title: Intra- and intersubunit changes accompanying thermal activation of the HtrA2(Omi) protease homotrimer.
abstract: HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limits access both to the PDZ peptide (carboxylate) binding site and to the PD catalytic center. The molecular mechanism of activation is not well understood. To further the knowledge of HtrA2 thermal activation we monitored the dynamics of the PDZ-PD interactions during temperature increase using tryptophan-induced quenching (TrIQ) method. The TrIQ results suggested that during activation the PDZ domain changed its position versus PD inside a subunit, including a prominent change affecting the L3 regulatory loop of PD, and also changed its interactions with the PD of the adjacent subunit (PD*), specifically with its L1* regulatory loop containing the active site serine. The α5 helix of PDZ was involved in both, the intra- and intersubunit changes of interactions and thus seems to play an important role in HtrA2 activation. The amino acid substitutions designed to decrease the PDZ interactions with the PD or PD* promoted protease activity at a wide range of temperatures, which supports the conclusions based on the TrIQ analysis. The model presented in this work describes PDZ movement in relation to PD and PD*, resulting in an increased access to the peptide binding and active sites, and conformational changes of the L3 and L1* loops.
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Miroslaw
authors.initials: M
authors.lastname: Jarzab
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Wenta
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Zurawa-Janicka
authors.affiliation: Department of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Polit
authors.affiliation: Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland.
authors.forename: Artur J
authors.initials: AJ
authors.lastname: Gieldon
authors.affiliation: Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wysocka
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Glaza
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Skorko-Glonek
authors.affiliation: Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ciarkowski
authors.affiliation: Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland. Electronic address: barbara.lipinska@biol.ug.edu.pl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipinska
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D020134:Catalytic Domain; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054731:PDZ Domains; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
chemical_list: D024101:Mitochondrial Proteins; D010455:Peptides; D014364:Tryptophan; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2
keywords: Allosteric regulation; HtrA2 activation cascade; HtrA2(Omi); PDZ domain; Proteolytic enzyme; TrIQ analysis