If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Protein tyrosine phosphatases (PTPs) play important roles in cellular signal transduction mediating cell growth, differentiation, transcription, and metabolism.1 PTPs have been regarded as a drug target for human disorders such as cancer, diabetes, and autoimmune disease according to their distinct mode of actions.1, 2 The human PTP family, which consists of 107 members, can be divided into three classes of Cys-based PTPs and a class of Asp-based PTPs according to their substrate specificity and structural characteristics.2 Cys-based PTPs include 38 classical PTPs and 61 dual-specific PTPs (DSPs), which can dephosphorylate both phosphoserine/threonine and phosphotyrosine substrates, whereas classical PTPs only can dephosphorylate phosphotyrosine substrates.2 Typical DSPs can remove phosphate groups of Erk, Jnk, and p38 at their dually phosphorylated pT-X-pY motif (X is any amino acid). Besides the catalytic domain, typical DSPs have an additional mitogen-activated protein (MAP) kinase–binding (MKB) domain that contributes to substrate binding and determination of substrate specificity.3 Atypical DSPs are distinct in that they lack the MKB domain and thereby constitute smaller enzymes than typical DSPs. Among 19 members of atypical DSPs known in the human genome, Vaccinia virus VH1-related dual-specific protein phosphatase (VHR) is well characterized structurally and biologically.4, 5 VH1-related member Y (VHY) is another member of atypical DSPs and quite recently has been characterized as a testis-specific DSP encoded by the DUSP15 gene.6 VHY is an active phosphatase in vitro and is readily detectable in spermatocytes and spermatids.6 Further, the subcellular location of VHY is the plasma membrane in a myristoylation-dependent manner. Thus, VHY may be involved in the regulation of meiotic signal transduction in testis cells. Here we report the crystal structure of human VHY catalytic domain (VHYc) at 2.4 Å. The detailed structural information of VHYc reveals distinct structural motif and surface properties compared to those of VHR, suggesting that VHY may have unique substrate specificity and a regulation mechanism.
Materials and Methods.
Expression and Purification.
VHY was cloned from human brain complementary DNA (cDNA; Clontech) and subcloned into pET28a. VHYc(C88S) (residues 1–157) lacking C-terminal domain was expressed in Escherichia coli BL21(DE3) strain. The C88S mutation was necessary to obtain well-diffracted crystals. Cells were grown at 18°C after induction with 0.1 mM isopropyl-thio-β-D-galactopyranoside (IPTG) for 24 h. Cells were harvested and resolved in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM phenyl-methylsulfonyl-fluoride (PMSF), 0.04% (v/v) 2-mercaptoethanol, and 5% (v/v) glycerol. After cell lysis by sonication, the His-tagged VHYc protein was purified by nickel-affinity chromatography. The His-tag was removed by thrombin protease digestion and the VHY protein was further purified by Q-Sepharose ion exchange chromatography. The purified protein was dialyzed against a buffer containing 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-NaOH (pH 7.5), 0.1 M NaCl, 2 mM dithiothreitol (DTT). In order to improve the solubility of the protein, 8 mM DTT and 0.2% (v/v) β-n-octyl-D-glucoside (β-OG) were also added, and the protein was concentrated to 10 mg/mL for use in crystallization.
Crystallization and Data Collection.
Crystallization was performed at 18°C using the hanging-drop vapor-diffusion method, and initial trials were carried out by using commercial screening kits (Hampton Research). The best crystals were grown by mixing 1.8 μL of protein (10 mg/mL) solution and equal volume of reservoir solution containing 0.1M HEPES-NaOH (pH 7.0), 0.6 M ammonium sulfate, and 1% (w/v) polyethylene glycol (PEG) 8000, 0.2% (w/v) β-OG. After 3 days, VHYc crystals were grown to their full size. X-ray diffraction data were collected using a Rigaku RU-300 generator with an R-AXIS IV++ image plate detector. The crystal in the droplet was transferred to a cryosolution containing the mother liquor supplemented with 20% (v/v) glycerol for 1 min and flash-frozen in a nitrogen gas stream at −180°C. The crystal diffracted to 2.4 Å resolution and belonged to the space group P212121 with unit cell parameters of a = 43.23 Å, b = 76.40 Å, and c = 101.43 Å. The collected diffraction data were processed and scaled with the programs Mosflm7 and Scala,8 respectively. The statistics for data collection and refinement are summarized in Table I.
Table I. Data Collection and Refinement Statistics
The values in parentheses (completeness and Rmerge) are for the highest resolution bin.
Rmerge = Σi|Ii − <I>|/Σ|<I>|, where I is the intensity for the ith measurement of an equivalent reflection with the indices h, k, and l.
Cell parameters (Å)
a = 43.23 Å, b = 76.40 Å, c = 101.43 Å, α = β = γ = 90°
The structure of VHYc was determined by molecular replacement method using VHR structure [Protein Data Bank (PDB) code: 1VHR] as a search model.4 The program EPMR9 placed two monomers in the asymmetric unit of crystal. Refinement was carried out using the program CNS.10 The randomly selected 5% of data were set aside for the Rfree calculation. Refinement included an overall anisotropic B factor and bulk solvent correction. The noncrystallographic symmetry (NCS) restraints have been applied on an initial stage of refinement. Rounds of refinements were performed with manual rebuilding using the program O.11 Two monomers in the asymmetric unit have essentially the same conformation and are related to each other by a two-fold NCS. However, these two monomers do not make significant dimer interactions, and VHYc appeared to act as a monomer, as analyzed by gel filtration chromatography and dynamic light scattering (data not shown). Thus, it is likely that dimer observed in the crystal is not biologically relevant. In addition, strong electron density was observed in the interface between two monomers in the asymmetric unit. We modeled it as a β-OG that was added for the crystallization. The Rcryst and Rfree are 21.8% and 26.9%, respectively. The Ramachandran plot drawn by the program PROCHECK12 shows that 87.3% and 12.7% of all residues fall within the most favored and additionally favored regions, respectively. There are no residues in the additionally allowed or disallowed regions. The final model includes residues 1–156 plus 3 preceding residues from the N-terminus for thrombin cleavage site in monomer A, the residues 2–157 in monomer B, 1 β-OG, 4 sulfate ions, and 95 water molecules, respectively. Figures were drawn by the programs RIBBONS,13 GRASP,14 BobScript,15 and MolScript.16
Results and Discussion.
VHYc (1–157) folds into a compact and globular domain of approximate dimensions of 40 × 35 × 30 Å. Each VHYc molecule contains a central twisted, five-stranded β-sheet (β1–β5) surrounded by six α helices (α1–α6) [Fig. 1(a)]. The topological structure of VHY is similar to that of VHR and the catalytic domain of MKP-3.4, 17 In a search for homologous structures by using the Dali server,18 several members of the PTPs, including VHR, MKP-3, and PTEN,19 are identified with similar z values ranging from 13 to 10. Structural similarities are mainly found in the region having secondary structural elements. When we aligned the structure of VHY with that of VHR, 132 Cα atoms could be superimposed with a root-mean-square deviation (RMSD) of 1.6 Å [Fig. 1(b)].
Although there are good alignments with VHR, substantial differences are also found in several regions. The most prominent differences are the absence of N-terminal helix α0 and loop α0–β1, and insertion of helix α6. Helix α0 and loop α0–β1 are known for substrate recognition motif of VHR and other phosphatases.4 Deletion of these moieties implicates a different substrate recognition mechanism of VHR. In addition, helix α6 in VHYc, which cannot be found in other PTPs, is located at the back side of the active site, having numerous hydrophobic interactions with helices α2 and α3. Helix α6 is followed by helix α5 by three residues' insertion, changing direction toward the middle of helices α2 and α3. Helix α6 in VHYc is a distinct motif and is not part of the DSP core structure, suggesting that helix α6 may be implicated for the interaction between the catalytic domain and the domain following the catalytic domain (residues 158–235). The second domain of VHY is not functionally well characterized. Other regions that cannot be aligned between VHY and VHR proteins are found in loop β3–β4 and loop β5–α2. The N-terminal region, which is responsible for myristoylation and thereby targeting to the plasma membrane,6 is away from the body of the catalytic domain, suggesting that myristoylation may not affect the enzyme activity of VHYc. The structure near the active site of VHYc exhibits the PTP active site conformation that can be found in VHR and other PTPs. Namely, amide atoms of the main-chain in the active site loop and guanidine group from Arg94 strongly interact with the oxygen atoms from sulfate ion, which is incorporated as a crystallizing agent. These interactions, mimicking intermediates between phosphorylated substrate and corresponding phosphatase, were also found in VHR4 and KAP20 structures. General acid residue Asp57 in VHYc occupies the similar position of Asp92 in VHR. Although catalytically important residues in VHYc are well aligned with those in VHR, sequence conservation of residues near the active site is quite low. Most prominent differences are found in the P loop, which resides in the active site sequence (C)X1X2GX4X5(R). The residues in the P loop of VHYc (Phe89-Ala90-Gly91-Ile92-Ser93) have more hydrophobic character than those of VHR (Arg125-Glu126-Gly127-Tyr128-Gly129). The protruding loops β3–α2 and α0–β1 in VHR result in rather deep and narrow active site pocket, whereas in VHYc, loop β3–α2 is shortened by three residues, and loop α0–β1 is absent, which results in flat and wide active site pocket [Fig. 1(b and c)]. In addition to surface differences, charge distribution on the VHYc active site surface is quite different from that of VHR [Fig. 1(c)]. Asp39 in the loop β3–α2 of VHYc is negatively charged, while Met69 in the corresponding loop of VHR is neutral. In addition, Phe89 and Ala90 in VHYc are neutral, while the corresponding residues, Arg125 and Glu126, in VHR are positively and negatively charged, respectively. These results suggest that VHYc constitute a unique three-dimensional structure and thus has distinct substrate specificity among other DSPs. The atomic coordinates of the final structure have been deposited in the PDB with the code 1YZ4.