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

Abstract

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

Differential Contribution of Tryptophans to the Folding and Stability of the Attachment Invasion Locus Transmembrane β-Barrel from Yersinia pestis

“…In low DPRs, W149F particularly possesses the most solvent-accessible Trp ( k q = 1.85 × 10 9  M −1 s −1 ; Table 1), while in high DPRs, the k q is lowered in all three proteins, with WT and W42F now possessing relatively less exposed Trp residues compared to W149F. However, since all the k q values are within the accepted range for moderately exposed indoles ( k q for buried Trp lies in the 0.6–1.0 × 10 9  M −1 s −1 range4546), we propose that all three proteins show the presence of interfacial Trp residue(s); differences arising from their respective interaction pockets give rise to the observed variation in K SV and <τ>. Put together, these properties provide the order WT ≥ W42F > W149F for increase in solvent exposure of the indole.…”

Design and synthesis of new substrates of HtrA2 protease

“…The activity of all synthesized substrates or its libraries was evaluated using human recombinant HtrA2 [16]its inactive analogue with mutated Ser, also proteolytical domain called HtrA2 (-)PDZ was used Additionally HTRA1 purchased from R&D systems and bacterial HTRA isolated from E.coli was used as a control. The assay was performed using 96-well black microtiter plates in a final volume of 200 µl.…”

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli

“…Spectroscopic Measurements-Far-UV circular dichroism spectra (185-260 nm) of the HtrA proteins (0.5 mg/ml) were recorded in 10 mM NaHPO 4 , pH 8.0, at 20°C in 1-mm path length cells and using a JASCO J-815 (Japan) spectropolarimeter as described in Zurawa-Janicka et al (29). Steady-state fluorescence, fluorescence quenching, and time-resolved fluorescence decay measurements were carried out as described in Sobiecka-Szkatula et al (12).…”