title: Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants.
abstract: HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not allow to explain an increased activity of HtrA2V226K.
authors: Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdansk, Poland.
authors: Artur
authors: A
authors: Gieldon
authors: Department of Biochemistry, Faculty of Biology, University of Gdansk, 80-308, Gdansk, Poland.
authors: Dorota
authors: D
authors: Zurawa-Janicka
authors: Department of Biochemistry, Faculty of Biology, University of Gdansk, 80-308, Gdansk, Poland.
authors: Miroslaw
authors: M
authors: Jarzab
authors: Department of Biochemistry, Faculty of Biology, University of Gdansk, 80-308, Gdansk, Poland.
authors: Tomasz
authors: T
authors: Wenta
authors: Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland.
authors: Przemyslaw
authors: P
authors: Golik
authors: Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland.|Malopolska Centre of Biotechnology, ul. Gronostajowa 7a, 30-387, Krakow, Poland.
authors: Grzegorz
authors: G
authors: Dubin
authors: Department of Biochemistry, Faculty of Biology, University of Gdansk, 80-308, Gdansk, Poland.
authors: Barbara
authors: B
authors: Lipinska
authors: Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdansk, Poland.
authors: Jerzy
authors: J
authors: Ciarkowski
mesh_terms: D018360:Crystallography, X-Ray; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D024101:Mitochondrial Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2