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 Forster resonance energy transfer showed lesser intramolecular PDZ-protease distance in trimeric HtrA2 compared to its inactive monomeric counterpart (approximate to 21 and approximate to 22.3 angstrom, respectively, at 37 degrees 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.