The mechanism of temperature-induced bacterial HtrA activation

High-temperature requirement A (HtrA) protein has been known as a moonlighting protein that plays dual roles as a molecular chaperone and as a protease. The proteolytic activity of HtrA is switched on at elevated temperatures, whereas the chaperone function predominates at normal temperatures. The temperature-regulated functional switch of HtrA appears to be critical for the control of the stability of cellular proteins, as well as for the elimination of denatured proteins in order to maintain viability. Although certain conformational changes are expected to be concurrent with the functional activation of HtrA proteolysis, the molecular mechanisms inherent to this process have yet to be elucidated. Spin labeling electron paramagnetic resonance and fluorescence spectroscopy experiments on the HtrA from Thermotoga maritima (Tm HtrA) have shown that a helical lid (H_L) that covers the active site is lifted up to expose the catalytic and substrate-binding sites to the solvent at elevated temperatures, whereas the overall structure is maintained over a wide temperature range. Results indicate that the proteolytic activity of Tm HtrA is turned on by the geometric change occurring around the H_L, resulting in a substrate-accessible path. In conclusion, the functional switch of Tm HtrA is embedded in the sentinel of the H_L in terms of substrate accessibility.     

Temperature-induced changes of HtrA2(Omi) protease activity and structure

HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95 kDa, disk large, and zonula occludens 1 (PDZ) regulatory domain and functions both as a protease and a chaperone. Based on the crystal structure of the HtrA2 inactive trimer, it has been proposed that PDZ domains restrict substrate access to the protease domain and that during protease activation there is a significant conformational change at the PDZ–protease interface, which removes the inhibitory effect of PDZ from the active site. The crystal structure of the HtrA2 active form is not available yet. HtrA2 activity markedly increases with temperature. To understand the molecular basis of this increase in activity, we monitored the temperature-induced structural changes using a set of single-Trp HtrA2 mutants with Trps located at the PDZ–protease interface. The accessibility of each Trp to aqueous medium was assessed by fluorescence quenching, and these results, in combination with mean fluorescence lifetimes and wavelength emission maxima, indicate that upon an increase in temperature the HtrA2 structure relaxes, the PDZ–protease interface becomes more exposed to the solvent, and significant conformational changes involving both domains occur at and above 30 °C. This conclusion correlates well with temperature-dependent changes of HtrA2 proteolytic activity and the effect of amino acid substitutions (V226K and R432L) located at the domain interface, on HtrA2 activity. Our results experimentally support the model of HtrA2 activation and provide an insight into the mechanism of temperature-induced changes in HtrA2 structure.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-012-0355-1) contains supplementary material, which is available to authorized users.     

Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains

Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.     

N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli: rapid purification of a proteolytically active HtrA2/Omi

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death; however, the underlying mechanism of HtrA2/Omi-mediated apoptosis remains to be elucidated. Using the pGEX bacterial expression system, we investigated the expression patterns of various forms of HtrA2/Omi. Full-length mouse HtrA2/Omi (mHtrA2/Omi) was successfully expressed in E. coli and purified as a proteolytically active protein. In contrast, the expression of full-length human HtrA2/Omi (hHtrA2/Omi) in E. coli was barely detected. On the basis of this result, we characterized further the expression patterns of N- or C-terminally truncated hHtrA2/Omi proteins. We found that three copies of the PRAXXTXXTP motif, which exist only in hHtrA2/Omi, might serve as a primary site that is highly susceptible to proteolytic degradation by host proteases. Removal of the N-terminal region containing the PRAXXTXXTP motifs produced a form resistant to proteolytic degradation during expression in E. coli and purification, consequently improving the production of a catalytically active, mature hHtrA2/Omi. Our study provides a method for generating useful reagents to investigate molecular mechanism by which HtrA2/Omi contributes to regulating apoptotic cell death and to identify natural substrates of HtrA2/Omi.     

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.     

PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide

The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protein:protein interactions. The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site. E. coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system. This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal. Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K(D) values of approximately 5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition. Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathione S-transferase) renders it sensitive to digestion by Tsp but not HtrA. These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathione S-transferase are interpreted in terms of the structure of HtrA.     

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.     

Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA

Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.     

Characterization of a novel human serine protease that has extensive homology to bacterial heat shock endoprotease HtrA and is regulated by kidney ischemia

We report the isolation and characterization of a cDNA encoding the novel mammalian serine protease Omi. Omi protein consists of 458 amino acids and has homology to bacterial HtrA endoprotease, which acts as a chaperone at low temperatures and as a proteolytic enzyme that removes denatured or damaged substrates at elevated temperatures. The carboxyl terminus of Omi has extensive homology to a mammalian protein called L56 (human HtrA), but unlike L56, which is secreted, Omi is localized in the endoplasmic reticulum. Omi has several novel putative protein-protein interaction motifs, as well as a PDZ domain and a Src homology 3-binding domain. Omi mRNA is expressed ubiquitously, and the gene is localized on human chromosome 2p12. Omi interacts with Mxi2, an alternatively spliced form of the p38 stress-activated kinase. Omi protein, when made in a heterologous system, shows proteolytic activity against a nonspecific substrate beta-casein. The proteolytic activity of Omi is markedly up-regulated in the mouse kidney following ischemia/reperfusion.     

PDZ domains determine the native oligomeric structure of the DegP (HtrA) protease

DegP (HtrA) is a periplasmic heat shock serine protease of Escherichia coli that degrades misfolded proteins at high temperatures. Biochemical and biophysical experiments have indicated that the purified DegP exists as a hexamer. To examine whether the PDZ domains of DegP were required for oligomerization, we constructed a DegP variant lacking both PDZ domains. This truncated variant, DegPDelta, exhibited no proteolytic activity but exerted a dominant-negative effect on growth at high temperatures by interfering with the functional assembly of oligomeric DegP. Thus, the PDZ domains contain information necessary for proper assembly of the functional hexameric structure of DegP.     

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.     

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in?vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.     

Application of the wheat-germ cell-free translation system to produce high temperature requirement A3 (HtrA3) proteases

Mammalian high temperature requirement A3 (HtrA3) is a serine protease of the HtrA family. It is an important factor for placental development and a tumor suppressor. The biochemical properties of HtrA3 are uncharacterized. One critical step in biochemical characterization is overexpressing and purifying the full-length recombinant protein. However, utility of cell-based expression systems is limited for a protease because of autocleavage. The wheat-germ cell-free translation system is highly efficient at producing "difficult" eukaryotic multidomain proteins and is easily modifiable for protein synthesis at different temperatures. In this study, we evaluated the potential of the wheat-germ cell-free translation system for producing human HtrA3. HtrA3 underwent autocleavage when synthesized at 17 °C. When the synthesis temperature was lowered to 4 °C, full-length HtrA3 was successfully produced and proteolytically active. Catalytic site serine substitution with alanine (S305A) stabilized HtrA3 while abolishing its protease activity. This mutant was readily synthesized and stable at 17 °C. When used with glutathione S-transferase (GST) pull-down assay, S305A HtrA3 was a valuable bait in searching for endogenous HtrA3 binding proteins. Thus, we demonstrated the unique utility of the wheat-germ cell-free translation system for producing and characterizing human HtrA3. These strategies will be likely applicable to a wide range of proteases.     

Characterization of the chaperone-like activity of HtrA (DegP) protein from Escherichia coli under the conditions of heat shock

The protective action of chaperone-like activity of HtrA protease against protein aggregation was studied. High levels of proteolytically inactive HtrAS210A (active center serine replaced by alanine) suppressed the temperature-sensitive phenotype of the htrA mutants. The ability of HtrAS210A to alleviate the lethality of htrA bacteria at high temperatures correlated well with the observed decrease of cellular level of large protein aggregates in cells overproducing HtrAS210A. The in vitro experiments proved that HtrA was very efficient in inhibiting the unfolded substrate (lysozyme) aggregation over a wide range of temperatures (30-45 °C). HtrA was able to bind to the denatured polypeptides and as a consequence limited their ability to form large aggregates. Our results suggest that HtrA may protect the bacterial cells from deleterious effects of heat shock not only by degrading the damaged proteins but by combination of the proteolytic and chaperoning activities.     

Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin

The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections.     

The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli

The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop’s region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections.     

Expression of human T-cell leukemia virus type I protease in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected. To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli. The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000. A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro. Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity. These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E. coli.     

Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.