Dual regulatory switch confers tighter control on HtrA2 proteolytic activity

Abstract

High‐temperature requirement protease A 2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C ‐terminal post‐synaptic density protein, D rosophila disc large tumor suppressor, zonula occludens‐1 protein ( PDZ ) domain of H trA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, H trA2 exhibits an additional level of functional modulation through its unique N ‐terminus, as is evident from ‘inhibitor of apoptosis proteins’ binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N ‐terminal ligand‐mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ ‐independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops ( L 3, LD , L 1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site‐specific effector molecules. Structured digital abstract HtrA2 cleaves beta casein by enzymatic study ( 1 , 2 , 3 ) HtrA2 binds to XIAP-BIR2 by surface plasmon resonance ( View interaction ) HtrA2 binds to XIAP-BIR2 pep by surface plasmon resonance ( View interaction ) HtrA2 binds to XIAP-BIR3 by surface plasmon resonance ( View interaction ) HtrA2 binds to XIAP-BIR3 pep by surface plasmon resonance ( View interaction ) XIAP binds to HtrA2 by pull down ( View interaction ) XIAP-BIR2 binds to HtrA2 by pull down ( View interaction ) ( View interaction ) XIAP-BIR3 binds to HtrA2 by pull down ( View interaction ) ( View interaction ) ( View interaction )