Crystal structures of protein phosphatase-1 bound to nodularin-R and tautomycin: a novel scaffold for structure-based drug design of serine/threonine phosphatase inhibitors

Abstract

Protein phosphatase 1 occurs in all tissues and regulates many pathways, ranging from cell-cycle progression to carbohydrate metabolism. Many naturally occurring, molecular toxins modulate PP1 activity, though the exact mechanism of this differential regulation is not understood. A detailed elucidation of these interactions is crucial for understanding the cellular basis of phosphatase function and signaling pathways but, more importantly, they can serve as the basis for highly specific therapeutics, e.g. against cancer. We report the crystal structures of PP1 in complex with nodularin-R at 1.63 A and tautomycin at 1.70 A resolution. The PP1:nodularin-R complex was used to demonstrate the utility of our improved PP1 production technique, which produces highly active, soluble PP1. Tautomycin is one of the few toxins that reportedly preferentially binds PP1>PP2A. Therefore, the PP1:tautomycin structure is the first complex structure with a toxin with preferred PP1 specificity. Furthermore, since tautomycin is a linear non-peptide-based toxin, our reported structure will aid the design of lead compounds for novel PP1-specific pharmaceuticals.

Figure 1

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.

Figure 2

Overview of nodularin-R and tautomycin binding to PP1. Stereo images of (a) 1.63 Å, simulated annealing omit map contoured at 3.0σ and (b) detailed active site interactions of nodularin-R. (c) 1.7 Å simulated annealing omit map 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.

Figure 3

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. 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; 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.

Figure 4

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.