Research ArticleStructural Biology

Structural basis for the preference of the Arabidopsis thaliana phosphatase RLPH2 for tyrosine-phosphorylated substrates

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Science Signaling  03 Apr 2018:
Vol. 11, Issue 524, eaan8804
DOI: 10.1126/scisignal.aan8804
  • Fig. 1 Ribbon diagram showing the path of the AtRLPH2 polypeptide chain and global arrangement of secondary structure elements.

    Domains 1, 2, and 3 are colored blue, green, and orange, respectively. The dashed line in the loop (magenta) between α-helices 7 and 8 indicates a region of disorder not defined by clear electron density. Metal ions 1 and 2 are pink, and the position of motif 1 (residues 262 to 267, VVSGHH) is indicated with a box.

  • Fig. 2 Coordination of divalent metal ions in the active sites.

    Metal ions 1 and 2 are coordinated in the active sites of (A) AtRLPH2 and (B) rabbit PP1 [Protein Data Bank (PDB) code 1FJM] as shown. Ions are colored gray, and coordination bonds are shown as red dashed lines. Highly conserved residues that are important for metal ion coordination, substrate recognition, or catalysis are shown as stick representations and labeled.

  • Fig. 3 Comparison of the three-dimensional structures of PPP-family phosphatases.

    Domain 1 shown in the same orientation in (A) AtRLPH2, (B) rabbit PP1 (PDB code 1FJM), (C) Shewanella CAPTP (PDB code 1V73), and (D) Shigella diadenosine tetraphosphatase (PDB code 2DFJ). Domain 1, blue; domain 2, green; domain 3, orange; metal ions, pink.

  • Fig. 4 AtRLPH2 preferentially dephosphorylates phosphotyrosine substrates that contain an N-terminal phosphothreonine.

    (A) Kinetic analysis of dephosphorylation of various concentrations of extracellular signal–regulated kinase 1 and 2 (ERK1/2) phosphopeptides by recombinant AtRLPH2. Assays were performed using ERK1/2 phosphopeptides HTGFLTEpYVATR (TEpY, squares) and HTGFLpTEpYVATR (pTEpY, circles), and kinetic analysis was used to determine Vmax and Km. Vmax was about the same for both peptides (~240 nmol min−1 mg−1). Km for peptide HTGFLTEpYVATR was 1.44 mM, and Km for HTGFLpTEpYVATR was 0.18 mM. Error bars represent ±SE (n = 3). (B to E) On-blot dephosphorylation assays. Singly phosphorylated (TEpY and pTEY) and dually phosphorylated (pTEpY) ERK1/2 phosphopeptides covalently coupled to bovine serum albumin (BSA) were spotted onto a nitrocellulose membrane and incubated with either BSA alone (B and D) or BSA plus recombinant AtRLPH2 (C and E). Final phosphorylation status was assessed by Western blot (WB) with an antibody specific for phosphotyrosine (B and C) or phosphothreonine (D and E). All blots were performed three times in parallel and developed at the same time, and one representative blot is shown.

  • Fig. 5 Coordination of tungstate ions and model of a substrate peptide bound to AtRLPH2.

    (A) Hydrogen-bonding interactions between tungstate (red bars) and amino acid side chains are indicated by red dashed lines. The anomalous difference electron density map is shown as a green mesh (contoured at 10 SDs). (B) The structure of AtRLPH2 is shown as a solvent-accessible surface representation colored according to electrostatic potential, with blue indicating positive potential, white indicating neutral potential, and red indicating negative potential. The peptide HTGFLpTEpYVATR was modeled into the substrate. (C) Higher-magnification view of the boxed area in (B). The peptide substrate was modeled with the phosphoryl group of the phosphothreonine residue positioned in the same place as the tungstate ion seen in the complex of AtRLPH2 cocrystallized with tungstate and the phosphoryl group of the phosphotyrosine residue positioned next to the divalent metal cations, also coincident with a bound tungstate ion in the tungstate cocrystal structure. Semitransparent surface representation shown over ribbon diagram and stick representation of the crystal structure.

  • Fig. 6 A double-arginine gating mechanism contributes to the substrate specificity preference for phosphotyrosine.

    (A) Stick and semitransparent surface model of AtRLPH2 (gray) bound to an ERK1/2 peptide (blue) with the peptide’s phosphotyrosine bound to the divalent metal ions coordinated in the active site of AtRLPH2. (B) Model of AtRLPH2 bound to an ERK1/2 peptide in which the peptide’s phosphotyrosine residue is replaced with phosphothreonine (pink). The arrangement of AtRLPH2 Arg51, Arg245, and Phe285 creates a binding pocket that allows the longer side chain and flat aromatic ring to extend the phosphoryl group of the phosphotyrosine residue toward the divalent metal ions (A). The shorter phosphothreonine residue does not allow the phosphoryl group to reach deep enough into the pocket to reach the divalent metal ions (B).

  • Table 1 Crystallographic statistics.
    CrystalNative INative IISodium tungstate
    PDB code5VJV5VJW
    Unit cell dimensions a = b, c (Å)102.23, 60.75102.60, 61.04102.66, 60.21
    Wavelength (Å)1.85321.033181.21416
    Resolution (Å)*40–2.20 (2.26–2.20)40–1.95 (2.00–1.95)40–1.80(1.85–1.80)
    Rsym0.092 (0.405)0.109 (1.15)0.098 (0.95)
    CC1/21.000 (0.975)0.998 (0.537)0.998 (0.641)
    II35.3 (8.9)12.1 (1.6)12.3 (1.9)
    Completeness (%)93.1 (75.2)99.5 (98.7)99.0 (99.0)
    Redundancy30.9 (27.1)5.5 (5.5)5.0 (5.0)
    Refinement
    Resolution (Å)40–1.9540–1.80
    Unique reflections25,38031,880
    Rwork§/Rfree0.184/0.2190.176/0.199
    Total number of atoms2,5552,561
    Protein atoms2,3432,343
    Phosphate/tungstate atoms48
    Water atoms208210
    Average B-factors (protein)26.420.5
    Average B-factors (water)34.129.0
    Average B-factors (metal ions)23.116.5
    Average B-factors (phosphate/tungstate ions)32.235.3
    RMSD from ideal geometry
    Bond lengths (Å)0.0080.007
    Bond angles (°)1.201.20
    Ramachandran outliers00
    Ramachandran favored (%)96.696.6
    Clash score**0.860.43
    MolProbity score (percentile)0.98 (100)0.87 (100)

    *Values from the outermost resolution shell are given in parentheses.

    Rsym = Σi |Ii − <I>| / Σi Ii, where Ii is the ith integrated intensity of a given reflection and <I> is the weighted mean of all measurements of I.

    ‡Percentage of correlation between intensities from random half–data sets (33).

    §Rwork = Σ||Fo| − |Fc|| / Σ|Fo| for 95% of reflection data used in refinement.

    Rfree = Σ||Fo| − |Fc|| / Σ|Fo| for 5% of reflection data excluded from refinement.

    ║Ramachandran plot analysis performed in MolProbity (34).

    **Number of serious steric overlaps (>0.4 Å) per 1000 atoms.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/11/524/eaan8804/DC1

      Fig. S1. Multiple sequence alignment of AtRLPH2 with other RLPH2 class members.

      Fig. S2. Multiple sequence alignment of AtRLPH2 with other PPP family members from A. thaliana.

      Fig. S3. Enzymatic activity of RLPH2 mutated in phosphothreonine-binding and gatekeeper residues.

      Fig. S4. Different rotamers may be adopted by phosphothreonine in the pTEpY peptide substrate versus threonine in the TEpY peptide substrate.

      Fig. S5. Location of motif 1 in RLPH2.

      Table S1. Accession numbers of proteins used for sequence alignments.

      Table S2. Primers used to create the AtRLPH2-V5-H6 mutants.

      Movie S1. AtRLPH2 substrate-binding groove.

    • Supplementary Materials for:

      Structural basis for the preference of the Arabidopsis thaliana phosphatase RLPH2 for tyrosine-phosphorylated substrates

      Anne-Marie Labandera, R. Glen Uhrig, Keaton Colville, Greg B. Moorhead,* Kenneth K. S. Ng*

      *Corresponding author. Email: moorhead{at}ucalgary.ca (G.B.M.); ngk{at}ucalgary.ca (K.K.S.N.)

      This PDF file includes:

      • Fig. S1. Multiple sequence alignment of AtRLPH2 with other RLPH2 class members.
      • Fig. S2. Multiple sequence alignment of AtRLPH2 with other PPP family members from A. thaliana.
      • Fig. S3. Enzymatic activity of RLPH2 mutated in phosphothreonine-binding and gatekeeper residues.
      • Fig. S4. Different rotamers may be adopted by phosphothreonine in the pTEpY peptide substrate versus threonine in the TEpY peptide substrate.
      • Fig. S5. Location of motif 1 in RLPH2.
      • Table S1. Accession numbers of proteins used for sequence alignments.
      • Table S2. Primers used to create the AtRLPH2-V5-H6 mutants.
      • Legend for movie S1

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.mp4 format). AtRLPH2 substrate-binding groove.

      © 2018 American Association for the Advancement of Science

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