title: 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.
authors.affiliation: Department of Biochemistry, University of Gdansk, Kladki 24, 80-952, Gdansk, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Zurawa-Janicka
authors.forename: Miroslaw
authors.initials: M
authors.lastname: Jarzab
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Polit
authors.forename: Joanna
authors.initials: J
authors.lastname: Skorko-Glonek
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.forename: Agata
authors.initials: A
authors.lastname: Gitlin
authors.forename: Artur
authors.initials: A
authors.lastname: Gieldon
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ciarkowski
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Glaza
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Lubomska
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipinska
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D007700:Kinetics; D008027:Light; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D054731:PDZ Domains; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism; D014867:Water / Q000737:chemistry
chemical_list: D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D014867:Water; D014364:Tryptophan; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2