G targets due to several advantages they have overFigure 7. HIV-RT inhibitor 1 biological activity Structural comparison of PDZ domain orientation. a. Structural alignment of E.coli DegS (PDB ID: 1SOZ) and the peptide bound HtrA2 showing PDZ domains for both the proteins (represented in blue and yellow respectively) are oriented differently but the peptides, P1 (blue) and P2 (pink) represented as sticks for the respective proteins seem to bind to a structurally similar region. The GLGF substrate binding motif is exposed for DegS while buried for HtrA2 as shown in pink and blue respectively. b. Alignment of the peptide bound (pink) and unbound (green) structures at the region around the YIGV groove shows outward movement of the loops spanning residues 67?3 and 263?77 shown in red for the bound structures which leads to opening up of the YIGV groove. doi:10.1371/journal.pone.0055416.gAllosteric Regulation of HtrAFigure 8. Allosteric model for HtrA2 protease activity. 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. doi:10.1371/journal.pone.0055416.gorthosteric ligands that include more diversity, less toxicity and absolute subtype selectivity [32,33]. Therefore, designing suitable SBP binding peptides or peptidomimetics of HtrA2 might be an excellent approach to modulate HtrA2 functions for devising therapeutic strategies against various diseases it is associated with.Out of 5 pockets predicted, the site that scored the best based on its size, hydrophobic and hydrophilic characters, degree to which ligand might donate or accept hydrogen bonds and exposure to solvent was selected for further analysis. This site selective binding pocket (SBP) encompasses PDZ-protease interface with the involvement of hinge region and a part of PDZ domain (Table 1).Materials and Methods Loop Modeling and 22948146 Site PredictionCrystal structure of HtrA2 (PDB ID: 1LCY) [4] obtained from Protein Data Bank [34] has missing N-terminal residues (AVPSP) and two flexible regions (211RGEKKNSSSGISGSQ 225 and 149 ARDLGLPQT 157). These missing structures were modelled and loops were refined using Prime 3.0 (Schrodinger, LLC, New ?York, 2011). which was later subjected to molecular dynamics simulation for 5 ns with GROMACS, version 4.5.1 [17] to obtain the lowest energy structure of HtrA2. The binding sites were then predicted using SiteMap 2.5 (Schrodinger, LLC, New York, 2011). ?Peptide Designing and Molecular DockingBased on properties of amino acids lining the binding site, Fexinidazole supplier fragment docking (Glide XP, Schrodinger, LLC, New York, 2011) ?[35] approach was used to dock 20 amino acids and 8 functional group replicas (N-methylacetamide, methanol, phenol, benzene, propane, acetate ion, methylammonium, methylguanidinium) at SBP [36]. Based on properties of the amino acids that form SBP, replicas were chosen and were used for generating fragments in combinations of four as shown in Table S1.G targets due to several advantages they have overFigure 7. Structural comparison of PDZ domain orientation. a. Structural alignment of E.coli DegS (PDB ID: 1SOZ) and the peptide bound HtrA2 showing PDZ domains for both the proteins (represented in blue and yellow respectively) are oriented differently but the peptides, P1 (blue) and P2 (pink) represented as sticks for the respective proteins seem to bind to a structurally similar region. The GLGF substrate binding motif is exposed for DegS while buried for HtrA2 as shown in pink and blue respectively. b. Alignment of the peptide bound (pink) and unbound (green) structures at the region around the YIGV groove shows outward movement of the loops spanning residues 67?3 and 263?77 shown in red for the bound structures which leads to opening up of the YIGV groove. doi:10.1371/journal.pone.0055416.gAllosteric Regulation of HtrAFigure 8. Allosteric model for HtrA2 protease activity. 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. doi:10.1371/journal.pone.0055416.gorthosteric ligands that include more diversity, less toxicity and absolute subtype selectivity [32,33]. Therefore, designing suitable SBP binding peptides or peptidomimetics of HtrA2 might be an excellent approach to modulate HtrA2 functions for devising therapeutic strategies against various diseases it is associated with.Out of 5 pockets predicted, the site that scored the best based on its size, hydrophobic and hydrophilic characters, degree to which ligand might donate or accept hydrogen bonds and exposure to solvent was selected for further analysis. This site selective binding pocket (SBP) encompasses PDZ-protease interface with the involvement of hinge region and a part of PDZ domain (Table 1).Materials and Methods Loop Modeling and 22948146 Site PredictionCrystal structure of HtrA2 (PDB ID: 1LCY) [4] obtained from Protein Data Bank [34] has missing N-terminal residues (AVPSP) and two flexible regions (211RGEKKNSSSGISGSQ 225 and 149 ARDLGLPQT 157). These missing structures were modelled and loops were refined using Prime 3.0 (Schrodinger, LLC, New ?York, 2011). which was later subjected to molecular dynamics simulation for 5 ns with GROMACS, version 4.5.1 [17] to obtain the lowest energy structure of HtrA2. The binding sites were then predicted using SiteMap 2.5 (Schrodinger, LLC, New York, 2011). ?Peptide Designing and Molecular DockingBased on properties of amino acids lining the binding site, fragment docking (Glide XP, Schrodinger, LLC, New York, 2011) ?[35] approach was used to dock 20 amino acids and 8 functional group replicas (N-methylacetamide, methanol, phenol, benzene, propane, acetate ion, methylammonium, methylguanidinium) at SBP [36]. Based on properties of the amino acids that form SBP, replicas were chosen and were used for generating fragments in combinations of four as shown in Table S1.
