Structural and functional characterization of the c-terminal domain of the ecdysteroid phosphate phosphatase from bombyx mori reveals a new enzymatic activity

Abstract

Here, we present the crystal structure of the ecdysone phosphate phosphatase (EPPase) phosphoglycerate mutase (PGM) homology domain, the first structure of a steroid phosphate phosphatase. The structure reveals an alpha/beta-fold common to members of the two histidine (2H)-phosphatase superfamily with strong homology to the Suppressor of T-cell receptor signaling-1 (Sts-1 PGM) protein. The putative EPPase PGM active site contains signature residues shared by 2H-phosphatase enzymes, including a conserved histidine (His80) that acts as a nucleophile during catalysis. The physiological substrate ecdysone 22-phosphate was modeled in a hydrophobic cavity close to the phosphate-binding site. EPPase PGM shows limited substrate specificity with an ability to hydrolyze steroid phosphates, the phospho-tyrosine (pTyr) substrate analogue para-nitrophenylphosphate ( pNPP) and pTyr-containing peptides and proteins. Altogether, our data demonstrate a new protein tyrosine phosphatase (PTP) activity for EPPase. They suggest that EPPase and its closest homologues can be grouped into a distinct subfamily in the large 2H-phosphatase superfamily of proteins.

Figure 1

Ribbon diagram of the EPPase_PGM dimer. In the top view, the dimer’s two-fold axis is perpendicular to the plane of the page while in the bottom view it is vertical in the plane of the page. Secondary structure elements and termini are indicated for one monomer. The side chains of His80 and His260 as well as the tungstate ions are shown in ball-and-stick representation to locate the active site. Prepared with MOLSCRIPT (39) and PYMOL (http://pymol.sourceforge.net/).

Figure 2

Comparison of B. mori EPP_PGM, SixA, and Sts-1_PGM. A- SixA (PDB ID 1UJC), EPPase_PGM, and mSts-1_PGM (PDB ID 2H0Q) monomers are shown in ribbon representation in similar orientations. Conserved catalytic residues are shown in red ball-and-stick: the histidines (H8/H108 in SixA, H80/H260 EPPase_PGM, and H380/H565 in mSts-1_PGM), the arginines (R7/R55 in SixA, R79/R83/R160 in EPPase_PGM, and R379/R383/R462 in mSts-1_PGM), and the glutamates (E188 in EPPase_PGM and E490 in mSts-1_PGM). The secondary structure elements that are conserved between the three proteins are in grey. The regions that deviate between SixA and EPPase_PGM are in crimson (insert 1), blue (insert 2), and yellow (insert 3). The C termini are in green. B- Stereoview of overlaid Cα atoms of EPPase_PGM (light grey) and mSts-1_PGM (dark grey). The three regions that deviate between EPPase_PGM (residues 192–197, 272–292) and mSts-1_PGM (residues 494–500, 575–593, 607–610) and shaded in grey in Supplementary Figure are colored in red and blue, respectively. Conserved active site residues are shown in gold to highlight the phosphate-binding site.

Figure 3

Dimerization and phosphate-binding site. A- EPPase_PGM is shown in surface representation in grey. Polypeptides and single residues (272 and 282) at the dimer interface are colored in shades of red and indicated. B- Superposition of the EPPase_PGM active site residues (yellow) with their equivalents in Sts-1_PGM (red). C- EPPase_PGM oriented as in Figure 1 top view and displayed in its Connolly surface is colored according to its electrostatic potential contoured from −15 (intense red) to 15 k_BT/e (intense blue). D- Interactions made by the tungstate (T) ion and EPPase_PGM. Active site residues within hydrogen-bonding distances of the tungstate ion are shown in ball-and-stick. Dashed lines represent hydrogen-bond interactions and red spheres water molecules (W).

Figure 4

Comparison of the surroundings of the phosphate-binding site in EPPase_PGM and Sts-1_PGM. EPPase_PGM (grey) and Sts-1_PGM (red) were superposed (33), overlaid and shown in stick or surface representations. The tungstate ion is shown in cyan. Hydrophobic residues of Sts-1_PGM that block the access to a cavity that is otherwise solvent exposed in EPPase_PGM are labeled in red.

Figure 5

Putative E22P binding site. Docking of E22P to EPPase_PGM orientated as in Figure 4, was done with Autodock (28). Hydrogen bond interactions between E22P and its proposed binding pocket shown in stick and surface representation are shown as dotted lines.

Figure 6

Phosphatase activity of recombinant B. mori EPPase_PGM. A- Assays were carried out with 50 nM EPPase_PGM wild-type, K196S, K196D or K292S mutants and pNPP at various concentrations. K_m and k_cat were obtained after fitting the data points to equation (1). B- Inhibition of EPPase_PGM activity by tungstate or C- phosphate. Initial velocities of pNPP hydrolysis by EPPase_PGM were measured and plotted at the indicated tungstate or phosphate concentrations (^). The data points were fitted to equation (2). All assays were conducted at pH 7.5 and 37 ºC. D- Phosphatase activity was carried out as in A- with prednisolone 21-phosphate as substrate.

Figure 7

PTP activity of EPPase_PGM. A- Dephosphorylation of a pTyr-containing peptide by wild type, H80S mutant EPPase_PGM, and Sts-1_PGM (50 and 500 nM). The concentration of the released phosphate was measured using the malachite green assay read at 650 nm. B- Plot of the initial rate of hydrolysis vs the pTyr-peptide concentration. C- Proteins from TCR-stimulated Jurkat cells were isolated by immunoprecipitation, eluted from the pTyr antibodies, and evaluated as EPPase_PGM substrates at the indicated concentration. Reaction products were evaluated by anti-phosphotyrosine western analysis. All assays were conducted at pH 7.5 and 37 °C. Each assay was repeated at least three times. Figures are representative of one experiment.

Scheme 1

Activation of ecdysone 22-phosphate by EPPase.

Scheme 2

Schematic representation of the proposed reaction mechanism of EPPase. Only catalytic residues are shown. See text for more details.