Abstract
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.
Highlights
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°C
Deconvoluted as well as second derivative spectra of wild-type HtrA and mutant HtrAS210 and HtrAH105 collected at 20°C were identical, indicating no differences in the secondary structure of these proteins
The earlier finding that mutational alterations of Ser210 to alanine and His105 to arginine eliminated enzymatic activity of HtrA suggested that Ser210 and His105 are the elements of the active site of the HtrA protease
Summary
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°C. 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 . 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). All the above mentioned heat shock proteins involved in proteolysis belong to the main heat shock regulon, regulated by the product of the rpoH gene, ifl factor, which enables RNA polymerase to recognize promoters of the heat shock genes [6] There is another heat shock regulon, positively regulated by a different heat shock sigma factor, 024 , which consists of two elements: the rpoH and htrA (degP) genes [15]. HtrA is synthesized as a 51-kDa unstable precursor protein and processed by removal of a 26-
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