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1.
Article in English | WPRIM | ID: wpr-922770

ABSTRACT

Nine new compounds, including five natural rarely-occurring 2, 3-dihydro-1H-indene derivatives named diaporindenes E-I (1-5), and four new benzophenone analogues named tenellones J-M (6-9) were isolated from the deep-sea sediment-derived fungus Phomopsis lithocarpus FS508. All the structures for these new compounds were fully characterized on the basis of spectroscopic data, NMR spectra, and ECD calculation and single-crystal X-ray diffraction analysis. The potential anti-tumor activities of compounds 1-9 against four tumor cell lines SF-268, MCF-7, HepG-2, and A549 were evaluated using the SRB method. Compound 7 exhibited cytotoxic activity against the SF-268 cell line with an IC


Subject(s)
Antineoplastic Agents/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Fungi , Molecular Structure , Phomopsis
2.
Protein & Cell ; (12): 877-888, 2021.
Article in English | WPRIM | ID: wpr-922482

ABSTRACT

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M


Subject(s)
Antiviral Agents/therapeutic use , Binding Sites , COVID-19/virology , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Drug Evaluation, Preclinical , Drug Repositioning , High-Throughput Screening Assays/methods , Humans , Imidazoles/therapeutic use , Inhibitory Concentration 50 , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Naphthoquinones/therapeutic use , Protease Inhibitors/therapeutic use , Protein Structure, Tertiary , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification
3.
J. venom. anim. toxins incl. trop. dis ; 25: e20190013, 2019. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1020024

ABSTRACT

In Brazil and in other tropical areas Zika virus infection was directly associated with clinical complications as microcephaly in newborn children whose mothers were infected during pregnancy and the Guillain-Barré syndrome in adults. Recently, research has been focused on developing new vaccines and drug candidates against Zika virus infection since none of those are available. In order to contribute to vaccine and drug development efforts, it becomes important the understanding of the molecular basis of the Zika virus recognition, infection and blockade. To this purpose, it is essential the structural determination of the Zika virus proteins. The genome sequencing of the Zika virus identified ten proteins, being three structural (protein E, protein C and protein prM) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Together, these proteins are the main targets for drugs and antibody recognition. Here we examine new discoveries on high-resolution structural biology of Zika virus, observing the interactions and functions of its proteins identified via state-of-art structural methodologies as X-ray crystallography, nuclear magnetic resonance spectroscopy and cryogenic electronic microscopy. The aim of the present study is to contribute to the understanding of the structural basis of Zika virus infection at an atomic level and to point out similarities and differences to others flaviviruses.(AU)


Subject(s)
Pharmaceutical Preparations , Vaccines , Magnetic Resonance Spectroscopy , Zika Virus , Zika Virus Infection , Crystallography, X-Ray
4.
Experimental Neurobiology ; : 658-669, 2019.
Article in English | WPRIM | ID: wpr-785791

ABSTRACT

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca²⁺ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca²⁺ binding. Because the Ca²⁺ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca²⁺ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca²⁺ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca²⁺-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca²⁺ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca²⁺. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca²⁺ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca²⁺ and modulate the activation by Ca²⁺ and voltage.


Subject(s)
Amino Acids, Acidic , Animals , Calcium , Calmodulin , Chloride Channels , Cryoelectron Microscopy , Crystallography, X-Ray , EF Hand Motifs , Mice , Models, Molecular , Mutagenesis , Nectria , Protein Isoforms
5.
Article in English | WPRIM | ID: wpr-776863

ABSTRACT

Three new phenazine-type compounds, named phenazines SA-SC (1-3), together with four new natural products (4-7), were isolated from the fermentation broth of an earwig-associated Streptomyces sp. NA04227. The structures of these compounds were determined by extensive analyses of NMR, high resolution mass spectroscopic data, as well as single-crystal X-ray diffraction measurement. Sequencing and analysis of the genome data allowed us to identify the gene cluster (spz) and propose a biosynthetic pathway for these phenazine-type compounds. Additionally, compounds 1-5 exhibited moderate inhibitory activity against acetylcholinesterase (AChE), and compound 3 showed antimicrobial activities against Micrococcus luteus.


Subject(s)
Animals , Anti-Bacterial Agents , Chemistry , Metabolism , Pharmacology , Bacterial Proteins , Genetics , Metabolism , Crystallography, X-Ray , Insecta , Microbiology , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Micrococcus luteus , Molecular Structure , Multigene Family , Phenazines , Chemistry , Metabolism , Pharmacology , Streptomyces , Chemistry , Genetics , Metabolism
6.
Braz. j. med. biol. res ; 51(3): e7050, 2018. tab, graf
Article in English | LILACS | ID: biblio-889043

ABSTRACT

A new microporous lanthanide metal-organic framework, {[Yb(BTB)(H2O) (DEF)2}n (1, DEF=N,N-Diethylformamide), with 1D nano-sized channels has been constructed by bridging helical chain secondary building units with 1,3,5-benzenetrisbenzoic acid (H3BTB) ligand. Structural characterization suggests that this complex crystallizes in the hexagonal space group P6122 and possesses 1D triangular channels with coordinated water molecules pointing to the channel center. In addition, anti-myocarditis properties of compound 1 were evaluated in vivo. The results showed that compound 1 can improve hemodynamic parameters of, and it may be a good therapeutic option for heart failure in the future.


Subject(s)
Animals , Male , Mice , Anti-Inflammatory Agents/chemistry , Crystallography, X-Ray , Lanthanoid Series Elements/chemistry , Metal-Organic Frameworks/chemistry , Myocarditis/therapy , Anti-Inflammatory Agents/therapeutic use , Metal-Organic Frameworks/therapeutic use , Models, Molecular , Powder Diffraction , Thermogravimetry , X-Ray Diffraction
7.
Braz. j. med. biol. res ; 51(1): e6858, 2018. tab, graf
Article in English | LILACS | ID: biblio-889001

ABSTRACT

A novel heterometallic metal-porphyrinic framework (MPFs) built from Y and K ions as nods and meso-tetra(4-carboxyphenyl)porphyrin as linkers has been successfully synthesized and characterized. The single crystal X-ray diffraction indicated that this complex 1 exhibited a bilayered architecture of the porphyrins, which is seldom seen in MPFs. In addition, in vitro anticancer activity of complex 1 on three human breast cancer cells (BT474, SKBr-3 and ZR-75-30) was further determined.


Subject(s)
Humans , Porphyrins/chemistry , Breast Neoplasms/drug therapy , Metal-Organic Frameworks/pharmacology , Metal-Organic Frameworks/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Reference Values , Tetrazolium Salts , Reproducibility of Results , Crystallography, X-Ray , Cell Line, Tumor , Coordination Complexes/pharmacology , Coordination Complexes/chemistry , Formazans
8.
Article in English | WPRIM | ID: wpr-773596

ABSTRACT

Nine new ent-kaurane diterpenoids, named scopariusols L-T (1-9), were isolated from the aerial parts of Isodon scoparius. Their structures were characterized mainly by analyzing the NMR and HR-ESI-MS data, and the absolute configuration of 1 was determined by single-crystal X-ray diffraction. Compound 1 was active against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480), and it also inhibited NO production in LPS-stimulated RAW264.7 cells, with an IC value of 0.6 μmol·L.


Subject(s)
Animals , Antineoplastic Agents, Phytogenic , Chemistry , Pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Diterpenes, Kaurane , Chemistry , Pharmacology , Drug Screening Assays, Antitumor , Drugs, Chinese Herbal , Chemistry , Pharmacology , HL-60 Cells , Humans , Isodon , Chemistry , Lipopolysaccharides , Pharmacology , Macrophages , Mice , Molecular Structure , Nitric Oxide , Nuclear Magnetic Resonance, Biomolecular , Plant Components, Aerial , Chemistry
9.
Article in English | WPRIM | ID: wpr-812385

ABSTRACT

Nine new ent-kaurane diterpenoids, named scopariusols L-T (1-9), were isolated from the aerial parts of Isodon scoparius. Their structures were characterized mainly by analyzing the NMR and HR-ESI-MS data, and the absolute configuration of 1 was determined by single-crystal X-ray diffraction. Compound 1 was active against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480), and it also inhibited NO production in LPS-stimulated RAW264.7 cells, with an IC value of 0.6 μmol·L.


Subject(s)
Animals , Antineoplastic Agents, Phytogenic , Chemistry , Pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Diterpenes, Kaurane , Chemistry , Pharmacology , Drug Screening Assays, Antitumor , Drugs, Chinese Herbal , Chemistry , Pharmacology , HL-60 Cells , Humans , Isodon , Chemistry , Lipopolysaccharides , Pharmacology , Macrophages , Mice , Molecular Structure , Nitric Oxide , Nuclear Magnetic Resonance, Biomolecular , Plant Components, Aerial , Chemistry
10.
Braz. j. med. biol. res ; 50(11): e6455, 2017. tab, graf
Article in English | LILACS | ID: biblio-888957

ABSTRACT

Series of novel coumarin derivatives [I (a-d) and II (a-d)] were successfully synthesized and their structures were determined based on infrared 1H-nuclear magnetic resonance (NMR), HRMS, and single crystal X-ray crystallography. Additionally, the new synthesized compounds were evaluated to identify the molecular characteristics that contribute to their cytotoxicity, which was tested against SK-LU-1, SPC-A-1 and 95D human lung cancer cell lines, using the MTT assay. The results of this study showed that compounds Ic, Id, IIc, and IId exhibited an efficient percentage of inhibition of cell proliferation.


Subject(s)
Humans , Antineoplastic Agents/pharmacology , Coumarins/pharmacology , Lung Neoplasms/drug therapy , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Coloring Agents , Coumarins/chemical synthesis , Coumarins/chemistry , Crystallography, X-Ray/methods , Drug Screening Assays, Antitumor , Lung Neoplasms/pathology , Magnetic Resonance Spectroscopy/methods , Reference Values , Reproducibility of Results , Tetrazolium Salts , Thiazoles
11.
Braz. j. med. biol. res ; 50(7): e6390, 2017. tab, graf
Article in English | LILACS | ID: biblio-951701

ABSTRACT

Using two flexible Schiff bases, H2L1 and H2L2, two new cobalt II (Co(II))-coordination compounds, namely, Py3CoL1 (1) and Py3CoL2 (2) (Py=pyridine, L1=3,5-ClC6H2(O)C=NC6H3(O)-4-NO2, L2=3,5-BrC6H2(O)C=NC6H3(O)-4-NO2) have been synthesized under solvothermal conditions. Single crystal X-ray structural analysis revealed that compounds 1 and 2 are both six-coordinate in a distorted octahedral geometry, and the 1D chain structure was formed by the π…π and C-H…O interactions or C-H…Cl interaction. The in vitro antitumor activities of 1, 2 and their corresponding organic ligands Py, L1, and L2 were studied and evaluated, in which three human skin cancer cell lines (A-431, HT-144 and SK-MEL-30) were used in the screening tests.


Subject(s)
Humans , Schiff Bases/pharmacology , Skin Neoplasms/drug therapy , Cobalt/pharmacology , Schiff Bases/chemistry , Molecular Structure , Cobalt/chemistry , Crystallography, X-Ray , Cell Line, Tumor
12.
Protein & Cell ; (12): 25-38, 2017.
Article in English | WPRIM | ID: wpr-757373

ABSTRACT

Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 (FUNDC1) was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with microtubule-associated protein light chain 3 beta (LC3B) through its LC3 interaction region (LIR). Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDC1 LIR peptide phosphorylated at Ser17 (pS), demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS. Alternatively, phosphorylated Tyr18 (pY) and Ser13 (pS) in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry (ITC) assays. Therefore, our structural and biochemical results reveal a working model for the specific recognition of FUNDC1 by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.


Subject(s)
Crystallography, X-Ray , Membrane Proteins , Chemistry , Metabolism , Microtubule-Associated Proteins , Chemistry , Metabolism , Mitophagy , Mitochondrial Proteins , Chemistry , Metabolism , Peptides , Chemistry , Metabolism , Phosphorylation , Protein Structure, Quaternary
13.
Protein & Cell ; (12): 590-600, 2017.
Article in English | WPRIM | ID: wpr-756983

ABSTRACT

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.


Subject(s)
Amino Acid Sequence , Animals , Antibodies, Monoclonal , Chemistry , Genetics , Metabolism , Binding Sites , Cell Line , Crystallography, X-Ray , Enterovirus A, Human , Genetics , Allergy and Immunology , Fibroblasts , Virology , Gene Expression , HEK293 Cells , Humans , Immunoglobulin Fab Fragments , Chemistry , Genetics , Metabolism , Lysosome-Associated Membrane Glycoproteins , Chemistry , Genetics , Allergy and Immunology , Mice , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Scavenger , Chemistry , Genetics , Allergy and Immunology , Receptors, Virus , Chemistry , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Chemistry , Genetics , Allergy and Immunology , Sequence Alignment , Sequence Homology, Amino Acid , Sf9 Cells , Spodoptera , Thermodynamics
14.
Protein & Cell ; (12): 675-685, 2017.
Article in English | WPRIM | ID: wpr-756968

ABSTRACT

The α3* nAChRs, which are considered to be promising drug targets for problems such as pain, addiction, cardiovascular function, cognitive disorders etc., are found throughout the central and peripheral nervous system. The α-conotoxin (α-CTx) LvIA has been identified as the most selective inhibitor of α3β2 nAChRs known to date, and it can distinguish the α3β2 nAChR subtype from the α6/α3β2β3 and α3β4 nAChR subtypes. However, the mechanism of its selectivity towards α3β2, α6/α3β2β3, and α3β4 nAChRs remains elusive. Here we report the co-crystal structure of LvIA in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at a resolution of 3.4 Å. Based on the structure of this complex, together with homology modeling based on other nAChR subtypes and binding affinity assays, we conclude that Asp-11 of LvIA plays an important role in the selectivity of LvIA towards α3β2 and α3/α6β2β3 nAChRs by making a salt bridge with Lys-155 of the rat α3 subunit. Asn-9 lies within a hydrophobic pocket that is formed by Met-36, Thr-59, and Phe-119 of the rat β2 subunit in the α3β2 nAChR model, revealing the reason for its more potent selectivity towards the α3β2 nAChR subtype. These results provide molecular insights that can be used to design ligands that selectively target α3β2 nAChRs, with significant implications for the design of new therapeutic α-CTxs.


Subject(s)
Animals , Aplysia , Binding Sites , Conotoxins , Chemistry , Crystallography, X-Ray , Humans , Protein Structure, Quaternary , Receptors, Nicotinic , Chemistry
15.
Medicina (B.Aires) ; 76(6): 343-348, dic. 2016. tab
Article in English | LILACS | ID: biblio-841607

ABSTRACT

Nephrolithiasis is one of the most frequent urologic diseases. The aim of this paper is to study the composition and frequency of 8854 patient kidney stones and in a subset of them their metabolic risk factors to be related to their type of calculi. Physicochemical and crystallographic methods were used to assess kidney stone composition. In a subset of 715 patients, we performed an ambulatory metabolic protocol with diagnostic purposes. From the total sample 79% of stones were made of calcium salts (oxalate and phosphate), followed by uric acid stones in 16.5%, calcium salts and uric acid in 2%, other salts in 1.9% and cystine in 0.6%. Male to female ratio was almost three times higher in calcium salts and other types of stones, reaching a marked male predominance in uric acid stones, M/F 18.8 /1.0. The major risk factors for calcium stones are idiopathic hypercalciuria, followed by unduly acidic urine pH and hyperuricosuria. In uric acid stones unduly acidic urine pH and less commonly hyperuricosuria are the most frequent biochemical diagnosis. Our results show that analysis of kidney stones composition and the corresponding metabolic diagnosis may provide a scientific basis for the best management and prevention of kidney stone formation, as well as it may help us to study the mechanisms of urine stone formation.


La litiasis renal es una de las enfermedades urológicas más frecuentes. El objetivo de este trabajo fue estudiar la composición y frecuencia de 8854 cálculos renales y evaluar en un subgrupo de ellos la relación de los factores de riesgo metabólicos con el tipo de cálculo hallado. Se utilizaron métodos fisicoquímicos y cristalográficos para evaluar la composición de los cálculos renales. En un subgrupo de 715 pacientes, se pudo realizar un protocolo metabólico ambulatorio con fines diagnóstico. De la muestra total, 79.0% de los cálculos fueron de sales de calcio (oxalato y fosfato), seguido por cálculos de ácido úrico en 16.5%, sales de calcio y ácido úrico en 2.0%, otras sales en 1.9% y cistina en 0.6%. La relación hombre/mujer fue casi tres veces mayor en las sales de calcio y otros tipos de cálculos, alcanzando un marcado predominio en varones con cálculos de ácido úrico, M/F 18.8/1.0. Los principales factores de riesgo para los cálculos de calcio fueron la hipercalciuria idiopática, seguida del pH urinario excesivamente ácido y la hiperuricosuria. En los cálculos de ácido úrico el pH urinario excesivamente ácido y con menor frecuencia la hiperuricosuria fueron los diagnósticos más frecuentes. Nuestros resultados muestran que el análisis de la composición de los cálculos renales y el correspondiente diagnóstico metabólico pueden proporcionar una base científica para el mejor manejo y prevención en la formación de cálculos renales, así como que nos puede ayudar a estudiar los mecanismos de formación de los mismos.


Subject(s)
Humans , Male , Adult , Middle Aged , Young Adult , Kidney Calculi/etiology , Kidney Calculi/metabolism , Kidney Calculi/epidemiology , Metabolic Diseases/complications , Metabolic Diseases/epidemiology , Argentina/epidemiology , Reference Values , Uric Acid/metabolism , Kidney Calculi/chemistry , Sex Factors , Calcium/metabolism , Risk Factors , Age Factors , Crystallography, X-Ray/methods , Risk Assessment , Kidney/metabolism
16.
Protein & Cell ; (12): 516-526, 2016.
Article in English | WPRIM | ID: wpr-757409

ABSTRACT

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.


Subject(s)
Amino Acid Motifs , Binding Sites , Cell Cycle Proteins , Chemistry , Crystallography, X-Ray , Humans , Multienzyme Complexes , Chemistry , Protein Phosphatase 2 , Chemistry , Protein Structure, Quaternary , Protein-Serine-Threonine Kinases , Chemistry
17.
Protein & Cell ; (12): 673-683, 2016.
Article in English | WPRIM | ID: wpr-757406

ABSTRACT

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117-131), Loop 2 (residues 192-201) and the substrate recognition peptide (residues 94-102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.


Subject(s)
Bacterial Proteins , Chemistry , Crystallography, X-Ray , Methyltransferases , Chemistry , Protein Domains , Protein Structure, Secondary , Pyrimidine Nucleosides , Streptomyces
18.
Protein & Cell ; (12): 562-570, 2016.
Article in English | WPRIM | ID: wpr-757402

ABSTRACT

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.


Subject(s)
Crystallography, X-Ray , Protein Domains , RNA Helicases , Chemistry , RNA, Viral , Chemistry , Viral Proteins , Chemistry , Zika Virus
19.
Protein & Cell ; (12): 792-803, 2016.
Article in English | WPRIM | ID: wpr-757369

ABSTRACT

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701). MRG701 forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.


Subject(s)
Amino Acid Sequence , Arabidopsis , Genetics , Metabolism , Arabidopsis Proteins , Chemistry , Genetics , Metabolism , Binding Sites , Chromosomal Proteins, Non-Histone , Chemistry , Genetics , Metabolism , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli , Genetics , Metabolism , Gene Expression , Histones , Chemistry , Genetics , Metabolism , Models, Molecular , Oryza , Genetics , Metabolism , Peptides , Chemistry , Genetics , Metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms , Chemistry , Genetics , Metabolism , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins , Chemistry , Genetics , Metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Viridiplantae , Genetics , Metabolism
20.
Protein & Cell ; (12): 46-62, 2016.
Article in English | WPRIM | ID: wpr-757162

ABSTRACT

Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.


Subject(s)
Algorithms , Computer Simulation , Cryoelectron Microscopy , Methods , Crystallography, X-Ray , Humans , Macromolecular Substances , Chemistry , Models, Molecular , Protein Conformation , Ribosomes , Chemistry
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