Browsing by Author "Wang, Wen-Ching"

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Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing--1, 3-glucanase
(2011-08-01)
Shrestha, Keshab Lal
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Liu, Sheng-Wen
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Huang, Chin-Ping
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Wu, Hsin-Mao
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Wang, Wen-Ching
Laminaripentaose-producing -1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of -1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to 90 homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residuesAsp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.
Core Site-Moiety Maps Reveal Inhibitors and Binding Mechanisms of Orthologous Proteins by Screening Compound Libraries
(2012-02-29)
Hsu, Kai-Cheng
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Cheng, Wen-Chi
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Chen, Yen-Fu
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Wang, Hung-Jung
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Li, Ling-Ting
Members of protein families often share conserved structural subsites for interaction with chemically similar moieties despite low sequence identity. We propose a core site-moiety map of multiple proteins (called CoreSiMMap) to discover inhibitors and mechanisms by profiling subsite-moiety interactions of immense screening compounds. The consensus anchor, the subsite-moiety interactions with statistical significance, of a CoreSiMMap can be regarded as a "hot spot" that represents the conserved binding environments involved in biological functions. Here, we derive the CoreSiMMap with six consensus anchors and identify six inhibitors (IC50 < 8.0 mu M) of shikimate kinases (SKs) of Mycobacterium tuberculosis and Helicobacter pylori from the NCI database (236,962 compounds). Studies of site-directed mutagenesis and analogues reveal that these conserved interacting residues and moieties contribute to pocket-moiety interaction spots and biological functions. These results reveal that our multi-target screening strategy and the CoreSiMMap can increase the accuracy of screening in the identification of novel inhibitors and subsite-moiety environments for elucidating the binding mechanisms of targets.
Crystal structure of Helicobacter pylori formamidase AmiF reveals a cysteine-glutamate-lysine catalytic triad
(2007-04-20)
Hung, Chiu-Lien
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Liu, Jia-Hsin
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Chiu, Wei-Chun
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Huang, Shao-Wei
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Hwang, Jenn-Kang
Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75 angstrom resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an alpha-beta-beta-alpha four-layer architecture characteristic of the nitrilase superfamily. One exterior alpha layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight alpha-beta-beta-alpha-alpha-beta-beta-alpha dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 angstrom, respectively. These structures reveal a small formamide-binding pocket that includes Cys(166), Glu(60), and Lys(133) catalytic residues, in which Cys166 acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl D-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.
Crystal Structure of Shikimate Dehydrogenase from Helicobacter Pylori.
(2009-01-01)
Cheng, Wen-Chi
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Lin, Shuang-Chih
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Wang, Hung-Jung
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Yang, Jinn-Moon
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Lin, Jong-Yih
Electrically surface-driven switchable wettability of liquid crystal/polymer composite film
(2010-03-29)
Chiu, Ya-Ping
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Shen, Cheng-Yu
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Wang, Wen-Ching
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Chu, Ting-Yu
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Lin, Yi-Hsin
This study demonstrates the electrical control of the surface wettability of liquid crystal and polymer composite film. The application of external voltages significantly affects the surface wettability of the film. This study uses atomic force microscopy to quantitatively characterize the fundamental mechanism responsible for the structurally driven changes in surface properties at various applied voltages. The surface wettability transitions of the film are electrically driven, as shown by reorganized liquid crystal molecules. Measurements of the voltage-dependent surface wettability of the composite film suggest approaches to supporting control applications of future electro-optical nanotechnology devices.
KDM4B as a Target for Prostate Cancer: Structural Analysis and Selective Inhibition by a Novel Inhibitor
(2014-07-24)
Chu, Chia-Han
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Wang, Ling-Yu
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Hsu, Kai-Cheng
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Chen, Chung-Chin
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Cheng, Hsing-Hung
The KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A-KDM4D), which selectively remove the methyl group(s) from tri/dimethylated lysine 9/36 of H3, modulate transcriptional activation and genome stability. The overexpression of KDM4A/KDM4B in prostate cancer and their association with androgen receptor suggest that KDM4A/KDM4B are potential progression factors for prostate cancer. Here, we report the crystal structure of the KDM4B.pyridine 2,4-dicarboxylic acid. H3K9me3 ternary complex, revealing the core active-site region and a selective K9/K36 site. A selective KDM4A/KDM4B inhibitor, 4, that occupies three subsites in the binding pocket is identified by virtual screening. Pharmacological and genetic inhibition of KDM4A/KDM4B significantly blocks the viability of cultured prostate cancer cells, which is accompanied by increased H3K9me3 staining and transcriptional silencing of growth-related genes. Significantly, a substantial portion of differentially expressed genes are AR-responsive, consistent with the roles of KDM4s as critical AR activators. Our results point to KDM4 as a useful therapeutic target and identify a new inhibitor scaffold.
Mapping Band Alignment across Complex Oxide Heterointerfaces
(2012-12-12)
Huang, Bo-Chao
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Chiu, Ya-Ping
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Huang, Po-Cheng
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Wang, Wen-Ching
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Vu Thanh Tra
In this study, direct observation of the evolution of electronic structures across complex oxide interfaces has been revealed in the LaAlO3/SrTiO3 model system using cross-sectional scanning tunneling microscopy and spectroscopy. The conduction and valence band structures across the LaAlO3/SrTiO3 interface are spatially resolved at the atomic level by measuring the local density of states. This study directly maps out the electronic reconstructions and a built-in electric field in the polar LaAlO3 layer. Results also clearly reveal the band bending and the notched band structure in the SrTiO3 adjacent to the interface. DOI: 10.1103/PhysRevLett.109.246807
Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways
(2013-07-01)
Hsu, Kai-Cheng
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Cheng, Wen-Chi
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Chen, Yen-Fu
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Wang, Wen-Ching
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Yang, Jinn-Moon
Many virtual screening methods have been developed for identifying single-target inhibitors based on the strategy of "one-disease, one-target, one-drug". The hit rates of these methods are often low because they cannot capture the features that play key roles in the biological functions of the target protein. Furthermore, single-target inhibitors are often susceptible to drug resistance and are ineffective for complex diseases such as cancers. Therefore, a new strategy is required for enriching the hit rate and identifying multitarget inhibitors. To address these issues, we propose the pathway-based screening strategy (called PathSiMMap) to derive binding mechanisms for increasing the hit rate and discovering multitarget inhibitors using site-moiety maps. This strategy simultaneously screens multiple target proteins in the same pathway; these proteins bind intermediates with common substructures. These proteins possess similar conserved binding environments (pathway anchors) when the product of one protein is the substrate of the next protein in the pathway despite their low sequence identity and structure similarity. We successfully discovered two multitarget inhibitors with IC50 of <10 mu M for shikimate dehydrogenase and shikimate kinase in the shikimate pathway of Helicobacter pylori. Furthermore, we found two selective inhibitors (IC50 of <10 mu M) for shikimate dehydrogenase using the specific anchors derived by our method. Our experimental results reveal that this strategy can enhance the hit rates and the pathway anchors are highly conserved and important for biological functions. We believe that our strategy provides a great value for elucidating protein binding mechanisms and discovering multitarget inhibitors.
SiMMap: a web server for inferring site-moiety map to recognize interaction preferences between protein pockets and compound moieties
(2010-07-01)
Chen, Yen-Fu
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Hsu, Kai-Cheng
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Lin, Shen-Rong
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Wang, Wen-Ching
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Huang, Yu-Chi
The protein-ligand interacting mechanism is essential to biological processes and drug discovery. The SiMMap server statistically derives site-moiety map with several anchors, which describe the relationship between the moiety preferences and physicochemical properties of the binding site, from the interaction profiles between query target protein and its docked (or co-crystallized) compounds. Each anchor includes three basic elements: a binding pocket with conserved interacting residues, the moiety composition of query compounds and pocket-moiety interaction type (electrostatic, hydrogen bonding or van der Waals). We provide initial validation of the site-moiety map on three targets, thymidine kinase, and estrogen receptors of antagonists and agonists. Experimental results show that an anchor is often a hot spot and the site-moiety map can help to assemble potential leads by optimal steric, hydrogen bonding and electronic moieties. When a compound highly agrees with anchors of site-moiety map, this compound often activates or inhibits the target protein. We believe that the site-moiety map is useful for drug discovery and understanding biological mechanisms. The SiMMap web server is available at http://simfam.life.nctu.edu.tw/.
Structural study of laminaripentaose-producing beta-1,3-glucanase from Streptomyces matensis DIC-108
(2008-01-01)
Wu, Hsin-Mao
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Hsu, Mina-Tsung
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Lai, Chun-Chieh
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Liu, Sheng-Wen
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Li, Yaw-Kuen
Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing beta-1,3-Glucanase
(2009-09-25)
Wu, Hsin-Mao
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Liu, Sheng-Wen
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Hsu, Ming-Tsung
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Hung, Chiu-Lien
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Lai, Chun-Chieh
Laminaripentaose-producing beta-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide beta-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 angstrom resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (alpha/beta) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 angstrom, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu(154) and Asp(170)) and four other sugar-binding residues (Thr156, Asn(158), Trp(163), and Thr(167)). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu(154) and Asp(170) as acid and base catalysts, respectively, whereas the side chains of Thr(156), Asn(158), and Trp(163) demarcate subsite +5. Site-directed mutagenesis of Glu(154) and Asp(170) confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu(154) and Asp(170) to cleave a beta-1,3-glucan into specific alpha-pentasaccharide oligomers.
Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism
(2012-03-16)
Cheng, Wen-Chi
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Chen, Yen-Fu
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Wang, Hung-Jung
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Hsu, Kai-Cheng
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Lin, Shuang-Chih
Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure-activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK center dot SO4, R57A, and HpSK center dot shikimate-3-phosphate center dot ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A.162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism.