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HAX-1, a multifunctional protein involved in cell proliferation, calcium homeostasis, and regulation of apoptosis, is a promising therapeutic target. It regulates apoptosis through multiple pathways, understanding of which is limited by the obscurity of its structural details and its intricate interaction with its cellular partners. Therefore, using computational modeling, biochemical, functional enzymology and spectroscopic tools, we predicted the structure of HAX-1 as well as delineated its interaction with one of it pro-apoptotic partner, HtrA2. In this study, three-dimensional structure of HAX-1 was predicted by threading and ab initio tools that were validated using limited proteolysis and fluorescence quenching studies. Our pull-down studies distinctly demonstrate that the interaction of HtrA2 with HAX-1 is directly through its protease domain and not via the conventional PDZ domain. Enzymology studies further depicted that HAX-1 acts as an allosteric activator of HtrA2. This ‘allosteric regulation’ offers promising opportunities for the specific control and functional modulation of a wide range of biological processes associated with HtrA2. Hence, this study for the first time dissects the structural architecture of HAX-1 and elucidates its role in PDZ-independent activation of HtrA2.

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