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. 2016 Jun 1;109:14-26.
doi: 10.1016/j.bcp.2016.03.011. Epub 2016 Mar 23.

Crystal Structures and Mutagenesis of PPP-family Ser/Thr Protein Phosphatases Elucidate the Selectivity of Cantharidin and Novel Norcantharidin-Based Inhibitors of PP5C

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Free PMC article

Crystal Structures and Mutagenesis of PPP-family Ser/Thr Protein Phosphatases Elucidate the Selectivity of Cantharidin and Novel Norcantharidin-Based Inhibitors of PP5C

Debasish Chattopadhyay et al. Biochem Pharmacol. .
Free PMC article

Abstract

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.

Keywords: Crystal structures; Inhibitors; PP5C; Phosphatase; Screening.

Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest with the contents of this article.

Figures

Fig. 1
Fig. 1
Structure inhibitors. (A) Natural toxins known to act as strong inhibitors of PP1C, PP2AC, and PP5C. (B) Novel norcantharidin based inhibitors numbered below. IC50 values for novel compounds are provided in Table 1.
Fig. 2
Fig. 2
Inability of cantharidin to inhibit the activity of native human PP4C. (A) Concentration response curves for the inhibition of PP4C and PP5C activity by cantharidin. The inhibition of native human PP4C and recombinant human PP5C by cantharidin was compared head-to-head. Highly purified PP5C (filled squares) and PP4C purified from HEK293T-derived cells by co-immunoprecipitation with FLAGPP4R2 (diamonds) were assayed over a range of inhibitor concentrations as described in Procedures. The data shown for PP4C represent 4 separate experiments (each indicated by a separate line connecting the symbols for each dataset) in which PP4C activity was measured in quadruplicate for each concentration of cantharidin shown (symbols and error bars denote mean and ± SD, respectively). (B) Western analysis of anti-FLAG immunoprecipitates demonstrating the PP4C used in A was not contaminated with PP2AC. Flp-In T-Rex HEK293T cells stably expressing the indicated FLAG-tagged constructs were treated with tetracycline to induce expression of FLAG-PP4C or FLAG-PP4R2 and the cells were lysed after 24 h. Immunoprecipitation with anti-FLAG (M2) conjugated agarose beads was then performed on lysates. Immune complexes were resolved by SDS-PAGE followed by transfer to Immobilon (PVDF). Co-precipitation of native PP4C or PP2AC protein was detected by immunoblotting (IB) for native PP4C or PP2AC with anti-PP4C (lanes 1and 2) or PP2AC (lanes 3–5) specific antibodies. CL = representative crude HEK293-cell lysate prior to IP. HC and LC represent heavy chain and light chain of the antibody use for IP, which is detected by the anti-PP2A antibody used.
Fig. 3
Fig. 3
Inhibitory effect of (2)/(2e) racemate, (6), and (7), and on the activities of purified PP1C (triangles) and PP5C (squares). (A) Reduction of C5–C6 double bond increases the potency against both PP1C and PP5C. (B) The addition of a propoxymethyl group provides a modest increase in the strength of inhibition and selectivity for PP5C compared to PP1C. Inhibition assays were conducted using highly purified PP1C or PP5C and using DiFMUP as a substrate (as described in Procedures). Each point represents the mean ± SD (n = 4). IC50 values are provided in Table1.
Fig. 4
Fig. 4
Detailed representation of contacts within the active site of PP5C, and a comparison of the structures of (4) and (5) bound to PP5C. (A) Active site contacts in the absence of inhibitor with bound substrate analog, phosphate (from [22] PDB code 1s95). Hydrogen bonds are shown as yellow dotted lines and metal–ligand bonds are shown as solid lines. The green dotted line between the hydroxyl (W1) ion and phosphorus atom (P) represents a close contact (3.0 Å) suggestive of a near-attack configuration between phosphorus and the nucleophile in the enzyme-catalyzed phosphomonoester hydrolysis reaction. (B) Active site contacts with bound inhibitor (4). Conventional hydrogen bonds are shown as yellow dotted lines and metal–ligand bonds are shown as solid lines. Hydrophobic contacts between the inhibitor and the side-chains of Val429, Phe446, and Tyr451 are represented as blue dotted lines (drawn to the β carbon of Val429 and the aromatic ring centroids, respectively). The small blue spheres represent aromatic ring centroids. Red dotted lines represent weak C–H⋯O hydrogen bonds. Figure was prepared with PyMol [62]. (C) Fo–Fc omit map at the location of the inhibitor [(4) left; (5) right] contoured at +3σ with the bound inhibitor structure from the refined model displayed as sticks. (D) Stick representation showing the coordination sphere of each metal ion in the (4) left, and (5) right, complexes. Residues are labeled, and catalytic metals are shown as spheres (labeled M1 and M2, respectively). (E) Comparison Stereo diagram showing superposition of amino acid residues near the active site in the phosphate complex (PDB code 1s95; [22]) and the (4) complex (present structure). Amino acid residues, phosphate, and (4) are shown as sticks. Carbon atoms are shown as cyan in the phosphate complex and green in the (4) complex. The metal-coordinated waters in the phosphate complex are shown as magenta spheres (W1 and W2). Metal ions are shown as ecru spheres.
Fig. 5
Fig. 5
Selection of C5-versus C6-substituted enantiomers by PP5C cocrystallization. (A) PP5C selects the C6-methyl enantiomer (4) from the C5/C6 racemate. Space-filling representations show that the hydrophobic space at right, bounded by Val429, Met455, and Tyr451, is just large enough to accommodate the C6-methyl group (red). The water associated with Val429’s backbone amide at left (dashed line) tolerates the presence of the new methyl group at C6, but would be displaced by a methyl moiety at the C5 position. (B) In reversal of preference, PP5C selects the C5-propoxymethyl enantiomer (5) from the C5/C6 racemate. Space-filling representations show navigation of the C5-propoxymethyl moiety (red) between the Cα hydrogen of Glu428 (circle) and Val429. The ether oxygen of the new sidechain displaces a water molecule and H-bonds with Val429’s backbone amide (dashed line). Here, the terminus of the propoxymethyl sidechain is shown to follow along Glu428 in one possible orientation, but the atom positions are undetermined by X-ray diffraction analysis. A propoxymethyl group at the C5 position would unfavorably encounter Tyr451 or Met455 at right. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Quantum-based modeling predictions of a steric clash between Trp257 of PP4C and the bis-acid form of norcantharidin (3). (A) Surface-filled diagram of the PP5C-(3) complex ([63]; PDB code 3H61) with (3) shown as a stick figure tightly bound within the catalytic site of PP5C (small depression, with catalytic metals shown as spheres). (B) Side view of the PP5C-(3) complex with inhibitor displayed as opaque sticks with a partially-transparent molecular surface. The carboxylates of (3) are coordinated to the metal ions (spheres). Phe446 (green surface) of PP5C abuts the bound inhibitor. The superposition of Trp257 (surface) from a homology model of PP4C (described in Procedures) indicates that binding to PP4C will be disrupted by a steric clash. (C) Close-up view of the PP5C-(3) complex showing key O⋯M1 distances (Å). The bridge oxygen (O7) of (3) interacts with one metal ion and is in contact with Hε of Phe446. (D) Optimized PP4C-(3) model system. The larger Trp257 of PP4C disrupts favorable interactions between (3) and the M1 M2 system, as compared to the PP5C-(3) complex; all carboxylate O⋯metal interaction distances (Å) are longer but one, and the O7⋯M1 distance is too far (>4 Å) to contribute significantly to binding. (E) Sequence similarity of the PPases within the catalytic core domain. Absolutely conserved residues of the compared PPases are indicated in boldface. Trp distinguishes PP4C and PP6C from the other PPases (highlight). (F) Model of a theoretical steric clash between Tyr311 of PPBC and carbons C2 and C6 of cantharidin and norcantharidin derived inhibitors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7
Mutation of PP1C, PP5C and PP6C alters sensitivity to inhibition by cantharidin. (A) Substitution of tryptophan with phenylalanine in PP6C [PP6C (W256F); open circles] increases sensitivity to inhibition by cantharidin. (B) In contrast, the substitution of phenylalanine (F) with tryptophan (W) in PP1C or PP5C [PP1C (F257W); PP5C (F446W)] reduces or ablates sensitivity to inhibition to cantharidin, respectively. Recombinant human PP1C, PP1C (F257W), PP5C and PP5C (F446W) were expressed in E. coli, and purified according to the same protocol. The activity was measured in the presence of the indicated amount of cantharidin as described in Fig. 2 and Procedures. Enzyme titrations curves are shown in Fig. 2S. The data are expressed as mean ± SD; n = 4.

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