(306a) Unraveling the Energy Transition Network That Dictates Allosteric Activation of Proapoptotic High-Temperature Requirement Protease A2 for Therapeutic Intervention

The human mitochondrial serine protease High-temperature requirement A2 (HtrA2) is associated with various diseases including neurodegenerative disorders and cancer. Each monomeric subunit of the trimeric enzyme comprises an N-terminal region that is responsible for its trimerization, a serine protease domain and a C-terminal regulatory domain. Despite the availability of structural details, the reports on HtrA2’s mechanistic allosteric regulation that varies with the type of activation signals still remain non-concordant . Therefore, to expound the role of the regulatory PDZ (Postsynaptic density-95/Discs large/Zonula occludens-1) domains in multimodal activation of HtrA2, we used a unique protein engineering approach to generate heterotrimeric HtrA2 variants comprising different numbers of PDZs (W denotes full length monomeric protease and Δ denotes variant that lacks PDZ-as shown in the figure) and/or active-site mutations. Sequential deletion of PDZs from the trimeric ensemble significantly affected its residual enzymatic activity that is studied using fluorescently-labeled biosensor probes. These experimental data helped proffer a hypothesis advocating inter-subunit allosteric cross-talk via PDZs in HtrA2. Furthermore, structural (x-ray crystallographic) and computational snapshots affirmed the role of PDZs in secondary structural element formation around the regulatory loops and coordinated reorganization of the N-terminal region. Therefore, apart from providing cues for devising structure-guided tailor-made therapeutic strategies, this study establishes a physiologically relevant working model of complex allosteric regulation through a trans-mediated cooperatively shared energy landscape.