Byung Il Lee

Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition

tRNA(m1G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S‐adenosyl‐L‐ methionine (AdoMet) to G37 within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3′ of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S‐adenosyl‐L‐homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G36G37 in the active site of TrmD and the catalytic mechanism. The N‐terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C‐terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD2–tRNA2 complex, which provides insights into recognition of the general tRNA structure by TrmD.

Ring-shaped architecture of RecR: implications for its role in homologous recombinational DNA repair

RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes . The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two‐domain structure: the N‐terminal domain with a helix–hairpin–helix (HhH) motif and the C‐terminal domain with a Cys4 zinc‐finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring‐shaped tetramer of 222 symmetry with a central hole of 30−35 Å diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding.

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

Quantitative measurement of anti-aquaporin-4 antibodies by enzyme-linked immunosorbent assay using purified recombinant human aquaporin-4.

Background: Antibodies to aquaporin-4 (AQP4-Ab), known as NMO-IgG, are a sensitive and specific marker for neuromyelitis optica (NMO). Methods: To develop an enzyme-linked immunosorbent assay (ELISA) for AQP4-Ab, we expressed M23 isoform of human AQP4 in a baculovirus system, and used it as an antigen. We measured AQP4-Ab in the sera of 300 individuals: 64 with definite NMO, 31 with high-risk NMO, 105 with multiple sclerosis (MS), 57 with other neurological diseases (ONDs), and 43 healthy controls. We also performed longitudinal measurements of AQP4–Ab in 787 samples collected from 51 patients with definite or high-risk NMO. Results: AQP4-Abs were positive in 72% with definite NMO, 55% with high-risk NMO, and 4% with MS, but none of the OND patients and the healthy individuals. The longitudinal measurement showed AQP4-Ab levels correlating with disease activity. Out of 38 initially seropositive patients, 21 became seronegative under effective immunosuppressive therapy. During most relapses, the serum AQP4-Ab levels were either high or rising compared with the previous value, although rising AQP4-Ab levels did not always lead to acute exacerbation. Two of the 13 initially seronegative patients converted to seropositive following acute exacerbations. Conclusions: We established an AQP4-Ab ELISA, which could be a potential monitoring tool of disease activity.

Crystal structure of human transglutaminase 2 in complex with adenosine triphosphate.

Transglutaminase 2 (TG2) is a calcium-dependent multifunctional protein associated with various human diseases. We determined the crystal structure of human TG2 in complex with adenosine triphosphate (ATP). The ATP molecule binds to the previously identified guanosine diphosphate (GDP) binding pocket but has different hydrogen bonds and ion interaction with protein. The four residues Arg476, Arg478, Val479 and Tyr583, all of which are involved in both ATP and GDP binding by hydrogen bonds, might play important roles in the stabilization of TG2 by ATP or GDP. However, Ser482 and Arg580, which are involved in GDP binding, do not form hydrogen bond with ATP. Additionally, we newly discovered an intramolecular disulfide bond between Cys230 and Cys370, which formation might regulate the enzymatic activity of TG2.

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).

Crystal structure of UDP-N-acetylglucosamine acyltransferase from Helicobacter pylori

Introduction. Lipid A is the hydrophobic anchor of lipopolysaccharide in Gram-negative bacteria and is required for growth of most Gram-negative bacteria. It is also necessary for maintaining the integrity of the outer membrane as a barrier to toxic chemicals. Therefore, the study of the enzymes involved in lipid A biosynthesis would be useful for the development of new antibacterial drugs against Gram-negative bacteria. UDP-N-acetylglucosamine acyltransferase (LpxA) is the first enzyme of the lipid A biosynthetic pathway. It catalyzes the transfer of an R-3-hydroxyacyl chain from R-3-hydroxy-acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDPGlcNAc) at the glucosamine 3-OH position. Among nine enzymes of lipid A biosynthetic pathway, information on the 3D structure is available on LpxA from Escherichia coli only. It is a trimer composed of three identical subunits of 262 residues and contains a left-handed parallel -helix motif. However, the crystal structure of E. coli LpxA determined at 2.6-Å resolution did not contain any bound ligand and provided little information on the active site. The interaction site of LpxA involved in binding ACP is also unknown. Therefore, further structural data on LpxA will be valuable for a better understanding of the active site and structure-based inhibitor design. Here, we present the crystal structure of LpxA from Heliobacter pylori refined using 2.1-Å data. The sequence identity between H. pylori LpxA (270 residues, 29,855 Da) and that from E. coli is 39.3% over the entire polypeptide chain. Thanks to higher resolution, we could assign solvent molecules as well as bound ions. Further, an extra electron density is present in the putative active site and we tentatively interpret this unknown ligand as a detergent molecule, which seems to mimic the acyl chain of the substrate or the product. On the basis of this observation, together with the location of strictly conserved residues and the highly positively charged surface of the C-terminal helical domain, we propose a model for the complex between LpxA and ACP.

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 structure of the type II 3-dehydroquinase from Helicobacter pylori

Introduction. Helicobacter pylori is a spiral-shaped, Gramnegative bacterium that lives in the stomach and duodenum. H. pylori infection is associated with peptic ulcer disease, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. The enzyme 3-dehydroquinate dehydratase or 3-dehydroquinase (DHQase; EC 4.2.1.10), which catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, is an attractive target for developing antibacterial compounds specific for H. pylori. DHQases fall into two groups: type I and type II. They have different biochemical and biophysical properties, and no sequence similarity exists between them. Type I enzymes are generally found in the biosynthetic shikimate pathway and use a Schiff base intermediate formed at the conserved lysine residue, and catalyze the elimination of water with syn stereochemistry. They have subunit molecular masses of 25 kDa, form dimers in the case of monofuntional enzymes such as Escherichia coli type I DHQase, and are thermally labile. Type II enzymes serve either the biosynthetic shikimate pathway or catabolic quinate pathway, or both. They have smaller subunit molecular masses than type I enzymes (16–18 kDa), oligomerize into dodecamers of 200 kDa, and are heat stable. H. pylori has only the type II enzyme (167 residues, 18,483 Da), in contrast with gut organisms such as E. coli, which have type I enzymes only. Crystal structures of type II DHQases from Mycobacterium tuberculosis and Streptomyces coelicolor, as well as type I DHQase from Salmonella typhi, have been reported. The type II DHQases from M. tuberculosis (147 residues) and S. coelicolor (157 residues) show 34.7% and 37.7% sequence identity, respectively, to that from H. pylori over the entire polypeptide chain. There are subtle differences among the type II DHQases, as indicated by the discrimination between different type II enzymes by rationally designed inhibitors and different interactions of type II DHQases from M. tuberculosis and S. coelicolor with phosphate and sulfate. Therefore, structural information on DHQase from H. pylori will be valuable for structure-based design of selective inhibitors against H. pylori. Here, we present its crystal structure, which reveals an electron density for a ligand bound in the active site.

Expression, purification and biochemical characterization of the N-terminal regions of human TIG3 and HRASLS3 proteins

Tarzarotene-induced gene 3 (TIG3) and HRAS-like suppressor (HRASLS3) are members of the HREV107 family of class II tumor suppressors, which are down-regulated in various cancer cells. TIG3 and HRASLS3 also exhibit phospholipase activities. Both proteins share a common domain architecture with hydrophilic N-terminal and hydrophobic C-terminal regions. The hydrophobic C-terminal region is important for tumor suppression. However, the function of the hydrophilic N-terminal region remains elusive. To facilitate biochemical characterizations of TIG3 and HRASLS3, we expressed and purified the N-terminal regions of TIG3 and HRASLS3, designated TIG3 (1-134) and HRASLS3 (1-133), in a bacterial system. We found that the N-terminal regions of TIG3 and HRASLS3 have calcium-independent phospholipase A(2) activities. Limited proteolysis revealed that TIG3 (1-132) is a structural domain in the N-terminal region of TIG3. Our data suggest that the hydrophobic C-terminal regions might be crucial for cellular localization, while the hydrophilic N-terminal regions are sufficient for the enzymatic activity of both TIG3 and HRASLS3.