Allosteric Regulation of Serine Protease HtrA2 through Novel Non-Canonical Substrate Binding Pocket

<div><p>HtrA2, a trimeric proapoptotic serine protease is involved in several diseases including cancer and neurodegenerative disorders. Its unique ability to mediate apoptosis via multiple pathways makes it an important therapeutic target. In HtrA2, C-terminal PDZ domain upon substrate binding regulates its functions through coordinated conformational changes the mechanism of which is yet to be elucidated. Although allostery has been found in some of its homologs, it has not been characterized in HtrA2 so far. Here, with an <em>in silico</em> and biochemical approach we have shown that allostery does regulate HtrA2 activity. Our studies identified a novel non-canonical selective binding pocket in HtrA2 which initiates signal propagation to the distal active site through a complex allosteric mechanism. This non-classical binding pocket is unique among HtrA family proteins and thus unfolds a novel mechanism of regulation of HtrA2 activity and hence apoptosis.</p> </div

Representative surface structures of peptide activator docked HtrA2.

<p>a. Peptide GSAWFSF -HtrA2 complex and b. Peptide GQYYFV-HtrA2 complex. The former peptide represents putative SBP binding peptide in Pea-15 and the latter is a peptide obtained from the literature. The common interacting residues from SBP for both the peptides are labelled and are shown as blue sticks. PD denotes serine protease domain in both the Figures.</p

Putative binding sites in HtrA2 identified by SiteMap tool.

<p>Putative binding sites in HtrA2 identified by SiteMap tool.</p

Ribbon model of HtrA2 structures (PDB ID: 1LCY).

<p>a. Domain organization of HtrA2 protease which comprises N-terminal region (blue), protease domain denoted as PD (yellow) and PDZ domain (red) at C-terminal end. b. Structural alignment of loop refined (light magenta) and unrefined (light green) structures of HtrA2 protein with modelled N-terminal AVPS, loop L3 (residues 142–162) and hinge region (residues 211–225) built with Prime (Schrödinger 2011). On refinement, loop L3 and hinge region are reorganized so as to define new regions at the protease and PDZ domain interface. c. Selective binding pocket (SBP) on HtrA2. The energy minimised structure of HtrA2 after modelling flexible regions in the protein is represented as a ribbon model. The binding site designated as SBP, selected on the basis of the Sitemap score and residue analyses, is located at the interface of PDZ and protease domain and shown as a multi-coloured mesh.</p

Peptide docking of HtrA2 and identification of interacting residues.

<p>The possible residues which are involved in hydrogen bonding and Vander Waal’s interactions along with Glide scores are mentioned.</p

Steady state kinetic parameters for HtrA2 wild type, variants and mutants with β-casein as the substrate.

<p>Steady state kinetic parameters for HtrA2 wild type, variants and mutants with β-casein as the substrate.</p

Allosteric model for HtrA2 protease activity.

<p>The substrate protein binds to relatively exposed part of SBP due to inaccessibility of the YIGV groove which triggers opening up of the PDZ domain. This reorientation makes the YIGV groove accessible for substrate interaction and the PDZ of a subunit moves closer to the protease domain of the adjacent subunit leading to formation of a proper active site and oxyanion hole. This complex allosteric signal propagation leads to subsequent substrate binding and catalysis at the active site pocket. Thus structural perturbations at these two distant sites (SBP and catalytic pocket) might be dynamically coupled to the canonical peptide binding groove through a complex allosteric mechanism.</p

Domain wise conformational changes induced on peptide binding at SBP.

<p>a. The structural alignment of minimum energy structure of the peptide bound GQYYFV-HtrA2 complex (light pink) and unbound structure (green) displays orientation of the movement of the hinge region and the α-helices of PDZ. b. The structural alignment of GSAWFSF-HtrA2 complex (light pink) and unbound structure (green). Graphical representations of the RMSD for the 30 ns MDS trajectory of the following: c. HtrA2–GQYYFV complex. d. unbound HtrA2 (negative control). e. HtrA2–GSAWFSF complex. The stretch of residues selected for each set of RMSD calculations are shown on the right of panel c.</p

Comparison of Kinetics, Toxicity, Oligomer Formation, and Membrane Binding Capacity of α‑Synuclein Familial Mutations at the A53 Site, Including the Newly Discovered A53V Mutation

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson’s disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to late-onset PD. In this study, we characterized for the first time the biophysical properties of A53V, including the aggregation propensities, toxicity of aggregated species, and membrane binding capability, along with those of all familial mutations at the A53 position. Our data suggest that the A53V mutation accelerates fibrillation of α-Syn without affecting the overall morphology or cytotoxicity of fibrils compared to those of the wild-type (WT) protein. The aggregation propensity for A53 mutants is found to decrease in the following order: A53T > A53V > WT > A53E. In addition, a time course aggregation study reveals that the A53V mutant promotes early oligomerization similar to the case for the A53T mutation. It promotes the largest amount of oligomer formation immediately after dissolution, which is cytotoxic. Although in the presence of membrane-mimicking environments, the A53V mutation showed an extent of helix induction capacity similar to that of the WT protein, it exhibited less binding to lipid vesicles. The nuclear magnetic resonance study revealed unique chemical shift perturbations caused by the A53V mutation compared to those caused by other mutations at the A53 site. This study might help to establish the disease-causing mechanism of A53V in PD pathology

Risk factors for unfavourable response at the end of treatment in new smear positive pulmonary TB patients [N = 1543].

<p>Risk factors for unfavourable response at the end of treatment in new smear positive pulmonary TB patients [N = 1543].</p