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Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

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Science Advances  24 Mar 2017:
Vol. 3, no. 3, e1601692
DOI: 10.1126/sciadv.1601692
  • Fig. 1 Solution-based assays coarse-grain reaction mechanisms for the tyrosine phosphatase SHP-1.

    (A) Schematic of the domain structure of SHP-1 and a subset of reactions that may occur in solution with peptide substrates. (B) Standard solution–based enzymatic assay showing the production of inorganic phosphate (product) over time for the indicated concentration of SHP-1 mixed with the PEG12-ITIM substrate (data are representative of two independent experiments). Progress curves are fit with a mathematical model to provide an estimate for Embedded Image (see Materials and Methods).

  • Fig. 2 An SPR assay recovers five independent biophysical/biochemical constants characterizing tethered enzymatic reactions by SHP-1.

    (A) Representative SPR trace (black dots) for SHP-1 injected over a surface immobilized with 48.5 RU of an ITIM peptide derived from LAIR-1 on a 28-repeat PEG linker (PEG28-ITIM). A fit of the MPDPDE model (red line) provides estimates for the indicated parameters. Early time data and fit are shown on the right. Unprocessed data are shown in fig. S1. (B) Anti-phosphotyrosine antibody injected at the end of the experiment shows reduced binding in the experimental flow cell (SHP-1) compared to a buffer-injected flow cell with equivalent peptide levels (control). (C) Schematic of reactions taking place when SHP-1 is injected over a surface of immobilized phosphorylated peptides. Note that peptide anchoring is displayed in one dimension for clarity, but because peptides are randomly coupled to a dextran matrix, which extends 100 to 200 nm above the surface, they are anchored in three dimensions.

  • Fig. 3 Mathematical models capture the physics and chemistry in tethered enzymatic SPR.

    (A) Levels of unphosphorylated peptide, phosphorylated peptide, and phosphorylated peptide bound to SHP-1 over time determined using the stochastic simulation (solid gray lines) or the MPDPDE model (dashed colored lines). Levels of SHP-1 bound to phosphorylated peptide are replotted for clarity (right panel). Good agreement is observed between the stochastic simulation and the MPDPDE model calculation. (B) Snapshots of the spatial distribution of the three molecular species at indicated time points from the stochastic simulation [colors as in (A)]. Initially, the phosphorylated peptides are randomly distributed on the surface (0 s), but as time progresses, clustered peptides are effectively dephosphorylated by tethered catalysis (50 s), ultimately resulting in phosphorylated peptides too far apart for efficient tethered catalysis (280 s). These two-dimensional spatial distributions are generated from the stochastic simulation by projecting 20 nm in the third dimension. See Materials and Methods for computational details. Parameters: [SHP-1] = 1 μM, [peptide] = 100 μM, kon = 0.1 μM−1 s−1, koff = 1 s−1, Embedded Image = 0.01 μM−1 s−1, L = 20 nm, and Embedded Image = 0.0005 μM−1 s−1.

  • Fig. 4 Fitted biophysical/biochemical parameters are independent of experimental variables.

    Representative SPR traces (black dots) and MPDPDE model fits (solid color) for (A) two SHP-1 concentrations and (B) two initial peptide concentrations with the right panels showing early time data and fit. (C) Plots of fitted parameters versus SHP-1 concentration (upper row) and peptide concentration (lower row) with linear regression fits (R2 and P values are indicated) reveal a lack of correlation, indicating that the fitted parameters do not depend on the experimental variables. Averages of fit parameters with SEMs from all experiments are shown in boxes (n = 15). All experiments are performed using wild-type SHP-1 and phosphorylated PEG28-ITIM peptides. Exclusion criteria for experiments exhibiting long time scale artifacts, such as nonspecific binding and/or differential flow cell drift, are discussed in Materials and Methods (Quality control) and fig. S6.

  • Fig. 5 Reduction in tether lengths reduces the reach parameter and introduces configurational steric hindrance reducing kon and Embedded Image.

    (A) SPR traces (black circles) and MPDPDE model fits (red lines) of SHP-1 injected over peptides with 28 (PEG28), 12 (PEG12), 3 (PEG3), and 0 (PEG0) PEG linker repeats. (B) Average fit parameters for PEG28-ITIM (n = 15), PEG12-ITIM (n = 3), PEG3-ITIM (n = 3), and PEG0-ITIM (n = 2) show reduced values of L, kon, and Embedded Image for shorter linkers. Two-way analysis of variance (ANOVA) with a Bonferroni multiple comparison correction is used to determine the P values (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05).

  • Fig. 6 Binding by either SH2 domain allosterically activates SHP-1, but the reach parameter is larger for N-SH2 binding.

    (A) SPR traces (black dots) and MPDPDE model fits (solid lines) of the N- and C-terminal SH2 domain binding–null mutants and wild-type SHP-1 injected over PEG28-ITIM. (B) Average fit parameters for wild-type (WT) SHP-1 (n = 15), N-terminal SH2 (N-SH2) mutant SHP-1 (n = 3), and C-terminal SH2 (C-SH2) mutant SHP-1 (n = 9) show weak binding and reduced reach when SHP-1 binds via the C-terminal SH2 domain compared to the N-terminal SH2 domain, but allosteric activation is observed in both cases. Two-way ANOVA with a Bonferroni multiple comparison correction is used to determine the P values (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05).

  • Fig. 7 Steric penalty to binding and catalysis by short tethers.

    (A) Shorter cytoplasmic tails (tethers) result in decreased kon for binding and Embedded Image for catalysis as a result of steric hindrance (indicated by thickness of black arrow). (B) Histograms of the contour length (number of amino acids × 0.3 nm per amino acid) to phosphotyrosine residues on inhibitory receptors (ITIM/ITSM; mean length of 21.2 nm, red histogram) and activatory receptors (ITAM/ITTM/YxxM; mean length of 11.3 nm, green histogram) for human receptors. Most inhibitory receptor tails are longer than PEG28 where steric penalties are lower, whereas most activatory receptor tails are shorter than PEG28 where steric penalties are higher.

  • Fig. 8 Clustering and allosteric activation increase the activity of tethered SHP-1 by 900-fold.

    (A) The concentration of SHP-1 experienced by the substrate when SHP-1 is tethered to clustered receptors (left), tethered to nonclustered receptors (center), and free in the cytoplasm (right). The concentration of SHP-1 increases 45-fold as it recruited from solution to clustered receptors. (B) The catalytic rate (Embedded Image) increases 20-fold when SHP-1 is tethered (bound) to a receptor compared to when it is in solution. This allosteric activation of SHP-1 upon binding is consistent with a dynamic transition between closed low-activity and open high-activity states while in solution. The combination of increased concentration (45-fold) and increased catalytic activity (20-fold) leads to a 900-fold increase in the overall dephosphorylation rate because SHP-1 is recruited from solution to clustered receptors.

  • Table 1 Peptides used in study (phosphotyrosines are denoted as Y*).
    NameSequenceContour length
    PEG28-ITIMBiotin-(PEG)28-DLQEVTY*IQLDHH12.1 nm
    PEG12-ITIMBiotin-(PEG)12-DLQEVTY*IQLDHH6.5 nm
    PEG3-ITIMBiotin-(PEG)3-DLQEVTY*IQLDHH3.3 nm
    PEG0-ITIMBiotin-GDLQEVTY*IQLDHH2.7 nm

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/3/e1601692/DC1

    fig. S1. Unprocessed SPR traces for the data in Fig. 2A showing the binding trace for the experimental flow cell (black) and the control flow cell (red).

    fig. S2. Point mutations to both SH2 domains of SHP-1 result in minimal binding but appreciable dephosphorylation.

    fig. S3. Comparison of the standard and MPDPDE model fits.

    fig. S4. Theoretical SPR traces generated by the MPDPDE model.

    fig. S5. MCMC analysis of the experimental data in Fig. 2A highlights that all five parameters can be determined independently of each other.

    fig. S6. Quality control of experimental data.

    fig. S7. Surface tethering markedly increases the rate of dephosphorylation.

    fig. S8. Calculation of local concentration, σ(r), based on two polymers a distance of r apart that can be approximated by worm-like chains with parameter LA for the free phosphorylated peptide and LB for the SHP-1–bound phosphorylated peptide.

    Supplementary code

    Supplementary data (Microsoft Excel format)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Unprocessed SPR traces for the data in Fig. 2A showing the binding trace for the experimental flow cell (black) and the control flow cell (red).
    • fig. S2. Point mutations to both SH2 domains of SHP-1 result in minimal binding but appreciable dephosphorylation.
    • fig. S3. Comparison of the standard and MPDPDE model fits.
    • fig. S4. Theoretical SPR traces generated by the MPDPDE model.
    • fig. S5. MCMC analysis of the experimental data in Fig. 2A highlights that all five parameters can be determined independently of each other.
    • fig. S6. Quality control of experimental data.
    • fig. S7. Surface tethering markedly increases the rate of dephosphorylation.
    • fig. S8. Calculation of local concentration, σ(r), based on two polymers a distance of r apart that can be approximated by worm-like chains with parameter LA for the free phosphorylated peptide and LB for the SHP-1–bound phosphorylated peptide.

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    Other Supplementary Material for this manuscript includes the following:

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