Hyoun Sook Kim

Crystal structure of human nucleophosmin-core reveals plasticity of the pentamer-pentamer interface

Crystal structure of human nucleophosmin-core reveals plasticity of the pentamer–pentamer interface Hyung Ho Lee, Hyoun Sook Kim, Ji Yong Kang, Byung Il Lee, Jun Yong Ha, Hye Jin Yoon, Seung Oe Lim, Guhung Jung, and Se Won Suh* 1 Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea 2 Department of Biological Science, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea

Structural basis for the inhibition of Mycobacterium tuberculosis L,D-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains

The crystal structure of M. tuberculosis l,d-transpeptidase (LdtMt2; Rv2518c) has been determined in both ligand-free and meropenem-bound forms. The detailed view of the interactions between meropenem and LdtMt2 will be useful in structure-guided discovery of new antituberculosis drugs.

Crystal Structure of 2-Nitropropane Dioxygenase Complexed with FMN and Substrate: identification of the catalytic base

Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.

Helicobacter pylori proinflammatory protein up-regulates NF-κB as a cell-translocating Ser/Thr kinase

There has been considerable interest in virulence genes in the plasticity region of Helicobacter pylori, but little is known about many of these genes. JHP940, one of the virulence factors encoded by the plasticity region of H. pylori strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-κB in cultured macrophages. Here we have characterized the structure and function of JHP940 to provide the framework for better understanding its role in inflammation by H. pylori. Our work demonstrates that JHP940 is the first example of a eukaryotic-type Ser/Thr kinase from H. pylori. We show that JHP940 is catalytically active as a protein kinase and translocates into cultured human cells. Furthermore, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP940 in inflammation and subsequent pathogenesis caused by H. pylori. We propose to rename the jhp940 gene as ctkA (cell translocating kinase A).

Reliability and validity of data obtained from alcohol, cannabis, and gambling populations on Amazon’s Mechanical Turk.

Researchers recently have begun using Mechanical Turk (MTurk), an online crowdsourcing platform, to recruit addiction populations. However, whether the data obtained from substance users and gamblers on MTurk are reliable and valid is unknown. Herein, we assessed the internal and retest reliability of and concurrent and convergent validity of data obtained from addiction populations on MTurk. Current drinkers (N = 208), cannabis users (N = 200), and gamblers (N = 200) residing in the United States completed measures of alcohol, cannabis, and gambling severity, psychological constructs (e.g., impulsivity) related to addictions, overt and subtle measures of valid responding, and motivations for completing MTurk studies. Of the original sample, 88–92% of participants who provided informed consent for recontact completed a reassessment 1 week later. The internal consistency of the addiction severity measures ranged from &agr; = .75 to .93. The stability over 1 week ranged from &kgr; = .57 to .70 for categorical classification, and intraclass correlation coefficient (ICC) = .71 to .86 for continuous measures. The addiction measures were significantly correlated with each other and with other constructs related to addictive behaviors. Overall, 80–85% of participants provided valid responses. They reported attending and answering questions honestly, with financial motives being the most frequently endorsed motivation. After invalid responses were excluded, results remained the same for alcohol and gambling, but significant differences emerged for the cannabis sample. The results suggest that the self-report data obtained from alcohol and gambling populations are of high quality, however, caution is warranted with cannabis populations. MTurk shows promise as a recruitment tool for some addictive behaviors.

Crystal structure of Tpa1 from Saccharomyces cerevisiae, a component of the messenger ribonucleoprotein complex

Tpa1 (for termination and polyadenylation) from Saccharomyces cerevisiae is a component of a messenger ribonucleoprotein (mRNP) complex at the 3′ untranslated region of mRNAs. It comprises an N-terminal Fe(II)- and 2-oxoglutarate (2OG) dependent dioxygenase domain and a C-terminal domain. The N-terminal dioxygenase domain of a homologous Ofd1 protein from Schizosaccharomyces pombe was proposed to serve as an oxygen sensor that regulates the activity of the C-terminal degradation domain. Members of the Tpa1 family are also present in higher eukaryotes including humans. Here we report the crystal structure of S. cerevisiae Tpa1 as a representative member of the Tpa1 family. Structures have been determined as a binary complex with Fe(III) and as a ternary complex with Fe(III) and 2OG. The structures reveal that both domains of Tpa1 have the double-stranded β-helix fold and are similar to prolyl 4-hydroxylases. However, the binding of Fe(III) and 2OG is observed in the N-terminal domain only. We also show that Tpa1 binds to poly(rA), suggesting its direct interaction with mRNA in the mRNP complex. The structural and functional data reported in this study support a role of the Tpa1 family as a hydroxylase in the mRNP complex and as an oxygen sensor.

Depletion of nucleophosmin via transglutaminase 2 cross-linking increases drug resistance in cancer cells

It has been suggested that nucleophosmin has an anti-apoptotic function via Bax binding. We found that nucleophosmin is a substrate of transglutaminase 2 (TGase 2) in cancer cells. Increased expression of TGase 2 expression is highly associated with drug resistance, and polymerization of nucleophosmin by TGase 2 also can be correlated with the drug resistance of cancer cells. In the present study, an accumulation of nucleophosmin in cytosol was detected when doxorubicin was treated to cancer cells, and it was found, moreover, that an increase of cytosolic nucleophosmin can result in drug-induced apoptosis. Nucleophosmin was polymerized by TGase 2, and the polymerization was inhibited with the TGase 2 inhibitor, cystamine, in vitro. The nucleophosmin level in the cytosolic cell fraction was reduced when TGase 2 was expressed, and the reduced nucleophosmin level was rescued by cystamine treatment. Moreover, nucleophosmin cross-linked by TGase 2 was eradicated in MCF7 cells via the ubiquitin-proteasomal pathway. In parallel with this nucleophosmin-level restoration, the pro-apoptotic Bax protein level was increased. Therefore, depletion of cytosolic nucleophosmin by TGase 2 can decrease Bax protein stability and lead to anti-apoptosis. Drug-resistant cancer cells became sensitive to doxorubicin treatment when nucleophosmin was expressed in cytosol. Taking these results together, it can be concluded that TGase 2 inhibits accumulation of cytosolic nucleophosmin through polymerization, which results in drug resistance in cancer cells.

Crystal structures of YwqE from Bacillus subtilis and CpsB from Streptococcus pneumoniae, unique metal-dependent tyrosine phosphatases.

Unique metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are present in Gram-positive bacteria. They are distinct from the Cys-based, low-molecular-weight phosphotyrosine protein phosphatases (LMPTPs). Two representative members of the PHP family tyrosine phosphatases are YwqE from Bacillus subtilis and CpsB from Streptococcus pneumoniae. YwqE is involved in polysaccharide biosynthesis, bacterial DNA metabolism, and DNA damage response in B. subtilis. CpsB regulates capsular polysaccharide biosynthesis via tyrosine dephosphorylation of CpsD, its cognate tyrosine kinase, in S. pneumoniae. To gain insights into the active site and possible conformational changes of the metal-dependent tyrosine phosphatases from Gram-positive bacteria, we have determined the crystal structures of B. subtilis YwqE (in both the apo form and the phosphate-bound form) and S. pneumoniae CpsB (in the sulfate-bound form). Comparisons of the three structures reveal conformational plasticity of two active site loops. Furthermore, in both structures of the phosphate-bound YwqE and the sulfate-bound CpsB, the phosphate (or sulfate) ion is bound to a cluster of three metal ions in the active site, thus providing insight into the pre-catalytic state.

Structural and functional characterization of Helicobacter pylori DsbG

DsbG and DsbG bind by X‐ray crystallography (View interaction).

Crystal structure of a metal ion-bound IS200 transposase.

IS200 transposases, present in many bacteria and Archaea, appear to be distinct from other groups of transposases. To provide a structural basis for understanding the action of IS200 transposases, we have determined the crystal structure of the SSO1474 protein from Sulfolobus solfataricus, a member of the IS200 family, in both Mn2+-bound and Mn2+-free forms. Its monomer fold is distinct from other classes of structurally characterized transposases. Two monomers form a tight dimer by exchanging the C-terminal α-helix and by merging the two central β-sheets into a large β-sheet. Glu55, His62, and four water molecules provide the direct coordination sphere of the catalytically essential metal ion in the Mn2+-bound structure. His16, Asp59, and His60 also play important roles in maintaining the metal binding site. The catalytic site is formed at the interface between monomers. The candidate nucleophile in the transposition mechanism, strictly conserved Tyr121 coming from the other monomer, is turned away from the active site, suggesting that a conformational change is likely to occur during the catalytic cycle.