Publications by authors named "Hye-Yeon Hwang"

22 Publications

  • Page 1 of 1

Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates.

Biochem Biophys Res Commun 2021 05 19;552:164-169. Epub 2021 Mar 19.

R&D Center, YidoBio, Inc., 123 Beolmal-ro, Anyang-si, Gyeonggi-do, 14056, South Korea. Electronic address:

Human mesenchymal stem cells (MSCs) are known to have anti-inflammatory and immunomodulatory functions; thus, several MSC products have been applied as cell therapy in clinical trials worldwide. Recent studies have demonstrated that MSC spheroids have superior anti-inflammatory and immunomodulatory functions to a single cell suspension. Current methods to prepare MSC spheroids include hanging drop, concave microwell aggregation, spinner flask, and gravity circulation. However, all these methods have limitations such as low scalability, easy cell clumping, low viability, and irregular size distribution. Here, we present a nano-patterned culture plasticware named PAMcell™ 3D plate to overcome these limitations. Nano-sized silica particles (700 nm) coated with RGD peptide were arrayed into fusiform onto the PLGA film. This uniform array enabled the seeded MSCs to grow only on the silica particles, forming uniform-sized semi-spheroids within 48 h. These MSC spheroids have been shown to have enhanced stemness, anti-inflammatory, and immunomodulatory functions, as revealed by the increased expression of stem cell markers (Oct4, Sox2, and Nanog), anti-inflammatory (IL-10, TSG6, and IDO), and immunomodulatory molecules (HGF, VEGF, CXCR4) both at mRNA and protein expression levels. Furthermore, these MSC spheroids demonstrated an increased palliative effect on glycemic control in a multiple low-dose streptozotocin-induced diabetes model compared with the same number of MSC single cell suspensions. Taken together, this study presents a new method to produce uniform-sized MSC spheroids with enhanced anti-inflammatory and immunomodulatory functions in vitro and in vivo.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.026DOI Listing
May 2021

Design of a RANK-Mimetic Peptide Inhibitor of Osteoclastogenesis with Enhanced RANKL-Binding Affinity.

Mol Cells 2016 Apr 26;39(4):316-21. Epub 2016 Feb 26.

Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

The receptor activator of nuclear factor κB (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function.
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http://dx.doi.org/10.14348/molcells.2016.2286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844938PMC
April 2016

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

Biochem Biophys Res Commun 2015 May 6;461(1):122-7. Epub 2015 Apr 6.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea. Electronic address:

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode.
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http://dx.doi.org/10.1016/j.bbrc.2015.03.179DOI Listing
May 2015

Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ).

Nucleic Acids Res 2014 May 20;42(9):5937-48. Epub 2014 Mar 20.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.
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http://dx.doi.org/10.1093/nar/gku189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027156PMC
May 2014

Structural insights into the molecular mechanism of Escherichia coli SdiA, a quorum-sensing receptor.

Acta Crystallogr D Biol Crystallogr 2014 Mar 15;70(Pt 3):694-707. Epub 2014 Feb 15.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.
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http://dx.doi.org/10.1107/S1399004713032355DOI Listing
March 2014

Structural and functional characterization of an Isd-type haem-degradation enzyme from Listeria monocytogenes.

Acta Crystallogr D Biol Crystallogr 2014 Mar 15;70(Pt 3):615-26. Epub 2014 Feb 15.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.

Bacterial pathogens have evolved diverse types of efficient machinery to acquire haem, the most abundant source of iron in the human body, and degrade it for the utilization of iron. Gram-positive bacteria commonly encode IsdG-family proteins as haem-degrading monooxygenases. Listeria monocytogenes is predicted to possess an IsdG-type protein (Lmo2213), but the residues involved in haem monooxygenase activity are not well conserved and there is an extra N-terminal domain in Lmo2213. Therefore, its function and mechanism of action cannot be predicted. In this study, the crystal structure of Lmo2213 was determined at 1.75 Å resolution and its haem-binding and haem-degradation activities were confirmed. Structure-based mutational and functional assays of this protein, designated as an Isd-type L. monocytogenes haem-degrading enzyme (Isd-LmHde), identified that Glu71, Tyr87 and Trp129 play important roles in haem degradation and that the N-terminal domain is also critical for its haem-degrading activity. The haem-degradation product of Isd-LmHde is verified to be biliverdin, which is also known to be the degradation product of other bacterial haem oxygenases. This study, the first structural and functional report of the haem-degradation system in L. monocytogenes, sheds light on the concealed haem-utilization system in this life-threatening human pathogen.
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http://dx.doi.org/10.1107/S1399004713030794DOI Listing
March 2014

Size-controlled synthesis and characterization of CoPt nanoparticles using protein shells.

J Mater Chem B 2013 Mar 25;1(10):1453-1460. Epub 2013 Jan 25.

Sungkyunkwan Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746, Korea.

Nanostructured magnetic materials such as iron oxide and bimetallic nanoparticles can be potentially applied to a variety of fields, including electronics and nanomedicine. To develop these applications, it is important to control their particle size which affects their magnetic properties. In particular, it is a major challenge to synthesize small-sized nanoparticles with high reproducibility. In this study, we synthesized cobalt-platinum nanoparticles (CoPt NPs) in an ambient solution phase using PepA, a bacterial aminopeptidase, as a protein shell, and investigated the physicochemical and magnetic properties of NPs with and without encapsulating proteins. The size of CoPt NPs encapsulated by PepA was stringently controlled from 1.1 to 2.8 nm, and their magnetic property was related to the size. The CoPt NPs with the diameter of 1.1 nm showed a superparamagnetic behavior only at low temperatures, while 2.1 and 2.8 nm CoPt NPs were ferromagnetic below the blocking temperature. PepA had no deleterious effects on the coercivity of CoPt NPs, as evidenced by the marginal effect of PepA on the coercivity of CoPt NPs. This study demonstrated that the particle size and magnetic property of CoPt NPs can be controlled by using PepA as a protein shell. Encapsulation by PepA will aid the development of multifunctional magnetic materials, since the biocompatibility and modification capability of PepA can be synergistically combined with the advanced functionalities of CoPt NPs.
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http://dx.doi.org/10.1039/c2tb00290fDOI Listing
March 2013

Crystal structure of the LG3 domain of endorepellin, an angiogenesis inhibitor.

J Mol Biol 2011 Nov 4;414(2):231-42. Epub 2011 Oct 4.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

Endorepellin, the C-terminal region of perlecan, inhibits angiogenesis by disrupting actin cytoskeleton and focal adhesions. The C-terminal laminin-like globular domain (LG3) of endorepellin directs most of this antiangiogenic activity. To investigate the angiostatic mechanism and to identify structural determinants, we have solved crystal structures of the LG3 domain in both apo- and calcium-bound forms at resolutions of 1.5 Å and 2.8 Å, respectively. The conserved core has the jellyroll fold characteristic of LG domains. The calcium-induced structural changes seem very restricted, and the calcium binding site appears to be preformed, suggesting that the bound calcium ion, rather than structural rearrangements, contributes to antiangiogenesis. We have identified H4268 on the EF loop as a key residue for the biochemical function of LG3, since its mutation abolishes antiangiogenic activity, and mutant LG3 can no longer form a direct interaction with integrin. Taken together, we propose that these two distinct structural elements contribute to the angiostatic effect of endorepellin.
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http://dx.doi.org/10.1016/j.jmb.2011.09.048DOI Listing
November 2011

Structure-based development of a receptor activator of nuclear factor-kappaB ligand (RANKL) inhibitor peptide and molecular basis for osteopetrosis.

Proc Natl Acad Sci U S A 2010 Nov 8;107(47):20281-6. Epub 2010 Nov 8.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL, which belong to the tumor necrosis factor (TNF) receptor-ligand family, mediate osteoclastogenesis. The crystal structure of the RANKL ectodomain (eRANKL) in complex with the RANK ectodomain (eRANK) combined with biochemical assays of RANK mutants indicated that three RANK loops (Loop1, Loop2, and Loop3) bind to the interface of a trimeric eRANKL. Loop3 is particularly notable in that it is structurally distinctive from other TNF-family receptors and forms extensive contacts with RANKL. The disulfide bond (C125-C127) at the tip of Loop3 is important for determining the unique topology of Loop3, and docking E126 close to RANKL, which was supported by the inability of C127A or E126A mutants of RANK to bind to RANKL. Inhibitory activity of RANK mutants, which contain loops of osteoprotegerin (OPG), a soluble decoy receptor to RANKL, confirmed that OPG shares the similar binding mode with RANK and OPG. Loop3 plays a key role in RANKL binding. Peptide inhibitors designed to mimic Loop3 blocked the RANKL-induced differentiation of osteoclast precursors, suggesting that they could be developed as therapeutic agents for the treatment of osteoporosis and bone-related diseases. Furthermore, some of the RANK mutations associated with autosomal recessive osteopetrosis (ARO) resulted in reduced RANKL-binding activity and failure to induce osteoclastogenesis. These results, together with structural interpretation of eRANK-eRANKL interaction, provided molecular understanding for pathogenesis of ARO.
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http://dx.doi.org/10.1073/pnas.1011686107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996688PMC
November 2010

Structural basis for the negative regulation of bacterial stress response by RseB.

Protein Sci 2010 Jun;19(6):1258-63

Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

The sigmaE-dependent stress response in bacterial cells is initiated by the DegS- and RseP-regulated intramembrane proteolysis of a membrane-spanning antisigma factor, RseA. RseB binds to RseA and inhibits its sequential cleavage, thereby functioning as a negative modulator of this response. In the crystal structure of the periplasmic domain of RseA bound to RseB, the DegS cleavage site of RseA is unstructured, however, its P1 residue is buried in the hydrophobic pocket of RseB, which suggests that RseB binding blocks the access of DegS to the cleavage site.
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http://dx.doi.org/10.1002/pro.393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895250PMC
June 2010

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase.

Mol Cells 2010 Apr 15;29(4):397-405. Epub 2010 Mar 15.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746, Korea.

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg(2+)-bound forms at 2.9 A and 2.3 A resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg(2+) ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.
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http://dx.doi.org/10.1007/s10059-010-0047-6DOI Listing
April 2010

Z-DNA binding proteins as targets for structure-based virtual screening.

Curr Drug Targets 2010 Mar;11(3):335-44

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea.

Z-DNA, the alternative form of double-stranded DNA involved in a variety of nucleotide metabolism, is recognized and stabilized by specific Z-DNA binding proteins (ZBPs). Three ZBPs known in vertebrates -ADAR1, DAI and PKZ- modulate innate immunity, particularly, the IFN-induced immune response. The E3L protein of the vaccinia virus appears to compete with the host ZBP for Z-DNA binding, thereby suppressing the host immune system. ZBPs are, therefore, considered to be attractive therapeutic targets for infectious and immune diseases. Recent advances in computer-aided drug development combined with the high-resolution crystal and NMR structures of ZBPs have enabled us to discover novel candidates as ZBP inhibitors. In this study, we present an overview of Z-DNA and known ZBPs as drug targets, and summarize recent progress in the structure-based identification of ZBP inhibitors.
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http://dx.doi.org/10.2174/138945010790711905DOI Listing
March 2010

Structural and functional characterization of soluble endoglin receptor.

Biochem Biophys Res Commun 2009 Jun 5;383(4):386-91. Epub 2009 Mar 5.

Department of Molecular Cell Biology, Samsung Biomedical Research institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Suwon 440-746, Republic of Korea.

Endoglin, an accessory membrane receptor of transforming growth factor-beta (TGF-beta)1, modulates the cellular response to TGF-beta via its interaction with type I and II TGF-beta receptors. It has been considered a promising target for the development of therapeutics and cancer markers. We have established stable CHO cell lines that efficiently secrete soluble endoglin (s-endoglin) fused with human growth hormone. Two oligomeric forms were observed in a homogeneous preparation of s-endoglin, as a dimer and a tetramer. The dimeric s-endoglin enhanced TGF-beta responsiveness in U937 cells, thus proving its potential for therapeutic applications. Small angle X-ray scattering (SAXS) experiments revealed elongated conformations of both dimeric and tetrameric s-endoglins in solution, suggesting that s-endoglin might undergo conformational adaptations upon TGF-beta binding. The current results provide important references and material for high-resolution structural studies and for medical applications of s-endoglin.
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http://dx.doi.org/10.1016/j.bbrc.2009.02.162DOI Listing
June 2009

Crystallization and preliminary X-ray crystallographic studies of the Z-DNA-binding domain of a PKR-like kinase (PKZ) in complex with Z-DNA.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2009 Mar 26;65(Pt 3):267-70. Epub 2009 Feb 26.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.

PKZ, a PKR-like eIF2alpha kinase, consists of a Z-DNA-specific binding domain (Zalpha) and an eIF2alpha kinase domain. The kinase activity of PKZ is modulated by the binding of Zalpha to Z-DNA. The mechanisms underlying Z-DNA binding and the subsequent stimulation of PKZ raise intriguing questions. Interestingly, the Z-DNA-binding domain of PKZ from goldfish (Carassius auratus; caZalpha(PKZ)) shows limited sequence homology to other canonical Zalpha domains, suggesting that it may have a distinct Z-DNA-recognition mode. In this study, the Z-DNA-binding activity and stoichiometry of caZalpha(PKZ) were confirmed using circular dichroism (CD). In addition, preliminary X-ray studies of the caZalpha(PKZ)-Z-DNA complex are reported as the first step in the determination of its three-dimensional structure. Bacterially expressed recombinant caZalpha(PKZ) was purified and crystallized with Z-DNA at 295 K using the microbatch method. X-ray diffraction data were collected to 1.7 A resolution with an R(merge) of 7.4%. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 55.54, b = 49.93, c = 29.44 A, beta = 96.5 degrees . Structural analysis of caZalpha(PKZ)-Z-DNA will reveal the binding mode of caZalpha(PKZ) to Z-DNA and its relevance to other Z-DNA-binding proteins.
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http://dx.doi.org/10.1107/S1744309109002504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650443PMC
March 2009

The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA.

Proc Natl Acad Sci U S A 2008 Dec 18;105(52):20671-6. Epub 2008 Dec 18.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZbeta(DAI)) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZbeta(DAI)/Z-DNA complex reveals that hZbeta(DAI) has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first beta-strand rather than residues in the beta-loop contributes to DNA binding, and part of the (alpha3) recognition helix adopts a 3(10) helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.
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http://dx.doi.org/10.1073/pnas.0810463106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634953PMC
December 2008

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

Nucleic Acids Res 2009 Feb 11;37(2):629-37. Epub 2008 Dec 11.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.
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http://dx.doi.org/10.1093/nar/gkn976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632926PMC
February 2009

Crystallization and preliminary X-ray studies of TON_0559, a putative member of the haloacid dehalogenase (HAD) superfamily from Thermococcus onnurineus NA1.

Protein Pept Lett 2008 ;15(2):235-7

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

To elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism of the enzymes belonging to the haloacid dehalogenase (HAD) superfamily, TON_0559, a putative HAD subfamily protein from a hyperthermophilic archaeon Thermococcus onnurineus NA1, was expressed, purified and crystallized. X-ray diffraction data were collected to 2.0 A resolution. The space group is C2, with unit cell parameters a = 121.2 A, b = 62.9 A, c = 37.5 A and beta= 106.5 degrees .
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http://dx.doi.org/10.2174/092986608783489625DOI Listing
May 2008

Biochemical characterization and preliminary X-ray crystallographic study of the domains of human ZBP1 bound to left-handed Z-DNA.

Biochim Biophys Acta 2006 Feb 18;1764(2):320-3. Epub 2006 Jan 18.

Department of Molecular Cell Biology, Center for Molecular Medicine, SBRI, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

ZBP1 is involved in host responses against cellular stresses, including tumorigenesis and viral infection. Structurally, it harbors two copies of the Zalpha domain containing the Zalpha motif, at its N terminus. Here, we attempted to characterize the Z-DNA binding activities of two Zalpha domains in the human ZBP1, hZalpha(ZBP1) and hZbeta(ZBP1), using circular dichroism (CD). Our results indicated that both hZalpha(ZBP1) and hZbeta(ZBP1) are viable Z-DNA binders, and their binding activities are comparable to those of previously-established Zalpha domains. Additionally, we crystallized hZbeta(ZBP1) in a complex with Z-DNA, d(TCGCGCG)2. The crystal diffracted to 1.45 angstroms, and belongs to the P2(1)2(1)2(1) space group, with the unit-cell parameters: a = 29.53 angstroms, b = 58.25 angstroms, and c = 88.61 angstroms. The delineation of this structure will provide insight into the manner in which diverse Zalpha motifs recognize Z-DNA.
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http://dx.doi.org/10.1016/j.bbapap.2005.12.012DOI Listing
February 2006

Structure of inorganic pyrophosphatase from Helicobacter pylori.

Acta Crystallogr D Biol Crystallogr 2005 Nov 19;61(Pt 11):1459-64. Epub 2005 Oct 19.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea.

Inorganic pyrophosphatase (PPase) is a ubiquitous cytosolic enzyme which catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to orthophosphate (Pi). The crystal structure of inorganic pyrophosphatase from Helicobacter pylori (H-PPase) has been solved by MAD and refined to an R factor of 20.6% at 2.6 A resolution. The crystallographic asymmetric unit contains a homohexameric H-PPase arranged as a dimer of trimers. While most of the structural elements of PPases are highly conserved in H-PPase, some unique structural features are localized in the flexible loops near the active site, suggesting that the structural flexibility of these loops is required for the catalytic efficiency of PPase.
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http://dx.doi.org/10.1107/S0907444905025667DOI Listing
November 2005

Efficient selection of stable chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon beta SAR element.

Biotechnol Prog 2005 May-Jun;21(3):933-7

Graduate School of Biotechnology, Kyung Hee University, Yongin City, Kyungki-Do 449-701, Korea.

We describe the development of an efficient expression system suitable for the stable expression of recombinant genes in Chinese hamster ovary (CHO) cells using the human interferon beta SAR element. The insertion of two copies of the human interferon beta SAR element at the 5' and 3' flanking regions of the beta-galactosidase reporter gene increased the frequency of beta-galactosidase positive colonies by up to 75% and enhanced beta-galactosidase expression by 15- to 20-fold after G418 selection or 30- to 40-fold at the initial stage of the MTX selection procedure. Deletion analysis showed that the whole DNA regions of the human interferon beta SAR element are required for beta-galactosidase expression enhancement. The developed expression system was also highly effective at enhancing the stable expression of two therapeutically important proteins, namely, erythropoietin (EPO) and hepatocyte growth factor (HGF). We isolated stable colonies with expression levels of 47 microg/10(6) cells/day for EPO and 13 microg/10(6) cells/day for HGF, suggesting that the developed expression system based on the human beta SAR element is suitable for expressing high levels of recombinant proteins in CHO cells.
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http://dx.doi.org/10.1021/bp049598vDOI Listing
September 2005

Crystal structure of the protease domain of a heat-shock protein HtrA from Thermotoga maritima.

J Biol Chem 2003 Feb 27;278(8):6543-51. Epub 2002 Nov 27.

Department of Molecular Cell Biology, Center for Molecular Medicine, SBRI, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

HtrA (high temperature requirement A), a periplasmic heat-shock protein, functions as a molecular chaperone at low temperatures, and its proteolytic activity is turned on at elevated temperatures. To investigate the mechanism of functional switch to protease, we determined the crystal structure of the NH(2)-terminal protease domain (PD) of HtrA from Thermotoga maritima, which was shown to retain both proteolytic and chaperone-like activities. Three subunits of HtrA PD compose a trimer, and multimerization architecture is similar to that found in the crystal structures of intact HtrA hexamer from Escherichia coli and human HtrA2 trimer. HtrA PD shares the same fold with chymotrypsin-like serine proteases, but it contains an additional lid that blocks access the of substrates to the active site. A corresponding lid found in E. coli HtrA is a long loop that also blocks the active site of another subunit. These results suggest that the activation of the proteolytic function of HtrA at elevated temperatures might occur by a conformational change, which includes the opening of the helical lid to expose the active site and subsequent rearrangement of a catalytic triad and an oxyanion hole.
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http://dx.doi.org/10.1074/jbc.M208148200DOI Listing
February 2003

Hyperhomocysteinemia due to methionine synthase deficiency, cblG: structure of the MTR gene, genotype diversity, and recognition of a common mutation, P1173L.

Am J Hum Genet 2002 Jul 30;71(1):143-53. Epub 2002 May 30.

Division of Medical Genetics, Department of Medicine, McGill University Health Centre, Montreal, Quebec, H3A 1A1, Canada.

Mutations in the MTR gene, which encodes methionine synthase on human chromosome 1p43, result in the methylcobalamin deficiency G (cblG) disorder, which is characterized by homocystinuria, hyperhomocysteinemia, and hypomethioninemia. To investigate the molecular basis of the disorder, we have characterized the structure of the MTR gene, thereby identifying exon-intron boundaries. This enabled amplification of each of the 33 exons of the gene, from genomic DNA from a panel of 21 patients with cblG. Thirteen novel mutations were identified. These included five deletions (c.12-13delGC, c.381delA, c.2101delT, c.2669-2670delTG, and c.2796-2800delAAGTC) and two nonsense mutations (R585X and E1204X) that would result in synthesis of truncated proteins that lack portions critical for enzyme function. One mutation was identified that resulted in conversion of A to C of the invariant A of the 3' splice site of intron 9. Five missense mutations (A410P, S437Y, S450H, H595P, and I804T) were identified. The latter mutations, as well as the splice-site mutation, were not detected in a panel of 50 anonymous DNA samples, suggesting that these sequence changes are not polymorphisms present in the general population. In addition, a previously described missense mutation, P1173L, was detected in 16 patients in an expanded panel of 24 patients with cblG. Analysis of haplotypes constructed using sequence polymorphisms identified within the MTR gene demonstrated that this mutation, a C-->T transition in a CpG island, has occurred on at least two separate genetic backgrounds.
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http://dx.doi.org/10.1086/341354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC384971PMC
July 2002
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