Discovery of antimicrobial agent targeting tryptophan synthase.

Antibiotic resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans. Drugs that can inhibit the bacterial biosynthesis of tryptophan offer a new class of antibiotics. In this work, we combined a structure-based strategy using in silico docking screening and molecular dynamics (MD) simulations to identify compounds targeting the α subunit of tryptophan synthase with experimental methods involving the whole-cell minimum inhibitory concentration (MIC) test, solution state NMR, and crystallography to confirm the inhibition of L-tryptophan biosynthesis. Screening 1,800 compounds from the National Cancer Institute Diversity Set I against α subunit revealed 28 compounds for experimental validation; four of the 28 hit compounds showed promising activity in MIC testing. We performed solution state NMR experiments to demonstrate that a one successful inhibitor, 3-amino-3-imino-2-phenyldiazenylpropanamide (Compound 1) binds to the α subunit. We also report a crystal structure of Salmonella enterica serotype Typhimurium tryptophan synthase in complex with Compound 1 which revealed a binding site at the αß interface of the dimeric enzyme. MD simulations were carried out to examine two binding sites for the compound. Our results show that this small molecule inhibitor could be a promising lead for future drug development.

Structural basis of the XPB helicase-Bax1 nuclease complex interacting with the repair bubble DNA.

Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal. StXPB and Bax1 together spirally encircle 10 base pairs of duplex DNA at the double-/single-stranded (ds-ss) junction. Furthermore, StXPB has its ThM motif intruding between the two DNA strands and gripping the 3'-overhang while Bax1 interacts with the 5'-overhang. This ternary complex likely reflects the state of repair bubble extension by the XPB and nuclease machine. ATP binding and hydrolysis by StXPB could lead to a spiral translocation along dsDNA and DNA strand separation by the ThM motif, revealing an unconventional DNA unwinding mechanism. Interestingly, the DNA is kept away from the nuclease domain of Bax1, potentially preventing DNA incision by Bax1 during repair bubble extension.

Structural and biochemical characterization of a glutathione transferase from the citrus canker pathogen Xanthomonas.

The genus Xanthomonas comprises several cosmopolitan plant-pathogenic bacteria that affect more than 400 plant species, most of which are of economic interest. Citrus canker is a bacterial disease that affects citrus species, reducing fruit yield and quality, and is caused by the bacterium Xanthomonas citri subsp. citri (Xac). The Xac3819 gene, which has previously been reported to be important for citrus canker infection, encodes an uncharacterized glutathione S-transferase (GST) of 207 amino-acid residues in length (XacGST). Bacterial GSTs are implicated in a variety of metabolic processes such as protection against chemical and oxidative stresses. XacGST shares high sequence identity (45%) with the GstB dehalogenase from Escherichia coli O6:H1 strain CFT073 (EcGstB). Here, XacGST is reported to be able to conjugate glutathione (GSH) with bromoacetate with a Km of 6.67 ± 0.77 mM, a kcat of 42.69 ± 0.32 s-1 and a kcat/Km of 6.40 ± 0.72 mM-1 s-1 under a saturated GSH concentration (3.6 mM). These values are comparable to those previously reported for EcGstB. In addition, crystal structures of XacGST were determined in the apo form (PDB entry 6nxv) and in a GSH-bound complex (PDB entry 6nv6). XacGST has a canonical GST-like fold with a conserved serine residue (Ser12) at the GSH-binding site near the N-terminus, indicating XacGST to be a serine-type GST that probably belongs to the theta-class GSTs. GSH binding stabilizes a loop of about 20 residues containing a helix that is disordered in the apo XacGST structure.

Structural basis of the XPB-Bax1 complex as a dynamic helicase-nuclease machinery for DNA repair.

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.

Síndrome de flutter ocular e ataxia de tronco pós-infecciosa / The post-infectious ocular flutter syndrome and truncal ataxia

OBJETIVO: Descrever um quadro clínico subagudo pós-infeccioso caracterizado por ataxia de tronco e flutter ocular. RELATO DE CASO: Um homem de 37 anos previamente hígido, pouco após um quadro sistêmico inespecífico que se resolveu espontaneamente, deu início a movimentos involuntários hipercinéticos dos olhos, da cabeça e desequilíbrio importante. Os sintomas evoluíram em poucos dias. Ao exame, apresentava ataxia de tronco e presença de flutter ocular. O líquor mostrou pleocitose discreta. Ressonância magnética (RM) de crânio e exames laboratoriais normais. Houve melhora espontânea e total dos sintomas em cerca de três semanas. CONCLUSÃO: A síndrome de flutter ocular e ataxia de tronco é rara e é destacado a benignidade do quadro.

OBJECTIVE: To describe a post-infectious subacute clinical picture characterized by truncal ataxia and ocular flutter. CASE REPORT: A healthy 37-year-old man, a few days after spontaneously resolved nonspecific systemic disease, initiated involuntary hyperkinetic movements of the eyes, head, and major imbalance. The symptoms progressed within a few days. He presented ataxia of the trunk and presence of ocular flutter. CSF showed pleocytosis. Magnetic resonance of the skull and laboratory tests were normal. There was spontaneous and total improvement of symptoms in about three weeks. CONCLUSION: The ocular flutter syndrome and truncal ataxia is rare and the benignity of the condition is highlighted.

Structure of the C-terminal half of human XPB helicase and the impact of the disease-causing mutation XP11BE.

XPB is a DNA-dependent helicase and a subunit of the TFIIH complex required for both transcription and DNA repair. XPB contains four domains: an N-terminal domain, two conserved helicase domains (HD1 and HD2) and a C-terminal extension. The C-terminal extension is important for DNA repair since the phosphorylation of Ser751 inhibits 5'-incision by ERCC1-XPF endonuclease. A disease-causing frameshift mutation (XP11BE) that changes the last 42 amino acids of XPB causes manifestations including impaired DNA repair and deficient transcription. Here, the crystal structure of the C-terminal half of XPB (residues 494-782) is reported at 1.8 Šresolution. The structure contained the conserved XPB HD2 and a C-terminal extension which shares structural similarity with RIG-I, leading to a structural model of the XPF-XPB-DNA complex for 5' incision during DNA repair. A mutation mimicking the XP11BE mutation produced the much less soluble mutant XPBm(494-781). Western blotting results confirmed that the intracellular levels of XPB and other TFIIH subunits in XP11BE patient cells were much lower than those from the healthy parents. Together, these results indicate that the XP11BE mutation not only divests the XPF-interaction motif, impairing DNA repair, but also reduces XPB solubility, leading to a lower intracellular level of TFIIH and deficient transcription.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from São Paulo State, Brazil.

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88% (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from São Paulo State, Brazil / Produção da proteína recombinante EMA-1 e sua aplicação para o diagnóstico baseadono imuno ensaio enzimático de Theileria equi em equinos do Estado de São Paulo, Brasil

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88 percent (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.

A proteína de superfície eritrocitária, Antígeno 1 do Merozoíta de Theileria equi (EMA-1), é um potencial candidato para o desenvolvimento de antígenos de valor diagnóstico para a piroplasmose equina. Com o objetivo de estabelecer um método de diagnóstico efetivo e prático, o gene EMA-1 da amostra Jaboticabal - SP de T. equi foi clonado e expresso em Escherichia coli contendo uma cauda de poli-histidina (His6-EMA1). O EMA-1 expresso reagiu com anticorpos específicos no Western blot e apresentou peso molecular aparente de 34 kDa, sendo altamente consistente com seu valor teórico. A sequência nucleotídica do gene EMA-1 da amostra Jaboticabal foi analisado comparativamente com outras sequências públicas, e os resultados indicam elevado grau de homologia com amostras de diversos países. A proteína recombinante purificada His6-EMA1 foi testada no ensaio imunoenzimático (ELISA) para a detecção de anticorpos anti-T. equi em equinos. O teste de ELISA diferenciou-se claramente entre soros de equinos infectados por T. equi, soros de animais infectados por Babesia caballi e soro normal de equino. Amostras de soros coletadas de equinos do Estado de São Paulo, sudeste do Brasil, foram examinadas para o diagnóstico da infecção por T. equi pelo ELISA. Das 170 amostras analisadas, 95,88 por cento (163/170) foram positivas para T. equi. Os resultados sugerem que a proteína His6-EMA1 expressa em E. coli pode ser um antígeno confiável para diagnóstico imunológico pelo teste de ELISA, e que T. equi merece grande atenção no Estado de São Paulo.

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae.

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition.