Fig 1.
Crystal structure of Tk-PTP(form I).
(A) Tk-PTP(form I) is presented as ribbon drawings with secondary structure labels. Green, α-helices; violet, β-strands; white, the remaining structures. The catalytic cysteine residue shown as a stick model is labeled as C93. (B) Stereo views of Cα traces of three superimposed DUSP proteins. (C) The P-loops of four DUSP proteins are shown in labeled sticks and compared. Arrows indicate the direction of main chain amides of four P-loop-constituting central residues. The PDB codes are 1VHR for DUSP3, 1MKP for DUSP6, and 3LJ8 for DUSP9, respectively. (D) Structural alignment of the P-loops of Tk-PTP(form I) and MES-bound DUSP3 shown in sticks.
Table 1.
Data collection and structure refinement statistics.
Fig 2.
Crystal structure of Tk-PTP(form II).
(A) Tk-PTP(form II) is presented as a secondary structure-labeled ribbon drawing. Cyan, α-helices; navy, β-strands; white, the remaining structures. The catalytic cysteine residue is labeled as C93 and the vanadate molecule bound to the active site pocket is shown in sticks. (B) Stereo views of Cα traces of two forms of Tk-PTP. The P-loop and α4−α5 loop regions are indicated by dotted circles and rectangles, respectively. (C) Stereo views of the superimposed P-loops of two forms of Tk-PTP shown in sticks. For clarity, the main chain and the side chains of Cys93 and Arg99 are shown. Vanadate-mediated hydrogen bonds and electrostatic interactions are presented as dashed lines. Dotted circles indicate the backbone carbonyl of Met94 that undergoes a peptide flip. The GG motif residues are marked with asterisks. A 2Fo-Fc electron density omit map of the vanadate molecule contoured at 1.5 σ is shown together. The temperature factor of vanadate bound to Tk-PTP(form II) is 18.2. (D) Stereo views of the superimposed P-loops of Tk-PTP(form II) and DUSP23a shown in sticks. The PDB code for DUSP23a bound to vanadate is 4ERC. (E) The P-loops of two forms of Tk-PTP are shown in labeled sticks along with the 2Fo-Fc electron density map contoured at 1.5 σ. The direction of main chain amides of four P-loop-constituting central residues are indicated by arrows. (F) The sequences of the P-loop of Tk-PTP and 28 DUSP members are aligned. DUSP1–28 are from human; TbpA is from Pseudomonas aeruginosa; VH1 is from Vaccinia virus. Conserved residues are shaded in green. The PTP signature motif is shown at the top, and the GG motif residues in the P-loop of Tk-PTP are marked in red.
Fig 3.
Structural comparison between two forms of Tk-PTP.
Stereo views of two forms of Tk-PTP for structural comparison. Dotted circles indicate the backbone carbonyl of Val131 undergoing a peptide flip. Hydrogen bonds described in the main text are presented as dashed lines. (A) The P-loop and α4−α5 loop of two forms of Tk-PTP are structurally compared. The GG motif residues are labeled in blue, while Arg124 and Arg127 are labeled in red. (B) The conformation of dual general acid/base residues (labeled in red) are structurally compared between two forms of Tk-PTP.
Fig 4.
Structural analysis of Tk-PTP(G95A).
(A) Stereo views of Cα trace of Tk-PTP(G95A) that are superimposed onto that of two forms of Tk-PTP. Dotted circles and rectangles indicate the P-loop and α4−α5 loop regions, respectively. (B) The P-loops of three Tk-PTP proteins are shown as labeled sticks and compared. Dashed lines represent hydrogen bonds and electrostatic interactions mediated by vanadate bound to Tk-PTP(form II) or by formate bound to Tk-PTP(G95A). (C) The P-loop and α4−α5 loop regions of Tk-PTP(G95A) are structurally aligned to those of the two forms of Tk-PTP.
Fig 5.
Characterization of enzymatic activity of Tk-PTP.
Phosphatase activity assays were carried out at 20°C and 60°C as described in the Materials and Methods section. (A–B) Enzymatic reactions were carried out using 100 μM DiFMUP for 1 h with 10 nM purified recombinant Tk-PTP proteins in the 100 μL reaction buffer at pH 5.0 (A) or at the indicated pH levels (B). Tk-PTP, but not Tk-PTP(C93S), has dephosphorylating activity (A), which is optimum at pH 4.5–5.0 (B). (C) Kinetic parameters of 11 types of Tk-PTP proteins are listed. Enzymatic reactions were carried out with purified recombinant Tk-PTP proteins in 100 μL reaction buffer (pH 5.0). Production of DiFMU was detected by measuring fluorescence at 2 min intervals for 10 min, with DiFMUP concentrations of 50, 100, 250, 500, 1000, and 1500 μM. Initial velocity data at each substrate concentration were obtained by detecting the release of DiFMU between 2 and 10 min after the start of reaction, which was calculated from the slope of the each progress curve. Using these data, Michaelis-Menten curves shown in S6 Fig were obtained using the program OriginPro 8.0, by fitting the initial velocities against each DiFMUP concentrations to the hill equation with hill coefficient of 1. Subsequently, Lineweaver-Burk plots shown in S7 Fig were interpreted for the determination of Vmax, kcat and KM values.
Fig 6.
Characterization of thermostability of Tk-PTP.
(A) Melting points of three different constructs of Tk-PTP and four PTP proteins. All the measurements were carried out twice. (B) Number of intramolecular carbon−carbon contacts within 4.5 Å mediated by side chain atoms of Tk-PTP and three human DUSP proteins are analyzed and compared. “Hydrophilic functional group” includes imidazole of histidine, guanidinium of arginine, carboxylates of aspartate or glutamate, and amides of asparagine or glutamine. (C) Intramolecular interactions within Tk-PTP(form II) and DUSP3. The three hydrophobic residues of Tk-PTP that were substituted in A and the corresponding residues of DUSP3 are marked in red.
Fig 7.
Structural comparison with SsoPTP.
(A) Sequence alignment of Tk-PTP and SsoPTP. The secondary structure of Tk-PTP is shown together. Aligned residues are shaded green. The P-loop residues are presented in orange, and the GG motifs are highlighted by rectangles. (B) Structural alignment of Tk-PTP(form II) and SsoPTP in stereo views. Cα trace (top) and P-loop in sticks (bottom) of Tk-PTP(form II) are superimposed on those of SsoPTP. The P-loop and α4−α5 loop regions are indicated by dotted circles or rectangles, respectively (top). (C) The P-loop and α4−α5 loop regions of Tk-PTP(form II) are structurally compared to those of SsoPTP. Dotted circles highlight the guanidinium group of Arg127 of Tk-PTP(form II) or that of Arg130 of SsoPTP. Hydrogen bonds described in the main text are presented as dashed lines.