Suk-Kyeong Jung

Dephosphorylation of the C-terminal tyrosyl residue of the DNA damage-related histone H2A.X is mediated by the protein phosphatase eyes absent.

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

Structure of human alpha-enolase (hENO1), a multifunctional glycolytic enzyme.

Aside from its enzymatic function in the glycolytic pathway, alpha-enolase (ENO1) has been implicated in numerous diseases, including metastatic cancer, autoimmune disorders, ischaemia and bacterial infection. The disease-related roles of ENO1 are mostly attributed to its immunogenic capacity, DNA-binding ability and plasmin(ogen) receptor function, which are significantly affected by its three-dimensional structure and surface properties, rather than its enzymatic activity. Here, the crystal structure of human ENO1 (hENO1) is presented at 2.2 A resolution. Despite its high sequence similarity to other enolases, the hENO1 structure exhibits distinct surface properties, explaining its various activities, including plasmin(ogen) and DNA binding.

Discovery of novel PRL-3 inhibitors based on the structure-based virtual screening

The inhibitors of phosphatase of regenerating liver-3 (PRL-3) have been shown to be useful as therapeutics for the treatment of cancer. We have been able to identify 12 novel PRL-3 inhibitors by means of the virtual screening with docking simulations under the consideration of the effects of ligand solvation in the scoring function. Because the newly identified inhibitors are structurally diverse and reveal a significant potency with IC(50) values ranging from 10 to 50muM, all of them can be considered for further development by structure-activity relationship or de novo design methods. Structural features relevant to the interactions of the newly identified inhibitors with the amino acid residues in the active site and the peripheral binding site of PRL-3 are discussed in detail.

Discovery of Novel Cdc25 Phosphatase Inhibitors with Micromolar Activity Based on the Structure-Based Virtual Screening

Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy because of the correlation of their overexpression with a wide variety of cancers. We have been able to identify five novel Cdc25 phosphatase inhibitors with micromolar activity by means of a computer-aided drug design protocol involving the homology modeling of Cdc25A and the virtual screening with the automated AutoDock program implementing the effects of ligand solvation in the scoring function. Because the newly discovered inhibitors are structurally diverse and reveal a significant potency with IC 50 values lower than 10 microM, they can be considered for further development by structure-activity relationship studies or de novo design methods. The differences in binding modes of the identified inhibitors in the active sites of Cdc25A and B are discussed in detail.

Crystal structure of ED-Eya2: insight into dual roles as a protein tyrosine phosphatase and a transcription factor

Eya proteins are transcription factors that play pivotal roles in organ formation during development by mediating interactions between Sine Oculis (SO) and Dachshund (DAC). Remarkably, the transcriptional activity of Eya proteins is regulated by a dephosphorylating activity within its Eya domain (ED). However, the molecular basis for the link between catalytic and transcriptional activities remains unclear. Here we report the first description of the crystal structure of the ED of human Eya2 (ED‐Eya2), determined at 2.4‐A resolution. In stark contrast to other members of the haloacid dehalogenase (HAD) family to which ED‐Eya2 belongs, the helix‐bundle motif (HBM) is elongated along the back of the catalytic site. This not only results in a structure that accommodates large protein substrates but also positions the catalytic and the SO‐interacting sites on opposite faces, which suggests that SO binding is not directly affected by catalytic function. Based on the observation that the DAC‐binding site is located between the catalytic core and SO binding sites within ED‐Eya2, we propose that catalytic activity can be translated to SO binding through DAC, which acts as a transcriptional switch. We also captured at two stages of reaction cycles‐acylphosphate intermediate and transition state of hydrolysis step, which provided a detailed view of reaction mechanism. The ED‐Eya2 structure defined here serves as a model for other members of the Eya family and provides a framework for understanding the role of Eya phosphatase mutations in disease.—Jung, S.‐K., Jeong, D. G., Chung, S. J., Kim, J. H., Park, B. C., Tonks, N. K., Ryu, S. E., Kim, S. J. Crystal structure of ED‐Eya2: insight into dual roles as a protein tyrosine phosphatase and a transcription factor. FASEB J. 24, 560–569 (2010). www.fasebj.org

Structural basis for the cold adaptation of psychrophilic M37 lipase from Photobacterium lipolyticum.

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 Å resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures. Proteins 2008.

Crystal structure of the catalytic domain of human MKP-2 reveals a 24-mer assembly

Crystal structure of the catalytic domain of human MKP-2 reveals a 24-mer assembly Dae Gwin Jeong, Suk-Kyeong Jung, Tae-Sung Yoon, Eui-Jeon Woo, Jae Hoon Kim, Byoung Chul Park, Seong Eon Ryu,* and Seung Jun Kim* 1 Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-Gu, Daejeon 305-333, Republic of Korea 2 Faculty of Biotechnology, College of Applied Life Science, Cheju National University, Jeju 690-756, Korea 3 Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-Gu, Daejeon 305-333, Republic of Korea 4 Department of Bio Engineering, Hanyang University, Seongdong-Gu, Seoul 133-791, Republic of Korea

Crystal structure of human slingshot phosphatase 2

Introduction. 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. 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). According to their structural and functional characteristics, 61 DSPs can be further divided into seven groups. MKPs and atypical DSPs groups can dephosphorylate mitogen-activated protein kinases, while CDC14 goup is involved in dephosphorylation of cell cyclin dependent kinases. Myotubularin and PTEN groups are reported to dephosphorylate phospholipid substrates but not protein substrates. PRL group whose substrates are still unknown is reported to be associated with cancer metastasis. 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. SSH can dephosphorylate cofilin at phospho-serine 3 whose activity disrupt actin reorganization induced by either LIMK or TESK. 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 (SSH2C) at 2.1 Å resolution. In this structure, SSH2-C adopts a fold similar to that known for Vaccinia virus VH1related dual-specific protein phosphatase (VHR; pdb code:1vhr) structure. 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. Materials and Methods. Expression and purification: 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 Histagged SSH2-C protein construct was purified by nickelaffinity 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 and data collection: 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 ll 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

Structure-Based Virtual Screening Approach to the Discovery of Novel Inhibitors of Eyes Absent 2 Phosphatase with Various Metal Chelating Moieties

Despite a series of persuasive experimental evidence for the involvement of eyes absent protein tyrosine phosphatases in various human cancers, no small‐molecule inhibitor has been reported so far. We have identified seven novel inhibitors of eyes absent homologue 2 (Eya2) with IC50 values ranging from 1 to 70 μm by the virtual screening with docking simulations and enzyme inhibition assay. Atomic charges of the active‐site Mg2+ ion complex are calculated to enhance the accuracy of docking simulations. The newly discovered inhibitors are structurally diverse and have various chelating groups for the Mg2+ ion. The interactions with the amino acid residues responsible for the stabilizations of the inhibitors in the active site of Eya2 are addressed in detail.

Discovery of VHR Phosphatase Inhibitors with Micromolar Activity based on Structure-Based Virtual Screening

Protein tyrosine phosphatases (PTPs) are a family of closely related key regulatory enzymes and are responsible for the dephosphorylation of phosphotyrosine residues in their protein substrates. So far much evidence has been reported in support of the correlation between malfunctions in PTP activity and various diseases including cancer, neurological disorders, and diabetes. This has made PTPs promising targets for therapeutic intervention. Among the variety of PTPs, vaccinia H1-related (VHR) phosphatase is a dual-specificity phosphatase (DSP) that dephosphorylates the activated ERK and JNK MAP kinases and thereby weakens the ERK signaling cascade in mammalian cells. 3] Recently Rahmouni et al. reported that the human VHR phosphatase was involved in the regulation of cell-cycle progression and was itself modulated during the cell cycle. Using RNA interference, they also showed that cells lacking VHR arrested at the G1-S and G2-M transitions of the cell cycle with a decreased telomerase activity. This line of experimental evidence indicates that VHR can serve as a therapeutic target for cancer. Furthermore, the VHR activity has been known to be promoted by ZAP-70, a spleen tyrosine kinase (Syk)-related tyrosine kinase, which plays a critical role in the immune response of activated T cells, or by the interaction with vaccinia-related kinase 3 (VRK3). The involvement of VHR in immune response also supports the possibility that it can be a potential target for drug discovery. Besides the pharmaceutical interest, specific inhibitors of VHR are expected to be useful for revealing the physiological functions of VHR and ERK signaling. The X-ray crystal structure of VHR exhibited a conserved structural scaffold for both DSPs and PTPs. A shallow active site pocket in VHR is consistent with its broad substrate specificity for the hydrolysis of phosphorylated serine, threonine, and tyrosine residues. Positively charged crevices near the active site were invoked to explain the preference of VHR for the substrates with two phosphorylated residues. In the crystal structure, the catalytic residue Cys124 resides in close proximity to the central sulfur atom of the substrate analogue at a distance of 3.65 >. This supports the possibility that Cys124 acts as a nucleophile attacking the phosphorus atom of a substrate to form a phosphoenzyme intermediate. The X-ray crystal structure also showed that the negatively charged oxygen atoms of the substrate analogue should be stabilized in the active site through the formation of multiple hydrogen bonds with backbone amide groups and the side chain of Arg130. These structural features may serve as key information for the discovery of small molecules modulating the catalytic activity of VHR. Despite a series of experimental evidence for the involvement of VHR in several human diseases, only a few VHR inhibitors have been discovered so far. These include natural products and synthetic compounds such as tetronic acid, RK682, and benzofuran. Recently, Shi et al. identified five new inhibitors of VHR based on structure-based virtual screening with docking simulations in combination with diffusionedited NMR spectroscopy experiments. Subsequent enzymatic assays showed that one of them could inhibit VHR at the micromolar level. The calculated binding mode of the inhibitor was consistent with that in the X-ray crystal structure of VHR in complex with a substrate analogue. In the present study, we identify the novel classes of VHR inhibitors by means of a structure-based drug design protocol involving the virtual screening with docking simulations and in vitro enzyme assay. Virtual screening with docking simulation has not always been successful because of the use of the inaccurate scoring functions. The characteristic feature that discriminates our virtual screening approach from the others lies in the implementation of an accurate solvation model in calculating the binding free energy between VHR and its putative ligands, which would have an effect of increasing the hit rate in the enzyme assay. The reason for this lies in that the overestimation of the binding affinity of a ligand with many polar atoms can be avoided by including its desolvation energy in the scoring function. It will be shown that the docking simulation with the improved scoring function can be a useful tool for elucidating the activities of the identified inhibitors, as well as for enriching the chemical library with molecules that are likely to have biological activities. Of the 85000 compounds subject to the virtual screening with docking simulations, 200 top-scored compounds were selected as virtual hits. 194 of them were available from the compound supplier and were tested for inhibitory activity against VHR by in vitro enzyme assay. The inhibition assay was performed in duplicates at all concentrations of the inhibitors and [a] Prof. H. Park Department of Bioscience and Biotechnology Sejong University 98 Kunja-Dong, Kwangjin-Ku, Seoul 143-747 (Korea) Fax: (+82)2-3408-4334 E-mail : hspark@sejong.ac.kr [b] S.-K. Jung, Dr. S. J. Kim Translational Research Center Korea Research Institute of Bioscience and Biotechnology 52 Eoeun-Dong, Yuseong-Gu, Daejeon 305-333 (Korea) Fax: (+82)42-860-4598 E-mail : ksj@kribb.re.kr [c] D. G. Jeong, Dr. S. E. Ryu Systemic Proteomics Research Center Korea Research Institute of Bioscience and Biotechnology 52 Eoeun-Dong, Yuseong-Gu, Daejeon 305-333 (Korea) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.