Dual regulatory switch confers tighter control on HtrA2 proteolytic activity.
High-temperature requirement protease A2 (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, Drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 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 (L3, LD, L1), 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.
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.
Nitu
N
Singh
Areetha
A
D'Souza
Anuradha
A
Cholleti
G Madhavi
GM
Sastry
Kakoli
K
Bose
D000494:Allosteric Regulation; D000818:Animals; D018360:Crystallography, X-Ray; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D007700:Kinetics; D008024:Ligands; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D054731:PDZ Domains; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry / Q000378:metabolism
D029721:Drosophila Proteins; D008024:Ligands; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; C530280:HtrA2 protein, Drosophila
IAP; N-terminus; PDZ; allostery; serine protease