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Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

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., (2010 Elsevier Inc. All rights reserved.)

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.

HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE, indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide. Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.

The HtrA (DegP) protein of Escherichia coli is a heat shock serine protease, essential for cell survival only at temperatures above 42 degrees C. It has been shown by genetic experiments that HtrA is an envelope protease, functioning in the periplasmic space. To clarify the cellular localization of HtrA, E. coli cells were fractionated, and HtrA was not detected by the immunoblotting technique in the periplasm or in the fraction of soluble proteins but was found in the inner membrane. The protein could be partially eluted from the total membrane fraction by a high ionic strength solution, whereas solutions affecting protein conformation released HtrA almost completely. These results, taken together with the evidence showing that HtrA functions in the periplasm, indicate that HtrA is a peripheral membrane protein, localized on the periplasmic side of the inner membrane. As the first step toward solving the problem of HtrA-membrane interactions, the structure of HtrA in the presence of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or cardiolipin (CL) was analyzed by fluorescence and Fourier-transform infrared spectroscopy. The infrared and fluorescence data indicated an interaction of HtrA with PG and CL but not with PE suspensions. Fluorescence spectroscopy revealed that this interaction was at the level of the polar head group of the phospholipid. In the PG/HtrA system, small changes were observed in the HtrA secondary structure and a remarkable decrease of the thermal stability of the protein, which suggested changes in HtrA tertiary structure. This suggestion was supported by fluorescence data that showed a shift of the fluorescence emission spectrum of HtrA tyrosine residues in the presence of PG and a reduced fluorescence intensity, phenomena not observed in the presence of PE or CL suspensions. Infrared data revealed also that the interaction of HtrA with PG leads to a protection of unfolded protein against aggregation at relatively low temperatures. The conformational changes of HtrA in the presence of PG influenced the proteolytic activity of HtrA by increasing it at the temperatures 37-45 degrees C and inhibiting it at 50-55 degrees C. CL inhibited HtrA activity at all of the temperatures tested.

The HtrA protease of Escherichia coli, identical with the DegP protease, is a 48-kDa heat shock protein, indispensable for bacterial survival only at temperatures above 42 degrees C. Proteolytic activity of HtrA is inhibited by diisopropyl fluorophosphate, suggesting that HtrA is a serine protease. We have recently found that mutational alteration of serine in position 210 of the mature HtrA or of histidine in position 105 totally eliminated proteolytic activity of HtrA. However, little was known about the consequences of the mutations on HtrA conformation. In this work, Fourier transform infrared spectroscopy has been used to examine the conformation in aqueous solution of wild-type HtrA and mutant HtrAS210 and HtrAH105 proteins. The spectra were collected at different temperatures in order to gain information also on the thermal stability of the three proteins. The analysis of HtrA protein spectrum, by resolution-enhancement methods, revealed that beta-sheet is the major structural element of the conformation of HtrA. Deconvoluted as well as second derivative spectra of wild-type HtrA and mutant HtrAS210 and HtrAH105 collected at 20 degrees C were identical, indicating no differences in the secondary structure of these proteins. The analysis of spectra obtained at different temperatures revealed a maximum of protein denaturation within 65-70 degrees C for wild-type HtrA as well as for the HtrAS210 and HtrAH105 mutant proteins. However, the thermal denaturation pattern of wild-type HtrA revealed a lower cooperativity in the denaturation process as compared to the mutant proteins which instead behaved similarly. These data suggest that the mutations in HtrA protein induced minor changes in the tertiary structure of the protein (most likely located at the mutation sites). Our results strongly support the idea that Ser210 and His105 may represent two elements of the active-site triad (Ser, His, and Asp), found in most serine proteases. We have also found that in vitro, in the range from 37 to 55 degrees C, the proteolytic activity of HtrA rapidly increased with temperature and that HtrA activity remained unchanged for at least 4 h at 45 degrees C.