D into the low resolution SANS model restricting the search to the part of the model that was not filled by the tRNA density using SUPCOMB. The normalized spatial discrepancy (NSD) value determined by SUPCOMB was 0.54, indicating a good fit between the two volumes (i.e., NSD below 1.0) [28]. In the resulting structure, Pth1 was oriented such that the positive patch and catalytic His20 residue were near the tRNA 3′ terminus. The high heterogeneity of the substrate resulted in a shape reflecting the various peptidyl-tRNA species and therefore, fitting the tRNA portion in the bead model has not been as straight forward as that of Pth1. In the end, the rigid tRNAPhe crystal structure was positioned manually leaving some unaccounted volume in the anticodon region. Variation in this region comes from plasticity of the tRNA molecule as a whole [29], mobility in the anticodon region [30], and heterogeneity of the peptidyl-tRNA used for data collection.Int. J. Mol. Sci. 2013,Figure 2. Model of Pth1:peptidyl-tRNA Complex. The overall shape of the Pth1H20R:peptidyl-tRNA complex is shown in gold spheres.25-Hydroxycholesterol E. coli Pth1 (PDBID: 2PTH) and tRNAPhe (PDBID:1EHZ) were fit into the mass density. Pictured in the inset (lower right) are the individual components: tRNAPhe in blue, Pth1 in red, and the calculated shape in gold spheres.2.3. Piperonylpiperazine Binding and Interaction with Pth1 From screening of a synthetic library of compounds for inhibitory activity against Pth1, we have found piperonylpiperazine is one of the prevailing common constituents of inhibitory compounds. The binding of piperonylpiperazine to wild type E. coli Pth1 was studied by NMR spectroscopy. Binding affinity was relatively low, with complete saturation not observable at a molar ratio of 64:1 (piperonylpiperazine:Pth1). Fast exchange on the NMR time scale was observed from migration of resonances to their bound positions. Piperonylpiperazine did not inhibit Pth1 activity and did not directly interact with the peptide binding site of the substrate, instead binding to the opposite side of the molecule, Figure 3. To further investigate the interaction of piperonylpiperazine with Pth1, molecular docking was pursued.Anti-Mouse CD44 Antibody The docking search space for piperonylpiperazine binding to Pth1 was centered on the Pth1 face indicated from NMR chemical shift perturbation mapping.PMID:23539298 Piperonylpiperazine was found to bind in a shallow depression with a calculated binding energy ranging from -3.8 and -4.4 kcal/mol. Significant interaction with the hydrophobic residues (Ala36 ro37 eu38) leading up to the edge of the central mixed -sheet were observed in all poses. Figure 3b shows the six lowest energy poses out of 36 calculated.Int. J. Mol. Sci. 2013,Figure 3. Interaction and docking of E. coli Pth1 with piperonylpiperazine. (a) Surface representation of E. coli Pth1 (PDBID:2PTH) shown with catalytically important His20 in orange. From NMR data, residues with 1H5N resonances affected by interaction with piperonylpiperazine are in blue; (b) Docking: The six lowest energy orientations of piperonylpiperazine are shown in yellow; (c) Structure of piperonylpiperazine; (d) An enlarged view of the piperonylpiperazine binding site.b) a)c)d)In bacterial culture, millimolar concentrations of piperonylpiperazine did not inhibit E. coli growth and no inhibition of Pth1 cleavage was observed from an in vitro activity assay [23,24] for concentrations exceeding 10 mM piperonylpiperazine. Thus, even though piperonylpipera.