PP1:Tautomycetin Complex Reveals a Path toward the Development of PP1-Specific Inhibitors

Abstract

Selective inhibitors for each serine/threonine phosphatase (PPP) are essential to investigate the biological actions of PPPs and to guide drug development. Biologically diverse organisms (e.g., cyanobacteria, dinoflagellates, beetles) produce structurally distinct toxins that are catalytic inhibitors of PPPs. However, most toxins exhibit little selectivity, typically inhibiting multiple family members with similar potencies. Thus, the use of these toxins as chemical tools to study the relationship between individual PPPs and their biological substrates, and how disruptions in these relationships contributes to human disease, is severely limited. Here, we show that tautomycetin (TTN) is highly selective for a single PPP, protein phosphatase 1 (PP1/PPP1C). Our structure of the PP1:TTN complex reveals that PP1 selectivity is defined by a covalent bond between TTN and a PP1-specific cysteine residue, Cys127. Together, these data provide key molecular insights needed for the development of novel probes targeting single PPPs, especially PP1.

Figure 1

Tautomycetin is a PP1-specific inhibitor. (a) Chemical structure of TTN, highlighting the diacid (green) and diene/alkene (purple) groups. (b) Inhibitory effect of TTN on the activities of purified PP1 (yellow squares), PP2A (blue diamonds), PP4 (orange circles), PP5 (green triangles), PP6 (magenta stars) and PP2B (red inverted triangles). Inhibition assays were conducted using purified enzymes with 100 μM DiFMUP as a substrate (see Methods). Each point is the mean ± SD (n = 4–8). (c) PP1 (■), PP2A (●) and PP5 (▲) were further tested using [³²P]-labeled phosphohistone (specific activity: 7.4 × 10⁶ cpm/nmol incorporated phosphate; 25 pM PP1, 30 pM PP5, ~50 pM PP2A). Each point is the mean ± SD (n = 4). IC50 values are reported in Table 1.

Figure 2

Tautomycetin binds the PP1 hydrophobic groove and occludes the active site. (a) The PP1-TTN complex (PP1, gray; TTN, yellow). PP1 residues that contact TTN are in lavender. The hydrophobic groove and active site are indicated. Right, active site with TTN shown as sticks. (b) Stereo image of the interactions between PP1 (lavender) and TTN (yellow). Ionic and hydrogen bonding interactions indicated by black dashed lines. PP1 Mn²⁺ ions, magenta spheres; two active site coordinated waters, blue spheres. TTN carbon numbering as in Figure 1a.

Figure 3

TTN forms a covalent bond with Cys127_PP1. (a) Simulated annealing composite omit map (blue mesh, 1σ; TTN omitted) of TTN illustrating the covalent bond with Cys127_PP1 (orange arrow). TTN is colored by atomic B-factor. (b) ESI-MS of PP1 (top) and PP1-TTN (bottom). PP1 expected MW, 34 124.19 Da. (c) PP1-TTN with PP1 color coded according to PPP family sequence conservation. TTN shown as yellow sticks.

Figure 4

TTN is selective for PP1 due to sequence variability in the hydrophobic groove. (a) Overlay of PP1-TTN, PP2A, PP2B and PP5, with key TTN interacting residues (and their PPP homologues) shown as sticks. (b) Sequence alignment of the Cys127_PP1 loop, colored as in panel a. (c) Model of PP2B bound to TTN (superposition of PP2B with PP1-TTN), highlighting that the replacement of Ser129_PP1 by the bulky His155_PP2B hinders access to Cys153_PP2B. (d) PP1-TTN complex illustrating the location of Ser129_PP1 and Asp197_PP1, which stabilizes the Cys127_PP1 thiolate ion.