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Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2
Lalith K Chaganti1, Raja Reddy Kuppili, Kakoli Bose
1Tata Memorial Centre, Navi Mumbai, India.
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.
Insights
Mitochondrial serine protease HtrA2 (High temperature requirement A2) regulates apoptosis and cancer. New findings reveal its N-terminal region, oligomerization, and intermolecular interactions are crucial for its function, redefining its activation model.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- HtrA2 (High temperature requirement A2) is a mitochondrial serine protease implicated in apoptosis and cancer.
- Its complex activation mechanisms and dynamics are not fully understood, with existing models focusing on intramolecular domain movements.
- HtrA2's role in critical biological functions and diseases makes it a significant therapeutic target.
Purpose of the Study:
- To elucidate the structure-function dynamics of HtrA2, particularly its activation mechanisms.
- To investigate the role of different HtrA2 domains, oligomerization, and intermolecular interactions in enzyme catalysis.
- To redefine the existing model of HtrA2 activation based on new structural and dynamic insights.
Main Methods:
- Structure-guided design, molecular biology, and protein biochemistry were employed to generate HtrA2 domain combinations and mutants.
- Molecular dynamics simulations and spectroscopic tools were used to characterize conformational changes and stability.
- Functional enzymology and quantitative Förster resonance energy transfer (qFRET) were utilized to assess enzyme catalysis and domain distances.
Main Results:
- Quantitative Förster resonance energy transfer revealed a smaller intramolecular PDZ-protease distance in trimeric HtrA2 (∼21 Å) compared to its monomeric form (∼22.3 Å) at 37°C.
- The study identified the N-terminal region, oligomerization, and intermolecular PDZ-protease interactions as critical for active-site formation and substrate stabilization.
- These findings challenge the existing activation model by emphasizing intermolecular contacts and domain coordination.
Conclusions:
- HtrA2 function is intricately regulated by its N-terminal region, oligomerization state, and precise intermolecular domain interactions.
- The study provides a redefined model for HtrA2 activation, highlighting the importance of interdomain coordination and plasticity.
- These insights offer a deeper understanding of HtrA2 structure, function, and dynamics, with implications for therapeutic strategies.
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