue were removed in the broken cage DegP mutant (39 to 78). Disordered residues (51 to 79) are shown as a dashed line.

Both mutants eluted in a filtration column at a position consistent with the hexameric form of the protein (Fig. 2A and B). As in the activity assays performed (see below), these mutants are incubated at different temperatures, we determined the stability of the proteins at these temperatures by repeating the injection of each mutant into the column after incubating the protein at 4, 37, or 43°C for 1 h. The amounts of protein and elution volumes were virtually identical for the three incubation conditions, suggesting that the hexamers formed by these DegP mutants remained stable. We also constructed a proteolytically inactive variant of DegP (DegPS210A) and the DegP mutants (reduced and collapsed cage DegPS210A). These variants also eluted as hexamers, regardless of the incubation conditions before injection into the column. For DegPS210A and the collapsed cage DegPS210A mutants, a second peak was observed at the expected elution volumes for a dodecamer (Fig. 2A and B). This second smaller peak indicated the presence of approximately 18% and 22% of dodecamers for these two mutants.


Figure 2
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  		FIG. 2. Oligomeric state of reduced and collapsed cage DegP mutants. (A) Elution profile of the reduced and collapsed cage DegP mutants from a Superdex-200 column after incubation at 4°C for 1 h. Elution profiles for the proteolytically inactive variants of these mutants (S210A) and DegPS210A are also displayed. The dashed lines in the plot on the right-hand side indicate the expected elution volume for the hexameric (11.5 ml) and the trimeric (13 ml) forms of these mutants. (B) Samples were withdrawn from the two fractions of the highest peak on each elution profile and resolved by SDS-PAGE (15%) and stained with Coomassie brilliant blue. (C) Sedimentation velocity results for the collapsed cage DegPS210A and DegPS210A mutants. The c(s) distributions were obtained using the program SEDFIT. Peaks corresponding to the hexameric (hx) and dodecameric (dc) forms are indicated. (D) Sedimentation equilibrium profiles shown in terms of A280 versus the radius, r, for samples DegPS210A (left) and the collapsed cage DegPS210A mutant (right) at loading A280 of 0.69 and 0.40, respectively. Data collected at 4 (blue), 6 (green), and 8 (red) krpm at 4.0°C are shown. Best fits are shown as solid lines, and the corresponding distributions of the residuals are shown above the plot.

Sedimentation velocity and sedimentation equilibrium experiments on the DegPS210A and the collapsed cage DegPS210A mutants confirmed the results obtained by size exclusion chromatography. An analysis of the sedimentation velocity data in terms of a continuous c(s) distribution yielded excellent fits. In both mutants, data was consistent with the presence of two well-resolved species having sedimentation coefficients of approximately 7S and 10.2S, respectively (Fig. 2C). The c(s) distribution reproducibly returned a slightly smaller sedimentation coefficient for the collapsed cage mutant hexamer, reflecting the reduced mass of this species. This shows that the overall shapes of the DegPS210A and collapsed cage DegPS210A mutant hexamers are very similar and that the structural integrity of the collapsed cage DegP is maintained. A previous study already showed that the S210A mutation only induces minor changes in the tertiary structure of DegP (22). Analysis of the sedimentation equilibrium data in terms of two noninteracting ideal solutes returned molecular masses consistent with the presence of DegP hexamers and DegP dodecamers (Fig. 2D and Table 2) with excellent fits. Even though the masses obtained for DegPS210A indicate hexameric and decameric stoichiometry (Table 2), biochemical studies and gel filtration support the presence of hexameric and dodecameric species. Based on these data, we conclude that the 7S species represents the DegP hexamer, whereas the 10.2S species represents the DegP dodecamer. Integration of the c(s) distribution indicates the presence of approximately 6% and 14% of the dodecamer for DegPS210A and the collapsed cage DegPS210A mutant, respectively. These values are consistent with the sedimentation equilibrium data, as well as the gel filtration profiles of the corresponding DegPS210A and collapsed cage DegPS210A mutants.


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  		TABLE 2. Sedimentation equilibrium results for DegPS210A, the cage, and {Delta}PDZ2 DegPS210A mutants

Reduction in the size of the DegP inner cavity does not abolish protease and chaperone activities. It was shown previously that DegP has a general molecular chaperone activity and is able to refold nonnatural substrates, such as CS (23). To compare the chaperone activity of the reduced and collapsed cage DegPS210A mutants with that of full-length DegPS210A, we tested the ability of these proteins to refold CS using an in vitro refolding assay based on purified components (2). The results show that the chaperone activity of the reduced cage DegPS210A mutant is decreased, but still very significant, whereas the collapsed cage DegPS210A mutant has an activity very similar to that of full-length DegPS210A (Fig. 3A). A refolding reaction in the absence of DegP or in the presence of a control protein such as bovine serum albumin (BSA) showed a very small amount of refolded CS (Fig. 3A).


Figure 3
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  		FIG. 3. Chaperone and protease activity of the reduced and collapsed DegP mutants. (A) Chemically denatured citrate synthase was refolded in the absence of any protein or in the presence of a fourfold molar excess of BSA, reduced, or collapsed cage DegPS210A mutants. Citrate synthase activity was measured at the indicated points and is given as a percentageof the highest specific activity value obtained for DegPS210A. DCS, denatured citrate synthase. (B to D) Proteolysis curves of MDH (B, left plots), ß-casein (C, left plots), and lysozyme (D, left plots) in the presence of reduced or collapsed cage DegP mutants. Proteolysis reactions were performed, and samples were resolved by SDS-PAGE. Gels were stained by Coomassie brilliant blue in the MDH and ß-casein (B and C) experiments or Deep Purple total protein stain in the lysozyme assays (D). Gels were scanned, and the substrate bands were quantified to obtain the hydrolysis curves. The rate of substrate hydrolysis by a specific mutant is shown as the ratio between the slope (slp) of the proteolysis curve for the mutant (solid line) and the wild-type (WT; dashed line) DegP protein. One representative proteolysis plot from each reaction type is shown. Each experiment was performed three times, and an average and standard deviation of the ratio of the rates (mutant to wild type) was calculated for each reaction type (B, C, and D, right plots). AU, arbitrary units.

To investigate whether reduction of the cage size affects the protease activity of DegP, we measured the ability of the reduced or collapsed DegP mutants to degrade three substrates (MDH, ß-casein, and lysozyme) compared to wild-type DegP. We found that both mutants had a reduced, but still significant protease activity against MDH (Fig. 3B). The reduced cage mutant degraded ß-casein very efficiently, but surprisingly the collapsed cage mutant showed low activity against this substrate (Fig. 3C). Unlike ß-casein, both mutants showed similar proteolysis rates for lysozyme when compared to the wild-type DegP (Fig. 3D). The proteolytically inactive DegPS210A variant and the corresponding cage mutants did not degrade any of the substrates tested (data not shown).

These data suggest that DegP does not require a cage of specific dimensions to perform either protease or chaperone activity.

Residues from positions 39 to 78 within the LA loops as well as the PDZ2 domains are essential to maintain the DegP hexameric cage. Based on the crystal structure of the DegP open conformation, it has been predicted that interactions between the LA loops of opposite monomers in the cage are mainly responsible for the stability of the hexamer (Fig. 1B). Furthermore, in the closed conformation of the cage, the PDZ2 domain of each monomer interacts with the PDZ1 and PDZ2 domains of the monomer at the opposite trimer in a zipper-like arrangement (11).

To determine which of these interactions are essential to maintain the integrity of the hexameric cage, we constructed three deletion mutants: the broken cage DegP ({Delta}39-78) (Fig. 1B), {Delta}PDZ1 DegP ({Delta}263-366), and {Delta}PDZ2 DegP ({Delta}356-446) mutants. We studied their oligomeric structure by size exclusion chromatography and sedimentation experiments.

An injection of purified broken cage DegP mutant in the size exclusion column produced an elution profile with two peaks at the expected elution volume for a trimer and a monomer, respectively (Fig. 4A and B). The proteolytically inactive variant of this mutant (broken cage DegPS210A) also eluted as a trimer and as a monomer. A third peak was also present in the elution profile with the elution volume corresponding to a dodecamer. This peak represents approximately 14% of the total protein (Fig. 4A). The elution profiles of these two mutants were virtually identical upon incubation at either 37°C or 43°C for 1 h before injection into the size exclusion column (data not shown).


Figure 4
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  		FIG. 4. Oligomeric state of the broken cage DegP mutant by size exclusion chromatography. (A) Elution profile of the broken cage DegP mutant and its proteolytically inactive variant (broken cage DegPS210A) from a Superdex-200 column after incubation at 4°C for 1 h. The panel on the left shows the elution profile for the DegPS210A mutant used as a control. The dashed lines in the plot on the right-hand side indicate the expected elution volume (Ve) for dodecameric (10.1 ml), hexameric (11.7 ml), trimeric (13.2 ml), and monomeric (15.5 ml) forms of these mutants. (B) One sample from each peak was withdrawn, resolved by SDS-PAGE (15% polyacrylamide), and stained with Coomassie brilliant blue. The lane number on the gels indicates from which peak the fraction was taken. (C) Sedimentation equilibrium profiles shown in terms of A280 versus the radius, r, for the broken cage DegPS210A mutant at a loading A280 of 0.54. Data were collected at 6 (blue), 8 (green), and 10 (red) krpm at 4.0°C and analyzed in terms of three noninteracting solutes. Best fits are shown as lines through the experimental points, and the corresponding distributions of the residuals are shown above the plot. (D) Sedimentation velocity results for the broken cage DegPS210A showing the c(s) distributions using SEDFIT.

Sedimentation equilibrium studies of the broken cage DegPS210A mutant were consistent with the size exclusion chromatography experiments. Data were analyzed globally in terms of three noninteracting ideal solutes, with the mass of the smallest species fixed to that of the monomer. At all loading concentrations, the data analysis returned masses of 122 ± 8 kDa (n = 2.9 ± 0.2) and 558 ± 70 kDa (n = 13 ± 2) (Table 2), consistent with the existence of trimers and dodecamers. We noted that excellent fits were obtained (Fig. 4C).

The c(s) profile of the broken cage DegPS210A mutant obtained by sedimentation velocity showed two well-resolved sedimenting species having sedimentation coefficients of approximately 1.8S and 2.8S (Fig. 4D), which correspond to monomers and trimers, respectively. No evidence for dodecamers was found in these experiments.

These experiments showed that both the broken cage DegP mutant and its proteolytically inactive variant are mostly trimers and monomers, indicating that residues 39 to 78 in DegP are essential to maintain the protein in the hexameric state.

The oligomeric state of the {Delta}PDZ1 and {Delta}PDZ2 DegP mutants and their S210A variants was also investigated by size exclusion chromatography. After 1 h of incubation of the mutants at either 4°C, 37°C, or 43°C, the mutants eluted at the expected elution volume for the trimeric form of the protein (Fig. 5A and B). In the elution profile of {Delta}PDZ2 DegPS210A, a small peak representing ~10% of the total injected protein was observed at the elution volume expected for a hexamer. Sedimentation equilibrium experiments were carried out with the {Delta}PDZ2 DegPS210A (Table 1) mutant, and a global data analysis in terms of a single ideal solute showed excellent fit consistent with the presence of a monodisperse species (Fig. 5C). The analysis returned a molecular mass of 123 ± 4 kDa (expected monomer mass = 37,267.3 Da; stoichiometry, n = 3.4 ± 0.1) (Table 2), consistent with the observation by size exclusion chromatography that this mutant is exclusively a trimer in solution.


Figure 5
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  		FIG. 5. {Delta}PDZ1 and {Delta}PDZ2 DegP mutants are trimers. (A) Elution profiles of {Delta}PDZ1 and {Delta}PDZ2 DegP mutants and their proteolytically inactive variants (S210A mutants) after incubation at 4°C for 1 h. The dashed lines in the plot on the right-hand side indicate the expected elution volume for the hexameric (12 ml) and the trimeric (13.5 ml) forms of these mutants. (B) Samples from each peak were resolved by SDS-PAGE (15% polyacrylamide) and stained with Coomassie brilliant blue. In the gel for {Delta}PDZ2 DegPS210A, the middle lane corresponds to a sample obtained from the peak eluting at 11.6 ml. (C) Sedimentation equilibrium profiles shown in terms of A280 versus the radius, r, for the {Delta}PDZ2 DegPS210A mutant at a loading A280 of 0.29. Data were collected at 6 (blue), 8 (green), and 10 (red) krpm at 4.0°C and analyzed in terms of a single ideal solute. Best fits are shown as black lines through the experimental points, and the corresponding distributions of the residuals are shown on the left-hand side of the plot.

All together, these mutants showed that both the residues 39 to 78 within the LA loop and the PDZ domains are essential to maintain DegP as a hexamer. The single interaction of each of these domains independently is not sufficient to stabilize the hexameric DegP cage. Consistent with the DegP X-ray structure (Fig. 1B), our results for {Delta}PDZ1 DegP suggest that the PDZ1 domain maintains the integrity of the hexameric cage by allowing the PDZ2 domain to reach the PDZ1 and PDZ2 domains of a monomer at the opposite end of the hexamer, rather than establishing interactions with another monomer.

Protease and chaperone activities are observed in DegP mutants without an enclosed cavity. Our finding that both the broken cage and the deleted PDZ domain mutants do not form hexamers gave us the opportunity to test whether the lack of an enclosed cavity prevents DegP from having molecular chaperone and protease activities.

The chaperone activity of the broken cage DegPS210A mutant was tested by the in vitro refolding assay, and we found that this mutant refolded CS with similar efficiency to the DegPS210A mutant (Fig. 6A). Similarly, {Delta}PDZ1 and {Delta}PDZ2 DegPS210A mutants were also able to refold CS (Fig. 7A),consistent with previous findings (23).


Figure 6
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  		FIG. 6. Chaperone and protease activity of the broken cage DegP mutant. (A) Refolding of denatured citrate synthase in the absence of proteins or in the presence of a fourfold molar excess of broken cage DegPS210A mutant. Citrate synthase activity is expressed as a percentage of the highest specific activity value observed in the DegPS210A reaction. (B) Representative proteolysis plots of the broken cage DegP mutant (solid line) and wild-type (WT) DegP (dashed line) for MDH, ß-casein, and lysozyme (left plots). The rates of hydrolysis for each substrate were measured and normalized to the rate observed for wild-type DegP. Proteolysis reactions for each substrate were repeated three times, and averages and standard deviations were calculated (right plot). AU, arbitrary units; slp, slope.


Figure 7
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  		FIG. 7. Chaperone and protease activity of the {Delta}PDZ DegP mutants. (A) Four molar excess of either {Delta}PDZ1 or {Delta}PDZ2 DegP mutants was mixed with unfolded citrate synthase under refolding conditions. Samples were taken at the indicated time points, and the CS specific activity was determined. Results are expressed as the percentage of the highest specific activity observed in the refolding reaction performed in the presence of DegPS210A. DCS, denatured citrate synthase. MDH (B), ß-casein (C), or lysozyme (D) was incubated with either {Delta}PDZ1 or {Delta}PDZ2 DegP mutants. Proteolysis plots were calculated (B, C, and D, left plots) as described. Mutant DegP is shown by the solid line, and the wild-type (WT) DegP protein is shown by the dashed line. Averages and standard deviations of the rates were obtained for each reaction condition (B, C, and D, right plots). AU, arbitrary units; slp, slope.

Next, we carried out protease assays with the proteolytically active variant of these three mutants. The broken cage DegP mutant showed even higher protease activity with the three tested substrates (MDH, ß-casein, and lysozyme) than that of wild-type DegP, as measured by the ratio of the proteolysis rates for the mutant and wild-type DegP (Fig. 6B). The {Delta}PDZ2 DegP mutant was also able to degrade these three substrates very efficiently. However, the {Delta}PDZ1 DegP mutant showed very little activity (Fig. 7B).

We conclude from these data that the PDZ2 domain is not required for protease activity, whereas the PDZ1 domain seems to be sufficient to render a protease as active as the wild-type DegP protein. However, the presence of the protease domain and any of the two PDZ domains was sufficient for DegP to maintain its molecular chaperone activity. As both {Delta}PDZ domain mutants were mostly trimers in solution and the broken cage mutant did not form a hexamer, we conclude that the presence of an enclosed cavity is not essential for DegP to perform its protease and molecular chaperone activities.


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DISCUSSION
 
The results presented in this paper indicate that residues 39 to 78 within the LA loops and either PDZ domain are all essential to maintain DegP as a hexamer with a central cavity that contains the catalytic triad for protease activity and possible binding sites for misfolded proteins. A DegP mutant lacking both PDZ domains was reported previously to exist in an equilibrium of dimers and tetramers, suggesting the involvement of the PDZ domains in the oligomerization process of this protein (19).

Due to the sequestration of the active sites, the DegP protein is an example of a self-compartmentalized protease. Access routes to the inner chamber lie between the pillars formed by the LA loops. However, these lateral windows and the inner chamber itself are only big enough to accommodate little more than individual secondary structure elements. In fact, previous unfolding of protein substrates is essential for hydrolysis by DegP (10), which has been interpreted as a suggestion that substrates are channeled into the inner chamber for proteolysis. Unfortunately, there is still no experimental evidence showing that substrates are delivered into the inner chamber for subsequent cleavage or refolding. To clarify the necessity of having an enclosed cavity for protease and molecular chaperone activity, we constructed several DegP deletion mutants having reduced inner cavity dimensions, along with mutants which formed trimers and thus were without the enclosed cavity. Our results demonstrate that neither a cavity of specific dimensions nor the presence of an enclosed cage is required to maintain protease and chaperone activity in DegP. These results are consistent with other trimeric members of the HtrA family, such as mammalian HtrA2 (12), that act as proteases (4, 20).

Interestingly, in the reduced cage DegP mutant, the proteolytic activity increased as we studied smaller substrates, suggesting that the size of the cage becomes less relevant for protease activity as the molecular mass of the substrate decreases. These data support the hypothesis that proteolysis takes place once the substrate has accumulated in the inner chamber. Therefore, reducing its size may decrease proteolysis rates for bigger substrates but not for small ones. In fact, our observation that the trimeric broken cage DegP is proteolytically more active than the wild type is consistent with this hypothesis as the catalytic sites are no longer confined within an enclosed cavity, and thus they are more readily accessible to the substrates.

DegP is a self-compartmentalized protease, and it has been hypothesized that unfolded polypeptides are threaded in an extended conformation into the cage to access the proteolytic sites. The height of the cavity in the collapsed cage mutant is predicted to be significantly smaller than 15 Å, and thus the size of its lateral openings, which grant access of the substrate to the inner chamber, is most likely not large enough to allow the threading of an unfolded protein. It was therefore surprising to observe significant protease activity in this mutant under the hypothesis that unfolded polypeptides are threaded in an extended conformation into the cage to access the proteolytic sites. Thus, these results raise the possibility that substrates may also access the catalytic sites through the transient disassembly of the hexameric cage. Interaction of DegP with a specific substrate may be the initial event promoting the disassembly of the hexamer. In this context, the functional reason for the stable hexameric form of DegP observed in the crystal structure in the absence of substrate would be to prevent indiscriminate substrate proteolysis by sequestering the catalytic sites. Therefore, the described X-ray structure of the DegP hexameric cage constitutes a double-layer safety mechanism protecting the cell from uncontrolled proteolysis, which can be hazardous to the cell. First, the cage physically limits the access of substrates to the catalytic sites, and second, the catalytic triad is in an inactive conformation. DegP interaction with a substrate protein may promote the disassembly of the DegP cage and further stimulate a conformational change that renders the catalytic triad in an active form, in a similar manner to the activation mechanism of another member of this family of proteins, DegS (26, 28).

There are also many similarities between the structure of the hexameric DegP and other well-characterized chaperones, such as GroEL. Both proteins form an enclosed cavity, and the walls of the cavity contain hydrophobic patches used for binding of unfolded proteins. However, there are important fundamental differences between the DegP and GroEL structures. Whereas GroEL has a large opening of about 45 Å through which nascent and unfolded proteins can access the chamber and be released upon reaching a native state, the lateral openings in the DegP hexamer are only 20 Å wide. Therefore, it is unclear how refolded substrates are released from the cage, assuming that substrate folding actually happens inside the chamber. Our data showing that an enclosed cavity is not required for DegP chaperone activity and that DegP mutant with a significantly smaller window size has similar chaperone activity to wild-type DegP suggest that DegP does not require enclosure of its substrates in the inner chamber for refolding as GroEL does. Alternatively, DegP may perform its chaperone activity by binding hydrophobic sequences of unfolded substrates (similarly to DnaK chaperone [8]), upon disassembly of the hexameric cage, which allows substrates to access the hydrophobic surfaces coating the ceilings of the inner chamber. Interestingly, it has been shown that a monomeric polypeptide containing residues 191 to 345 from GroEL has the same activity as the tetradecamer of GroEL in refolding rhodanase in vitro (29). Therefore, in GroEL as well, the presence of a central cavity is not essential for chaperone activity in vitro. However, it could still be important for refolding in vivo and to regulate the affinity of the protein for its various substrates.

A deletion analysis such as the one reported here has the intrinsic problem that the deletion mutants may produce atypical results simply because the deletion mutants either fail to fold correctly or are unstable. However, some of our data in fact indicate that the deletion mutants in this study are properly folded and are stable: (i) the various mutants showed both chaperone and protease activity; (ii) all deletion mutants behaved as expected in terms of their oligomeric size, including after incubation at 43°C for 1 h; and (iii) the collapsed cage DegPS210A and DegPS210A mutant showed S-values which correlate with their mass, indicative of similar overall shape. Furthermore, previous work has shown that PDZ domains are highly modular domains (7) and that it is relatively easy to change the PDZ domain binding specificity by mutations but without compromising the domain structure (25). Moreover, an infrared spectroscopic study has shown that mutational alterations in serine 210 and histidine 105 induced minor changes in the tertiary structure of DegP (22). Finally, with respect to the stability of our trimeric DegP mutants, there are members of the HtrA family of proteins that are functional trimers, as shown by the crystal structure of E. coli DegS (28) and human HtrA2 (12).

In conclusion, our results show that an enclosed cavity is not essential for the protease and chaperone activity of DegP. Future experiments will actually test whether DegP remains as a hexameric cage upon interaction with its substrates and whether DegP works similarly to other self-compartmentalized proteases and chaperones where the substrates are translocated to the inner chamber for hydrolysis or refolding.


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ACKNOWLEDGMENTS
 
We thank Alba Guarné for help in the initial stages of the cloning and purification of the DegP protein. We are also grateful to Alba Guarne and Cecelia Trainor for insightful comments and critical reading of the manuscript. We thank Tushar Shakya for constructing the broken cage DegP mutant.

This work was supported by grants from the Canadian Institutes of Health Research (CIHR), Canada Foundation for Innovation (CFI), and Ontario Innovation Trust (OIT). J.O. is a recipient of a CIHR salary award. R.G. was supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Biomedical Sciences, Health Sciences Centre, Room 4H24, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. Phone: 905-525-9140, ext. 22703. Fax: 905-522-9033. E-mail: ortegaj{at}mcmaster.ca. Back

{triangledown} Published ahead of print on 22 November 2006. Back


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Journal of Bacteriology, February 2007, p. 706-716, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01334-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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