Abstract
HtrA2 (high-temperature requirement 2) is a human mitochondrial protease that has a role in apoptosis and Parkinson’s disease. The structure of HtrA2 with an intact catalytic triad was determined, revealing a conformational change in the active site loops, involving mainly the regulatory LD loop, which resulted in burial of the catalytic serine relative to the previously reported structure of the proteolytically inactive mutant. Mutations in the loops surrounding the active site that significantly restricted their mobility, reduced proteolytic activity both in vitro and in cells, suggesting that regulation of HtrA2 activity cannot be explained by a simple transition to an activated conformational state with enhanced active site accessibility. Manipulation of solvent viscosity highlighted an unusual bi-phasic behavior of the enzymatic activity, which together with MD calculations supports the importance of motion in the regulation of the activity of HtrA2. HtrA2 is an unusually thermostable enzyme (TM=97.3 °C), a trait often associated with structural rigidity, not dynamic motion. We suggest that this thermostability functions to provide a stable scaffold for the observed loop motions, allowing them a relatively free conformational search within a rather restricted volume.
Highlights
HtrA proteins, named for the inability of deletion mutants to survive elevated temperatures, are widely distributed throughout nature
HtrAs are homo-trimeric enzymes, with each subunit composed of a serine protease domain and one or more regulatory PDZ domains.[1]
The structure of human HtrA2 catalytic serine mutant S306A, showed the PDZ packing against the protease domain.[14]
Summary
HtrA (high-temperature requirement) proteins, named for the inability of deletion mutants to survive elevated temperatures, are widely distributed throughout nature They maintain protein quality in the periplasmic space of Gram-negative bacteria or in the intermembrane space of the mitochondria in animals and plants.[1,2,3] HtrAs are homo-trimeric enzymes, with each subunit composed of a serine protease domain and one or more regulatory PDZ domains.[1] The protease domain, structurally conserved in this family, has a chymotrypsin-like fold and several flexible loops – including the surface loops 1-3 (L1-L3), and loops A-D (LA-LD)4 – that have critical roles in substrate specificity and allosteric regulation.[1,5] Unlike trypsin, which is activated by a proteolytic event that induces a disorder/order transition,[1] regulation of HtrA proteases has been linked to (i) ligand binding,[2] typically small hydrophobic peptides to the PDZ domain,[6,7,8] or (ii) increased temperature.[9,10]. Mutational studies, combined with structural and molecular dynamics analysis, and the effect of viscosity in HtrA2/ WT activity, suggest that motion in the LD and L1 loops is critical for HtrA2 proteolytic activity
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call