Structures of nodularin-R (a), tautomycin (b), tautomycetin (c) and cantharidin (d). Important interaction sites with PP1 identified in this study are highlighted in the figures. An asterisk marks the critical OH group in tautomycin. Details are given in the text.

Comparisons of the nodularin and tautomycin binding sites. (a) PP1:Nodularin-V (silver:blue) and PP1:Nodularin-R (green:yellow) PP1 active sites are overlaid. The location of a β-mercaptoethanol (BME) molecules of the PP1:Nodularin-V structure cause residues 194-199 to shift 4.0 Å away from the hydrophobic groove (movement depicted by black arrow) relative to those of PP1 bound to Nodularin-R. A second BME molecule, bound directly under the Adda residue of Nodularin-V, causes it to rotate 2.65 Å closer to α-helix 4. (b) Tautomycin from our PP1:tautomycin (green:yellow) crystal structure is overlaid with the hypothetical, molecular dynamics generated tautomycin model (purple) from Colby, et al.30 The general binding mode of the hypothetical tautomycin model is correct. The diacid binds the active site but is oriented in the opposite direction relative to the crystal structure. The C22 hydroxyl (noted by arrows) is involved in an intra-molecular hydrogen bond in the crystal structure, which explains its biological importance;26 this is not observed in the model. The spiroketal binds to the hydrophobic cleft; however, when compared to the crystal structure it’s rotated ~90° away from α-helix 4 such that atoms C1-C6 are no longer buried within the cleft. Mn⁺² atoms are silver spheres. All images in stereo. This figure was made with PyMol.52

Overview of nodularin-R and tautomycin binding to PP1. Stereo images of (a) 1.63 Å, simulated annealing omit map51 contoured at 3.0σ and (b) detailed active site interactions of nodularin-R. (c) 1.7 Å simulated annealing omit map51 contoured at 3.0σ map of tautomycin bound to the surface of PP1 and (d) detailed active site interactions of tautomycin. Dotted lines represent potential hydrogen bonding interactions. The tautomycin intramolecular hydrogen bond is shown as a solid black line. This figure was made with PyMol.52

PP1:canthardin model. (a) Hydrogen bond interaction network on cantharidin and the PP1 active site. PP1 residues Y272, R221 and R96 make critical stabilizing interactions. (b) Surface representation of PP1 of the PP1:canthardin model. Four surface pockets that can potentially be used to tether cantharidin-based inhibitors onto PP1 are highlighted.