title: Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2.
abstract: HtrA2, a complex trimeric pyramidal mitochondrial serine protease that regulates critical biological functions and diseases, including apoptosis and cancer, is a promising therapeutic target. It promotes apoptosis through multiple pathways, complex mechanisms of which are still elusive. The existing model of activation that emphasizes relative intramolecular movements between C-terminal PDZ and protease domains (PDZ-protease collapse in inactive and resting states) has not been able to unambiguously demonstrate dynamics of its actions. Using structure-guided design, molecular biology and protein biochemistry, we obtained various combinations of HtrA2 domains and mutants. Conformational changes and stability were characterized using molecular dynamics simulation and spectroscopic tools while functional enzymology delineated their roles in regulating enzyme catalysis. Quantitative Förster resonance energy transfer showed lesser intramolecular PDZ-protease distance in trimeric HtrA2 compared to its inactive monomeric counterpart (∼21 and ∼22.3 Å, respectively, at 37°C). Our findings highlight importance of N-terminal region, oligomerization, and intricate intermolecular PDZ-protease interaction in proper active-site formation, enzyme-substrate complex stabilization, and hence HtrA2 functions. These observations redefine the existing activation model and showcase a unique example of how precise interdomain coordination, plasticity, and intermolecular contacts lead to distinct functional properties and hence provide new insights into HtrA2 structure, function, and dynamics.
authors.affiliation: Tata Memorial Centre, Navi Mumbai, India.
authors.forename: Lalith K
authors.initials: LK
authors.lastname: Chaganti
authors.forename: Raja Reddy
authors.initials: RR
authors.lastname: Kuppili
authors.forename: Kakoli
authors.initials: K
authors.lastname: Bose
mesh_terms: D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002942:Circular Dichroism; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D007700:Kinetics; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D054731:PDZ Domains / Q000235:genetics; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D002364:Caseins; D024101:Mitochondrial Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2
keywords: PDZ; dynamics; serine protease domain