Identification and structure-function analyses of an allosteric inhibitor of the tyrosine phosphatase PTPN22

Abstract

Protein-tyrosine phosphatase nonreceptor type 22 (PTPN22) is a lymphoid-specific tyrosine phosphatase (LYP), and mutations in the PTPN22 gene are highly correlated with a spectrum of autoimmune diseases. However, compounds and mechanisms that specifically inhibit LYP enzymes to address therapeutic needs to manage these diseases remain to be discovered. Here, we conducted a similarity search of a commercial database for PTPN22 inhibitors and identified several LYP inhibitor scaffolds, which helped identify one highly active inhibitor, NC1. Using noncompetitive inhibition curve and phosphatase assays, we determined NC1's inhibition mode toward PTPN22 and its selectivity toward a panel of phosphatases. We found that NC1 is a noncompetitive LYP inhibitor and observed that it exhibits selectivity against other protein phosphatases and effectively inhibits LYP activity in lymphoid T cells and modulates T-cell receptor signaling. Results from site-directed mutagenesis, fragment-centric topographic mapping, and molecular dynamics simulation experiments suggested that NC1, unlike other known LYP inhibitors, concurrently binds to a "WPD" pocket and a second pocket surrounded by an LYP-specific insert, which contributes to its selectivity against other phosphatases. Moreover, using a newly developed method to incorporate the unnatural amino acid 2-fluorine-tyrosine and ¹⁹F NMR spectroscopy, we provide direct evidence that NC1 allosterically regulates LYP activity by restricting WPD-loop movement. In conclusion, our approach has identified a new allosteric binding site in LYP useful for selective LYP inhibitor development; we propose that the ¹⁹F NMR probe developed here may also be useful for characterizing allosteric inhibitors of other tyrosine phosphatases.

Keywords: WPD-loop; allosteric regulation; autoimmunity; enzyme; enzyme inhibitor; inhibitor; lymphoid-specific tyrosine phosphatase (LYP); nuclear magnetic resonance (NMR); protein-tyrosine phosphatase nonreceptor type 22 (PTPN22); tyrosine phosphatase inhibitor.

Figure 1.

Identification of a new noncompetitive LYP inhibitor. A, “ring-opening” strategy based on our previously reported competitive LYP inhibitors (A15 analogues) was used to identify new LYP inhibitors. B, chemical structure of compound NC1. C, kinetic study of the inhibition mode of NC1 toward LYP. The pNPP concentrations used were 1.17, 1.75, 2.63, 3.95, 5.93, 8.89, 13.33, and 20 mm. Lineweaver-Burk plots displayed a characteristic pattern of intersecting lines, which indicates noncompetitive inhibition.

Figure 2.

Effect of NC1 on anti-CD3 antibody-stimulated TCR signaling in Jurkat T cells. A, effects of NC1 on the anti-CD3 (OKT3)-induced phosphorylation of ERK (pThr-202 and pTyr-204) and LCK pTyr-394 in control siRNA-treated T cells or LYP–siRNA-treated T cells. A representative Western blotting selected from at least three independent experiments is shown. The GAPDH level was used as a control. B and C, statistical analysis of the phosphorylation of LCK Tyr-394 (B) and of ERK (C) in T cells preincubated with NC1. Statistical comparisons between two groups were performed with Student's t tests. *, p < 0.05 when the anti-CD3 antibody-treated cells were compared with the untreated cells. Statistical comparisons among the anti-CD3–treated groups were performed with two-way ANOVA analysis. Difference between NC1 groups and control (con) groups was significant (p < 0.001). Difference between siRNA-treated groups and siRNA-untreated groups was significant (p < 0.001); the interaction between these two factors was significant (p < 0.005). For all statistical analyses, data from at least three independent experiments were quantified and presented as the mean ± S.D. (error bars).

Figure 3.

Mutagenesis and sequence alignment reveal a potential unique molecular mechanism underlying the noncompetitive inhibition of LYP by NC1. A, structural representation of the locations of the selected mutations on the surface surrounding the active site of LYP, which may be involved in NC1–LYP interactions (PDB code 2QCJ). B, K_i values of NC1 toward WT LYP and a panel of selected mutants. C, structure-based sequence alignment of LYP mutations with more than 1.5-fold K_i values from different species together with other PTP members, including PTPN18, MEG1, MEG2, TCPTP, STEP, and HePTP. Residues located in the yellow background indicate mutations with more than 1.5-fold K_i values. Residues different from human LYP are colored in red. For all statistical analyses, data from at least three independent experiments were quantified and presented as the mean ± S.D.

Figure 4.

Molecular docking and MD simulation analyses reveal the molecular mechanism underlying the noncompetitive inhibition of LYP by NC1. A, pocket analysis of predicted binding mode of NC1 to pNPP-bound LYP using representative MD snapshot. The WPD pocket (colored in blue) and secondary pocket (colored in green) are represented as transparent surface and spheres. Compound NC1 is represented as orange sticks, and surrounding residues are represented as white sticks. B, individual residue contribution to the binding of compound NC1 with LYP. Data were calculated by the MM/GBSA-binding free energy decomposition analysis. C, calculated occupied spaces of compound NC1 in WPD pocket and secondary pocket during MD simulations.

Figure 5.

MD simulations of LYP–pNPP systems with or without NC1 bound reveal the details of NC1 inhibition. A, comparison of the distance between the catalytic residue Asp-195 and the substrate pNPP during MD simulations of LYP–pNPP systems with or without NC1 bound. B, representative MD snapshot of LYP–pNPP system. C, representative MD snapshot of LYP–pNPP–NC1 system.

Figure 6.

Comparison of the potential allosteric pockets in LYP with atypical-open WPD-loop (A, PDB code 3H2X), VHR with closed WPD-loop (B, PDB code 1J4X), PTP1B with open WPD-loop (C, PDB code 2HNP), STEP with open WPD-loop (D, PDB code 2CJT), PTPN18 with open WPD-loop (E, PDB code 2OC3), Glepp with open WPD-loop (F, PDB code 2GJT). The proteins are presented in transparent white surface with WPD-loop shown as red loop and substrate pNPP shown as yellow sticks. The predicted binding pose of NC1 was derived from representative MD simulation snapshot in Fig. 5A and shown as green sticks. Fragment-centric topographic mapping was performed using AlphaSpace. Good pockets (pocket score > 100) are presented with green spheres and auxiliary pockets (30 < pocket score < 100) are presented with blue spheres. Potential allosteric inhibitor binding pockets, which possess a series connected small pockets, are marked with yellow circles.

Figure 7.

¹⁹F NMR spectroscopy reveals suppression of WPD-loop conformational changes by NC1. A, crystal structures of LYP showing conformational changes in the WPD-loop and its adjacent residues with or without substrate. Left, residue Leu-281 is “buried” by the WPD-loop (PDB code 2P6X). Right, residue Leu-281 is “exposed” after substrate binding (PDB code 2QCJ). B, schematic flowchart of the incorporation of F2Y into LYP at position 281. C, purity of the protein was determined by electrophoresis (left panel). The purified protein was subjected to trypsin digestion and analyzed by MS/MS, which indicated the presence of the y12± F2Y-VYNAVLELFKR fragment (M_r 1550) and the y13± E-F2Y-VYNAVLELFKR fragment (M_r 1679). These results confirmed that F2Y was specifically incorporated into LYP at position 281. m/z, mass/charge ratio. D, upfield shift was observed in the ¹⁹F NMR spectrum of the LYP L281F2Y ¹⁹F NMR probe in response to Na₃VO₄ binding (upper panel). The ¹⁹F NMR spectrum of the LYP-L281F2Y probe in response to Na₃VO₄ binding after preincubation with compound NC1 (lower panel).