Structural modeling and role of HAX-1 as a positive allosteric modulator of human serine protease HtrA2.

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.

PDZscape: a comprehensive PDZ-protein database.

PDZ-containing proteins comprise one of the most widely distributed protein families playing major role in localization and membrane receptor clustering. They are hence important regulators of signal transduction in cellular pathways. Although knowledge on these proteins has increased exponentially, the existing database 'PDZBase' is limited by presence of only 339 proteins as it dates back to 2004 when very little data was available. Thus, lack of exclusive information on this protein family led us to develop PDZscape. 'PDZscape' encompasses the complete available information on 58,648 PDZ-containing proteins with their known and putative binding partners on one platform. It has a user-friendly web interface that can be easily queried with external protein identifiers. With unique integration of prominent databases including NCBI, UniProtKB, Swiss-Prot, Pubmed, PDB, STRING, IntAct, KEGG, Pfam and Protein Mutant Database, it provides detailed information on PDZ interactome apart from the customized BLAST option. Most importantly, this database encompasses the mutations and diseases associated with PDZ containing proteins manually curated by our group, thus making it a comprehensive compilation. It also features tools to query the database using sequence (PDZ-Blast) and to find if protein of interest is a PDZ-binding protein. PDZscape is freely available at http://www.actrec.gov.in:8080/pdzscape .

Intricate structural coordination and domain plasticity regulate activity of serine protease HtrA2.

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.