Share this post on:

Of the crystal structure with partially missing active site loops and flexible PDZprotease linker has been unable to unambiguously determine the role of dynamics and allostery if any in HtrA2 activation and specificity. Therefore, to understand the molecular details of its mechanism of action, dynamics study at the substrate binding site and active site pocket becomes imperative. HtrA2 belongs to a serine protease family that is conserved from prokaryotes to humans [12] where allostery is a common mechanism for protease activation in some of its homologs. DegS, a bacterial counterpart of HtrA2, allosterically stabilizes the active site pocket upon substrate binding at the distal PDZ domain [13]. DegP, the most extensively studied protein of the family, has a cage-like hexameric structure whose activation is regulated by allostery and oligomerization. Peptide binding to distal PDZAllosteric Regulation of HtrAdomain leads to rearrangement of the catalytic pocket into Gracillin manufacturer enzymatically competent form that readily oligomerizes and renders stability to the active conformation [14]. With an aim at 18325633 understanding the conformational changes and structural plasticity that govern HtrA2 activity and specificity, we took an in silico approach to study the movements of flexible regions of the protein upon ligand binding. The PDZ domain of HtrA2 has a known hydrophobic substrate binding YIGV pocket (similar to GLGF motif) which is deeply embedded within the trimeric protein structure with P225 and V226 from the serine protease domain occupying the groove [4,15]. This structural arrangement makes it impossible for substrate protein to bind without significant conformational changes. Thus, to examine whether allosteric modulation through an alternative site is involved in substrate binding and catalysis of HtrA2, molecular dynamics simulation (MDS) approach with a bound peptide activator was used to look into the structural rearrangements that occur in nanosecond time scale. Although the information usually obtained from MDS is restricted primarily to movements in the accessible and flexible regions of a protein, it nonetheless contributes significantly towards understanding of the overall structural rearrangement and dynamics during its allosteric activation. In our study, we modelled the entire mature protease by filling in the missing regions using Prime 3.0 [16], followed by energy minimization with GRoningen MAchine for Chemical Simulation or GROMACS [17]. Identification of the putative binding site(s) on HtrA2 was done using SiteMap 2.5 [18] and the selective binding pocket (SBP) for the ligand was chosen based on optimum energy parameters. Peptides at SBP were Licochalcone-A web docked from our peptide library that was generated based on available literature reports [19,20,21] and structural complementarities. MDS of the docked structures was done using Desmond 2010 [22] which provided critical information on loop and linker movements in HtrA2. These results combined with mutational and enzymology studies show that upon activator binding at the novel allosteric pocket, SBP, the linker at the PDZ-protease interface and loops L1, LA and LD around the catalytic groove undergo rearrangements in a coordinated manner so as to form an efficient active site pocket. Moreover, the PDZ domains mediate intersubunit interactions which stabilize the oxyanion hole. These observations highlight the importance of allostery which might be an important prerequisite for an active conform.Of the crystal structure with partially missing active site loops and flexible PDZprotease linker has been unable to unambiguously determine the role of dynamics and allostery if any in HtrA2 activation and specificity. Therefore, to understand the molecular details of its mechanism of action, dynamics study at the substrate binding site and active site pocket becomes imperative. HtrA2 belongs to a serine protease family that is conserved from prokaryotes to humans [12] where allostery is a common mechanism for protease activation in some of its homologs. DegS, a bacterial counterpart of HtrA2, allosterically stabilizes the active site pocket upon substrate binding at the distal PDZ domain [13]. DegP, the most extensively studied protein of the family, has a cage-like hexameric structure whose activation is regulated by allostery and oligomerization. Peptide binding to distal PDZAllosteric Regulation of HtrAdomain leads to rearrangement of the catalytic pocket into enzymatically competent form that readily oligomerizes and renders stability to the active conformation [14]. With an aim at 18325633 understanding the conformational changes and structural plasticity that govern HtrA2 activity and specificity, we took an in silico approach to study the movements of flexible regions of the protein upon ligand binding. The PDZ domain of HtrA2 has a known hydrophobic substrate binding YIGV pocket (similar to GLGF motif) which is deeply embedded within the trimeric protein structure with P225 and V226 from the serine protease domain occupying the groove [4,15]. This structural arrangement makes it impossible for substrate protein to bind without significant conformational changes. Thus, to examine whether allosteric modulation through an alternative site is involved in substrate binding and catalysis of HtrA2, molecular dynamics simulation (MDS) approach with a bound peptide activator was used to look into the structural rearrangements that occur in nanosecond time scale. Although the information usually obtained from MDS is restricted primarily to movements in the accessible and flexible regions of a protein, it nonetheless contributes significantly towards understanding of the overall structural rearrangement and dynamics during its allosteric activation. In our study, we modelled the entire mature protease by filling in the missing regions using Prime 3.0 [16], followed by energy minimization with GRoningen MAchine for Chemical Simulation or GROMACS [17]. Identification of the putative binding site(s) on HtrA2 was done using SiteMap 2.5 [18] and the selective binding pocket (SBP) for the ligand was chosen based on optimum energy parameters. Peptides at SBP were docked from our peptide library that was generated based on available literature reports [19,20,21] and structural complementarities. MDS of the docked structures was done using Desmond 2010 [22] which provided critical information on loop and linker movements in HtrA2. These results combined with mutational and enzymology studies show that upon activator binding at the novel allosteric pocket, SBP, the linker at the PDZ-protease interface and loops L1, LA and LD around the catalytic groove undergo rearrangements in a coordinated manner so as to form an efficient active site pocket. Moreover, the PDZ domains mediate intersubunit interactions which stabilize the oxyanion hole. These observations highlight the importance of allostery which might be an important prerequisite for an active conform.

Share this post on:

Author: bet-bromodomain.