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Kim, Su Jin,Cho, Jinhong,Song, Eun Joo,Kim, Soo Jin,Kim, Ho Min,Lee, Kyung Eun,Suh, Se Won,Kim, Eunice EunKyeong American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.18
<P>Valosin-containing protein (VCP), also known as p97, is an AAA<SUP>+</SUP> ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXL<SUB>OTU1</SUB>) complexed to the N-terminal domain of VCP (N<SUB>VCP</SUB>) at 1.8-Å resolution reveals that UBXL<SUB>OTU1</SUB> adopts a ubiquitin-like fold and binds at the interface of two subdomains of N<SUB>VCP</SUB> using the <SUP>39</SUP>GYPP<SUP>42</SUP> loop of UBXL<SUB>OTU1</SUB> with the two prolines in <I>cis</I>- and <I>trans</I>-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a <I>K<SUB>D</SUB></I> of 0.71 μ<SMALL>m</SMALL>. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.</P>
Kim, Seung Min,Shin, Sang Chul,Kim, Eunice EunKyeong,Kim, Sang-Heon,Park, Kwideok,Oh, Seung Ja,Jang, Mihue American Chemical Society 2018 ACS NANO Vol.12 No.8
<P>Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for <I>in vivo</I> applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing <I>in vivo</I>. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) <I>via in vitro</I> and <I>in vivo</I> editing of the <I>KRAS</I> gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening.</P> [FIG OMISSION]</BR>
Unique binding mode of Evogliptin with human dipeptidyl peptidase IV
Lee, Hyung Ki,Kim, Mi-Kyung,Kim, Ha Dong,Kim, Heung Jae,Kim, Ji Won,Lee, Jie-Oh,Kim, Chan-Wha,Kim, Eunice EunKyeong Elsevier 2017 Biochemical and biophysical research communication Vol. No.
<P><B>Abstract</B></P> <P>Evogliptin ((<I>R</I>)-4-((<I>R</I>)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(<I>tert-</I>butoxymethyl) piperazine-2-one)) is a highly potent selective inhibitor of dipeptidyl peptidase IV (DPP4) that was approved for the treatment of type 2 diabetes in South Korea. In this study, we report the crystal structures of Evogliptin, DA-12166, and DA-12228 (<I>S,R</I> diastereomer of Evogliptin) complexed to human DPP4. Analysis of both the structures and inhibitory activities suggests that the binding of the trifluorophenyl moiety in the S<SUB>1</SUB> pocket and the piperazine-2-one moiety have hydrophobic interactions with Phe357 in the S<SUB>2</SUB> extensive subsite, and that the multiple hydrogen bonds made by the (<I>R</I>)-β-amine group in the S<SUB>2</SUB> pocket and the contacts made by the (<I>R</I>)-<I>tert</I>-butyl group with Arg125 contribute to the high potency observed for Evogliptin.</P>
Lee, Choongman,Park, Joon Kyu,Youn, Yeoan,Kim, Joo Hyoung,Lee, Kyo-Seok,Kim, Nak-kyoon,Kim, Eunji,Kim, Eunice Eunkyeong,Yoo, Kyung-Hwa American Chemical Society 2017 ANALYTICAL CHEMISTRY - Vol.89 No.4
<P>We employed modified glass nanocapillaries to investigate interactions between the RNA-binding protein, known as cell carcinoma antigen recognized by T cells-3 (SART3), and the noncoding spliceosome component, U6 small nuclear RNA (snRNA), at the single-molecule level. We functionalized the nanocapillaries with U6 snRNA fragments, which were hybridized to DNA molecules and then covalently attached to the nanocapillary surface. When transported through the modified nanocapillaries, two different SART3-derived constructs, HAT-RRM1-RRM2 and RRM1-RRM2, exhibited resistive ionic current pulses with different dwell times, which represented their different binding affinities to tethered U6 snRNAs. The dissociation constants (K-D), estimated from the bias voltage dependence of translocation events, were approximately 1.9 mu M and 201 mu M for HAT-RRM1-RRM2 and RRM1-RRM2, respectively. These values were comparable to corresponding values obtained with isothermal titration calorimetry, demonstrating that the modified glass nanocapillaries are applicable to analyses of protein ligand interactions at the single-molecule level.</P>
Kim, Kook-Han,Lee, Won-Kyu,Choi, Kyung-Jae,Kim, Eunice EunKyeong The Korean Society for Applied Biological Chemistr 2014 Applied Biological Chemistry (Appl Biol Chem) Vol.57 No.5
Bacterial resistance to many existing antibiotics is a growing health concern worldwide. There is an urgent need to identify new antibiotics with unexploited modes of action. Peptide deformylase (PDF) is an essential enzyme involved in N-terminal protein processing in eubacteria but not in higher organisms. Therefore, PDF is considered an attractive target for the development of novel antibiotics. Here, we report the structures of the PDFs from Enterococcus faecalis (EfPDF) and Streptococcus pyogenes (SpyPDF) complexed with actinonin at 1.4 and $2.1{\AA}$ resolutions, respectively. Actinonin, a naturally occurring, highly potent inhibitor, is bound tightly at the active site. The conformation of actinonin in the EfPDF and SpyPDF complexes was similar to those of all others. The detailed information from this study will facilitate the development of novel antibacterial molecules.
Kim, Young Kwan,Mizutani, Kenji,Rhee, Kyung-Hee,Nam, Ki-Hyun,Lee, Won Ho,Lee, Eun Hye,Kim, Eunice Eunkyeong,Park, Sam-Yong,Hwang, Kwang Yeon American Society for Microbiology 2007 Journal of Bacteriology Vol.189 No.22
<B>ABSTRACT</B><P>In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA intron-splicing endonuclease from <I>Thermoplasma acidophilum</I> DSM 1728 (EndA<I>Ta</I>) have been solved to 2.5-Å and 2.7-Å resolution by molecular replacement, using the 2.7-Å resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndA<I>Ta</I> is a homodimer and that it has overall folding similar to that of other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.</P>
Lee, Jihye,Kim, Dongwook,Park, Jaehyeon,Kim, Eunice EunKyeong,Lah, Myoung Soo,Kim, Aeri The American Chemical Society 2018 CRYSTAL GROWTH AND DESIGN Vol.18 No.1
<P>Three different hydrates of LB30870, a new direct thrombin inhibitor, were identified during the screening of solid form, and their interconversion relationship and relative thermodynamic stabilities were investigated. Form I (hexahydrate) changes to Form II (dihydrate) or Form III (tetrahydrate) by dehydration, while Form II becomes Form I by hydration, and both Form II and Form III change to Form I in the aqueous slurry. Single crystals obtained from two different crystallization conditions, wet or air-dried, were found to be isostructural with a difference in the solvent channels, and based on the simulated and experimental powder X-ray diffraction patterns, the air-dried crystals are assigned as Form I and Form III, respectively. In all crystal structures, LB30870 is in a folded conformation forced by the presence of strong hydrogen bonds by two structural water molecules. The solvent channel formed can hold up to six additional hydration water sites per each LB30870, and the one-dimensional solvent channel facilitates the interconversion among the hydrates and rapid conversion to Form I in water. Although all hydrate forms would not differ in oral bioavailability as Form I predominates in the aqueous phase, considering the stable water content at 40–75% relative humidity Form III would be the most suitable for further development.</P><P>Three different hydrates of LB30870, a new direct thrombin inhibitor, were identified, and their interconversion relationship and relative thermodynamic stabilities were investigated. LB30870 is in a folded conformation forced by the presence of strong hydrogen bonds by two structural water molecules, and the one-dimensional solvent channel facilitates the interconversion among the hydrates and rapid conversion to Form I in water.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/cgdefu/2018/cgdefu.2018.18.issue-1/acs.cgd.7b00927/production/images/medium/cg-2017-00927h_0016.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/cg7b00927'>ACS Electronic Supporting Info</A></P>
Phosphorylation of USP15 and USP4 Regulates Localization and Spliceosomal Deubiquitination
Das, Tanuza,Kim, Eunice EunKyeong,Song, Eun Joo Elsevier 2019 Journal of molecular biology Vol.431 No.19
<P><B>Abstract</B></P> <P>Deubiquitinating enzymes have key roles in diverse cellular processes whose enzymatic activities are regulated by different mechanisms including post-translational modification. Here, we show that USP15 is phosphorylated, and its localization and activity are dependent on the phosphorylation status. Nuclear-cytoplasmic fractionation and mass spectrometric analysis revealed that Thr149 and Thr219 of human USP15, which is conserved among different species, are phosphorylated in the cytoplasm. The phosphorylation status of USP15 at these two positions alters the interaction with its partner protein SART3, consequently leading to its nuclear localization and deubiquitinating activity toward the substrate PRP31. Treatment of cells with purvalanol A, a cyclin-dependent kinase inhibitor, results in nuclear translocation of USP15. USP4, another deubiquitinating enzyme with a high sequence homology and domain structure as USP15, also showed purvalanol A-dependent changes in activity and localization. Collectively, our data suggest that modifications of USP15 and USP4 by phosphorylation are important for the regulation of their localization required for cellular function in the spliceosome.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Two phosphorylation sites of USP15 are identified. </LI> <LI> Phosphorylation regulates the localization of USP15. </LI> <LI> Phosphorylation status of USP15 alters the interaction with SART3. </LI> <LI> Deubiquitination of PRP31 is regulated by phosphorylation of USP15. </LI> <LI> CDK inhibitor induces the nuclear localization of USP15. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>