Ole and catalytic triad residues represented as sticks for peptide GQYYFV bound (magenta) and unbound (green) structures. PD denotes serine protease domain of HtrA2. b. Overlay of the oxyanion hole and catalytic triad residues represented as sticks for peptide GSAWFSF bound (red) and unbound (limon green) structures. c. Role of PDZ in the formation of proper active site formation. The structural superposition of GQYYFV bound (pink) and unbound (green) structures shows a5 helix of PDZ of one subunit moves towards the LD loop and oxyanion hole of the adjacent subunit. The positions of the residues in the oxyanion hole are denoted as 0, 21, 22 and 23. doi:10.1371/More emphasis on pathways whose member genes show fold changes that journal.pone.0055416.gdecrease in catalytic efficiency in SBP mutants. This observation suggests interaction of substrate protein with SBP brings about rearrangement around the active site of the enzyme by positively influencing its activity thus behaving as an allosteric regulator. The SBP mutants (N216A/S219A and E292A) show apparent decrease in Vmax without significantly altering the apparent Km (with L2 specificity pocket mostly unaltered) and hence follow the `V system’ of allosteric modulation [29]. In this system, both the relaxed (R) and the tensed (T) states bind the substrate at the active site with similar affinity while the peptide (activator) at SBP binds the R and T states with different affinity. This differential affinity of the peptide towards SBP along with R state stabilization shifts the equilibrium towards R state thus positively influencing its turnover rate and hence catalytic efficiency which has been observed in case of HtrA2. In N-SPD, where the PDZ domain is absent, apparent decrease in Km can be attributed to greater accessibility of the substrate to the active site. However, since the change in binding affinity is notlarge, the specificity pocket might be mostly unaltered compared to the wild type which is confirmed through our MD studies where the loop L2 remains mostly unaltered. Interestingly, 22948146 kcat value in N-SPD has been found to decrease significantly which is suggestive of either a malformed oxyanion hole and/or decrease in overall protein stability which might be due to absence of supporting PDZ domain. However, similar studies with F16D (monomeric full length HtrA2 mutant) also show significant decrease in turnover rate and catalytic efficiency which accentuates the importance of intermolecular and not intramolecular PDZ-protease crosstalk in Title Loaded From File trimeric HtrA2. Our MDS supports this observation by demonstrating that in the peptide bound form of HtrA2, a5* of PDZ* moves towards LD loop of protease domain of adjacent subunit thus pushing phenyl ring of F170 of the oxyanion hole towards H65 of the catalytic triad (Figure 6a). This reorientation in the oxyanion hole makes the protease poised for catalysis as seen in other HtrA family members as well [12] thus significantly enhancing the turnover rate. Therefore, intermolecular crosstalkAllosteric Regulation of HtrAstabilizes the active site and makes it catalytically competent establishing the requirement of complex trimeric architecture of the protease. The GLGF motif (YIGV in HtrA2) is the canonical peptide binding site [2,4] in PDZ domains. However, in HtrA2, it is deeply embedded within a hydrophobic groove where the residues are intertwined with each other through several intramolecular interactions making the site highly inaccessible to the binding of peptide [4]. Thus, peptide binding to YIGV i.Ole and catalytic triad residues represented as sticks for peptide GQYYFV bound (magenta) and unbound (green) structures. PD denotes serine protease domain of HtrA2. b. Overlay of the oxyanion hole and catalytic triad residues represented as sticks for peptide GSAWFSF bound (red) and unbound (limon green) structures. c. Role of PDZ in the formation of proper active site formation. The structural superposition of GQYYFV bound (pink) and unbound (green) structures shows a5 helix of PDZ of one subunit moves towards the LD loop and oxyanion hole of the adjacent subunit. The positions of the residues in the oxyanion hole are denoted as 0, 21, 22 and 23. doi:10.1371/journal.pone.0055416.gdecrease in catalytic efficiency in SBP mutants. This observation suggests interaction of substrate protein with SBP brings about rearrangement around the active site of the enzyme by positively influencing its activity thus behaving as an allosteric regulator. The SBP mutants (N216A/S219A and E292A) show apparent decrease in Vmax without significantly altering the apparent Km (with L2 specificity pocket mostly unaltered) and hence follow the `V system’ of allosteric modulation [29]. In this system, both the relaxed (R) and the tensed (T) states bind the substrate at the active site with similar affinity while the peptide (activator) at SBP binds the R and T states with different affinity. This differential affinity of the peptide towards SBP along with R state stabilization shifts the equilibrium towards R state thus positively influencing its turnover rate and hence catalytic efficiency which has been observed in case of HtrA2. In N-SPD, where the PDZ domain is absent, apparent decrease in Km can be attributed to greater accessibility of the substrate to the active site. However, since the change in binding affinity is notlarge, the specificity pocket might be mostly unaltered compared to the wild type which is confirmed through our MD studies where the loop L2 remains mostly unaltered. Interestingly, 22948146 kcat value in N-SPD has been found to decrease significantly which is suggestive of either a malformed oxyanion hole and/or decrease in overall protein stability which might be due to absence of supporting PDZ domain. However, similar studies with F16D (monomeric full length HtrA2 mutant) also show significant decrease in turnover rate and catalytic efficiency which accentuates the importance of intermolecular and not intramolecular PDZ-protease crosstalk in trimeric HtrA2. Our MDS supports this observation by demonstrating that in the peptide bound form of HtrA2, a5* of PDZ* moves towards LD loop of protease domain of adjacent subunit thus pushing phenyl ring of F170 of the oxyanion hole towards H65 of the catalytic triad (Figure 6a). This reorientation in the oxyanion hole makes the protease poised for catalysis as seen in other HtrA family members as well [12] thus significantly enhancing the turnover rate. Therefore, intermolecular crosstalkAllosteric Regulation of HtrAstabilizes the active site and makes it catalytically competent establishing the requirement of complex trimeric architecture of the protease. The GLGF motif (YIGV in HtrA2) is the canonical peptide binding site [2,4] in PDZ domains. However, in HtrA2, it is deeply embedded within a hydrophobic groove where the residues are intertwined with each other through several intramolecular interactions making the site highly inaccessible to the binding of peptide [4]. Thus, peptide binding to YIGV i.
