Crystal structure of human slingshot phosphatase 2 (2007)

Dual specificity protein tyrosine phosphatases (DSPs) play an important role in controlling various cellular processes, including cell growth, differentiation, transcription, and metabolism by catalyzing the hydrolysis of phosphorylated protein substrates.1 To meet the demand for distinct functions implicated in diverse cellular signaling, the human genome is estimated to encode 61 DSPs among 107 protein tyrosine phosphatases (PTPs).2 According to their structural and functional characteristics, 61 DSPs can be further divided into seven groups.2 MKPs and atypical DSPs groups can dephosphorylate mitogen-activated protein kinases, while CDC14 goup is involved in dephosphorylation of cell cyclin dependent kinases.3 Myotubularin and PTEN groups are reported to dephosphorylate phospholipid substrates but not protein substrates.4, 5 PRL group whose substrates are still unknown is reported to be associated with cancer metastasis.6 Finally slingshot (SSH) group is recently discovered and the least known. SSH was initially identified as a cofilin phosphatase through genetic studies in Drosophila, where its dysfunction was noted to cause disorganized epidermal cell morphogenesis, including splintered hair bristles.7 SSH can dephosphorylate cofilin at phospho-serine 3 whose activity disrupt actin reorganization induced by either LIMK or TESK.8 Three human SSH homologues were identified so far, denoted SSH1, SSH2, and SSH3. SSH is the only group whose structural information is not available among seven DSP groups. Here, we have determined the crystal structure of catalytic domain of human slingshot 2 phosphatase (SSH2-C) at 2.1 Å resolution. In this structure, SSH2-C adopts a fold similar to that known for Vaccinia virus VH1-related dual-specific protein phosphatase (VHR; pdb code:1vhr) structure.9 However, SSH2-C possesses a shallower and wider pocket, relative to VHR, which is well consistent with small size of phosphoserine in cofilin substrate. Furthermore, this is the first structure from SSH group to be determined. Hence, together with those from other six DSP groups, it will provide a unique opportunity to compare structures across seven DSP groups. SSH2-C was cloned from human brain cDNA (Clontech) and subcloned into pET28a. SSH2-C (C392S) (residues 305-450) was expressed in Escherichia coli strain BL21(DE3). The C392S mutation was necessary to obtain well-diffracting crystals, possibly because the cysteine residue in the active site is susceptible to oxidation. Cells were grown at 291 K after induction with 0.1 mM IPTG for 24 h. Cells were harvested and suspended in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM PMSF, 0.04% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol. After cell lysis by sonication, the His-tagged SSH2-C protein construct was purified by nickel-affinity chromatography. The His-tag was removed by trypsin digestion and the protein was further purified by Q-Sepharose ion exchange chromatography. The catalytic activity of SSH2-C was checked by monitoring hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate using a spectro-fluorometric assay. The purified protein was dialyzed against a buffer containing 20 mM HEPES (pH 7.0), 0.2M NaCl, 2 mM DTT, and 5% glycerol. The protein was concentrated to 20 mg/ml for use in crystallization. Crystallization was performed at 291 K using the hanging-drop vapor-diffusion method. Initial crystallization trials were carried out by using commercial screening kits (Hampton Research). The best crystals were grown by mixing 1.8 μl of protein (20 mg/ml) solution with an equal volume of reservoir solution containing 0.1M Tris-HCl (pH 8.5), 25% PEG3350, and 8% ethanol at 291 K. After three days, SSH2-C crystals grew to their full size. X-ray diffraction data were collected at the Pohang Accelerator Laboratory, 4A Macromolecular Crystallography Wiggler Beamline. The crystal in the droplet was transferred to a cryo solution containing the mother liquor supplemented with 20% (v/v) glycerol for 1 min and flash-frozen in a nitrogen gas stream at 93 K. The diffraction data were processed and scaled with the programs Mosflm10 and Scala.11 SSH2-C crystals belonged to space group P32 with unit cell parameters of a = b = 94.58 Å, c = 38.77 Å, α = β = 90°, γ = 120° and diffracted to 2.1Å resolution. The statistics for data collection and refinement are summarized in Table I. The structure of SSH2-C was determined by molecular replacement using VHR structure (PDB code: 1VHR) as a search model.9 The program PHASER12 placed three catalytic domains of SSH2-C in the asymmetric unit of crystal. The crystals tended to show merohedral twinning. To detect merohedral twinning and estimate the twinned fraction, a merohedral crystal twinning server was used.13 The value of twinned fraction was 0.45, indicating that SSH2-C crystal was perfectly twinned with twin operator of h + k, −k, −l. The model was refined with the program SHELX97,14 which could deal with perfectly twinned data for the refinement. The subsequent model building were carried out using the program O.15 Randomly selected 5% of data were set aside for the Rfree calculation. Tight noncrystallographic symmetry restraints were applied to three protomers, as implemented in SHELX97. Strong electron densities, not part of the protein, were found near the cavities of active sites of three protomers. We modeled these as sulfate ions from the crystallization condition. Water molecules were gradually added in the model throughout the refinement. The quality of model was inspected by using the program PROCHECK.16 The Ramachandran plot, prepared using the program PROCHECK, shows that 89.0% and 10.3% of all residues fell within the most favored and additionally favored regions, respectively. Two of three catalytic residues C392S were in the generously allowed region as they were consistent with well defined electron densities. The final model included the residues 307–448 for three protomers A, B, and C, three sulfate ions, and 91 water molecules. Figures were drawn by the programs RIBBONS,17 Bobscript,18 and Molscript.19 The atomic coordinates of the final structure were deposited in the PDB with the code 2NT2. Other SSH catalytic domains are likely to adopt similar fold with SSH2-C considering that sequence alignment of three SSHs (SSH1, SSH2, and SSH3) show more than 80% sequence similarity. The three protomers (A–C) in the asymmetric unit are nearly in identical conformation. SSH2-C contains a central twisted five-stranded β sheet (β1–β5) surrounded by six α helices (α1–α6) [Fig. 1(a)]. One side of the β sheet is covered by two α-helices (α1–α2) and the other by the remaining four α-helices (α3–α6). SSH2-C has virtually the same overall conformation with that of other DSPs. The structure near the active site of SSH2-C exhibits the canonical PTP conformation that can be found in VHR and other DSPs.5, 21-23 The common active site conformation of all DSPs can be defined by the direction of side-chain of the active-site cysteine, the interaction of the P-loop with substrate, and the hydrogen-bonding interaction of general acid.24 Namely, C392S is optimally positioned for nucleophilic attack by pointing to backbone amide atoms from the P-loop with interaction distance of 2.7–4.7 Å. However, the residue responsible for the general acid is somewhat unclear because the general acid loop contains three consecutive carboxylate group containing sequences (Tyr360-Asp361-Glu362-Glu363-Ala364) in SSH2. A structural superposition between SSH2-C and VHR reveals that Asp361 is aligned to the general acid residue of VHR, indicating that Asp361 is likely to function as general acid in SSH2. Amide atoms of the main chain in the P-loop and the guanidinium moiety of Arg398 strongly interact with the oxygen atoms from sulfate ion, which was used as a precipitant in the crystallization. The interaction distances between the amides of Lys393-Arg398 and one of the sulfate oxygen atoms are 2.7–3.1 Å. (a) A ribbon diagram of the SSH2-C. Secondary structural elements (helix, orange; strand, blue; loop, grey), which were assigned with the program PROCHECK,16 are labeled. Catalytic triads (D377, C392S, and R398) and bound sulfate ion are shown as a ball-and-stick representation. Boundaries of secondary structural elements are β1 (309–312), β2(315–318), β3(334–339), β4(353–356), β5(387–391), α1(320–323), α2(326–331), α3(371–383), α4(398–411), α5(415–425), and α6(433–447). (b) The superpositions of seven DSP structures representing each subfamily. A Cα trace of the DSP18 is superposed with that of VHR. Worm model is in blue for SSH2-C, orange for MKP-3 (pdb code:1mkp), green for VHR (pdb code:1vhr), cyan for PRL-1 (pdb code:1xm2), yellow for CDC14B (pdb code:1ohe), red for PTEN (pdb code:1d5r), and magenta for myotubularin2 (pdb code:1m7r), respectively. The superposition statistics against SSH2-C is 1.5/142 for MKP-3, 1.3/143 for VHR, 1.7/121 for PRL-1, 1.6/121 for CDC14B, 1.6/124 for PTEN, and 2.2/113 for Myotubularin2 where the former value refers to rms deviations and that the latter one refers to the number of superposed Cα atoms. The position of catalytic cysteine is indicated as a grey ball. (c) Cα trace of SSH2-C is superimposed with that of the VHR. The region cannot be aligned are colored green, whereas those of SSH2-C are missing are colored red. The point of view is the same as Figure 1(a). (d) The sliced view of active sites for SSH2-C and VHR. The molecular surface diagrams, which were produced using the program VOIDOO,20 are drawn as a basket-weaved model. It shows the depth and width of the active site pocket for SSH-2C and VHR. The positions of catalytic cysteines are labeled in the figure. To date, ∼20 structures of mammalian DSPs have been reported in the PDB, which belonged to six out of seven DSP groups. However, the structure of SSH group has not yet revealed. With the present structure, it is now possible to compare the structures of the all seven DSP groups. We selected seven human DSPs representing each group and compared structures [Fig. 1(b)]. They include VHR,9 MKP3,25 CDC14B,26 PTEN,27 PRL-1,28 Myotubularin229 and SSH2-C. The superpositions were performed using the program O using the criteria of being less than 3.8 Å for more than three consecutive Cα atoms. As shown in comparison, they all share the common core structure. Myotubularin2 has the most deviated structure and is much larger than SSH2-C because it contains a SID motif that is well conserved in the myotubularin group.29 Judging from the number of superposed Cα atoms with the least rms deviations, VHR has the most similar structure with SSH2 among seven DSP groups, suggesting atypical DSP group is structurally the closest one to SSH group. A search for homologous structures by using the Dali server30 also revealed that VHR (pdb code: 1VHR; z-score = 14.3), is the closest structure to SSH2-C. Although there are high degrees of similarities between the two structures, they are not well aligned in two regions [Fig. 1(c)]. The first region is due to the absence of an N-terminal helix and a loop in SSH2-C, which we refer to as helix α0 and following loop α0–β1 in VHR, respectively. The helix α0 and the following loop α0–β1 of VHR-related phosphatases are involved in substrate recognition.9 Deletion of this substrate recognition motif in SSH2-C implies that the substrate recognition mechanism of SSHs is different from that of VHR-related phosphatases. The second region that cannot be aligned between SSH2-C and VHR are found in loop β3–β4 [Fig. 1(c)]. The loop β3–β4 of SSH2-C is shortened by nine residues compared to that of VHR. The absence of helix α0 and the shorter β3–β4 loop in SSH2-C result in a flat and shallow active-site pocket [Figs. 1(c,d)]. The substrate preference for phosphotyrosine over phosphothreonine is thought to be determined by the pocket depth of VHR.9, 31 As shown in Figure 1(d), the pocket depth of SSH-2C is shallower when compared with that of VHR. The shallower pocket depth may facilitate the access of phospholylated group of phospho-Ser3 in cofilin substrate. Although cofilin structure has been determined, the extensive analysis regarding substrate specificity is difficult owing to the disorder at N-terminal region including Ser-3.32 In summary, we have determined for the first time a structure of human SSH group. Structural analysis revealed that atypical DSP group related to VHR is the closest one with the SSH group. The planar and shallow active site pocket might enable to accommodate small phosphoserine substrates. The precise mechanism of substrate specificity of SSH group needs to be elucidated with the complex structure with cofilin substrates in the future. Finally, the comprehensive understanding of SSH2-C structure should lead to the design of selective inhibitors for each DSPs and provide clues to their cellular functions. Authors thank Dr. H. S. Lee and the staffs at Pohang Accelerator Laboratory 4A Macromolecular Crystallography Wiggler Beamline for their help with data collection.