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The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3A resolution, clearly revealing the amino acid residues involved in shikimate binding. In MtSK, the Glu61 strictly conserved in SK forms a hydrogen bond and salt-bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81, and Arg136, and hydroxyl groups with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for elucidation of the mechanism of SK-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

Parmodel is a web server for automated comparative modeling and evaluation of protein structures. The aim of this tool is to help inexperienced users to perform modeling, assessment, visualization, and optimization of protein models as well as crystallographers to evaluate structures solved experimentally. It is subdivided in four modules: Parmodel Modeling, Parmodel Assessment, Parmodel Visualization, and Parmodel Optimization. The main module is the Parmodel Modeling that allows the building of several models for a same protein in a reduced time, through the distribution of modeling processes on a Beowulf cluster. Parmodel automates and integrates the main softwares used in comparative modeling as MODELLER, Whatcheck, Procheck, Raster3D, Molscript, and Gromacs. This web server is freely accessible at http://www.biocristalografia.df.ibilce.unesp.br/tools/parmodel.

Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 A resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants. In order to pave the way for structural and functional efforts towards antimicrobial agent development, here we describe the molecular modeling of seven enzymes of the shikimate pathway of X. fastidiosa. The structural models of shikimate pathway enzymes, complexed with inhibitors, strongly indicate that the previously identified inhibitors may also inhibit the X. fastidiosa enzymes.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:04:53Z No. of bitstreams: 1 S1415-47572004000300003.pdf: 72604 bytes, checksum: 45ab620bc2bf787f67627b4d95bf66be (MD5) Made available in DSpace on 2013-08-22T19:04:53Z (GMT). No. of bitstreams: 1 S1415-47572004000300003.pdf: 72604 bytes, checksum: 45ab620bc2bf787f67627b4d95bf66be (MD5) Previous issue date: 2004-01-01 Made available in DSpace on 2013-09-30T20:08:19Z (GMT). No. of bitstreams: 2 S1415-47572004000300003.pdf: 72604 bytes, checksum: 45ab620bc2bf787f67627b4d95bf66be (MD5) S1415-47572004000300003.pdf.txt: 18159 bytes, checksum: 28a4224487a0da93b5d3f358380ca0c6 (MD5) Previous issue date: 2004-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:00:44Z No. of bitstreams: 2 S1415-47572004000300003.pdf: 72604 bytes, checksum: 45ab620bc2bf787f67627b4d95bf66be (MD5) S1415-47572004000300003.pdf.txt: 18159 bytes, checksum: 28a4224487a0da93b5d3f358380ca0c6 (MD5) Made available in DSpace on 2014-05-20T14:00:44Z (GMT). No. of bitstreams: 2 S1415-47572004000300003.pdf: 72604 bytes, checksum: 45ab620bc2bf787f67627b4d95bf66be (MD5) S1415-47572004000300003.pdf.txt: 18159 bytes, checksum: 28a4224487a0da93b5d3f358380ca0c6 (MD5) Previous issue date: 2004-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. In the present study, a total of 55 unrelated NF1 patients were screened for mutations in the GAP-related domain/GRD (exons 20-27a) by single-strand conformation polymorphism (SSCP). Four different mutations were identified and, taken together, they comprise one nonsense substitution (Q1189X), one deletion (3525-3526delAA), one missense substitution (E1356G) and one mutation in the splice acceptor site (c.4111-1G>A). One novel polymorphism (c.4514+11C>G) and other three putative polymorphisms were also found (c.3315-27G>A, V1146I and V1317A). Genotype-phenotype correlations were investigated, but no particular association was detected. Universidade Estadual Paulista Instituto de Biociências, Letras e Ciências Exatas Departamento de Biologia Faculdade de Medicina de São José do Rio Preto (FAMERP) Universidade Estadual Paulista Instituto de Biociências, Letras e Ciências Exatas Departamento de Física Universidade Estadual Paulista Instituto de Biociências, Letras e Ciências Exatas Departamento de Biologia Universidade Estadual Paulista Instituto de Biociências, Letras e Ciências Exatas Departamento de Física

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N -ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. More recently, the 3-D structure of human PNP has been refined to 2.3 A resolution, which allowed a redefinition of the residues involved in the substrate-binding sites and provided a more reliable model for structure-based design of inhibitors. This work reports crystallographic study of the complex of Human PNP:guanine (HsPNP:Gua) solved at 2.7 A resolution using synchrotron radiation. Analysis of the structural differences among the HsPNP:Gua complex, PNP apoenzyme, and HsPNP:immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.

Crystal structure of hemoglobin isolated from the Brazilian maned wolf (Chrysocyon brachyurus) was determined using standard molecular replacement technique and refined using maximum-likelihood and simulated annealing protocols to 1.87A resolution. Structural and functional comparisons between hemoglobins from the Chrysocyon brachyurus and Homo sapiens are discussed, in order to provide further insights in the comparative biochemistry of vertebrate hemoglobins.

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N -ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP–immucillin-H (HsPNP–ImmH) solved at 2.6 A resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP–ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP.

Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 A. Crystallographic refinement led to an R factor of 0.161 at 2.45 A resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.

A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8%(w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 A resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.

A chitin-binding protein named PPL-2 was purified from Parkia platycephala seeds and crystallized. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 A, and grew over several days at 293 K using the hanging-drop method. Using synchrotron radiation, a complete structural data set was collected to 1.73 A resolution. The preliminary crystal structure of PPL-2, determined by molecular replacement, presents a correlation coefficient of 0.558 and an R factor of 0.439. Crystallographic refinement is in progress.

Inhibition of Trypanosoma brucei and Leishmania spp. sirtuins has shown promising antiparasitic activity, indicating that these enzymes may be used as targets for drug discovery against trypanosomatid infections. In the present work we carried out a virtual screening focused on the C pocket of Sir2 from Trypanosoma cruzi. Using this approach, the best ligand found was nicotinamide. In vitro tests confirmed the anti-T. cruzi activity of nicotinamide on epimastigote and trypomastigote forms. Moreover, treatment of T. cruzi-infected macrophages with nicotinamide caused a significant reduction in the number of amastigotes. In addition, alterations in the mitochondria and an increase in the vacuolization in the cytoplasm were observed in epimastigotes treated with nicotinamide. Analysis of the complex of Sir2 and nicotinamide revealed the details of the possible ligand-target interaction. Our data reveal a potential use of TcSir2 as a target for anti-T. cruzi drug discovery.

This work describes for the first time the structure of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) in complex with sulfate and its natural substrate, 2'-deoxyguanosine, and its application to virtual screening. We report docking studies of a set of molecules against this structure. Application of polynomial empirical scoring function was able to rank docking solutions with good predicting power which opens the possibility to apply this new criterion to analyze docking solutions and screen small-molecule databases for new chemical entities to inhibit MtPNP.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:24:37Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:41:16Z : No. of bitstreams: 1 2-s2.0-77349112482.pdf: 4467381 bytes, checksum: 99f2eb25ed82204035ff1cc9cf8bb8f0 (MD5) Made available in DSpace on 2014-05-27T11:24:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-01-07 Background: The functional and structural characterisation of enzymes that belong to microbial metabolic pathways is very important for structure-based drug design. The main interest in studying shikimate pathway enzymes involves the fact that they are essential for bacteria but do not occur in humans, making them selective targets for design of drugs that do not directly impact humans.Description: The ShiKimate Pathway DataBase (SKPDB) is a relational database applied to the study of shikimate pathway enzymes in microorganisms and plants. The current database is updated regularly with the addition of new data; there are currently 8902 enzymes of the shikimate pathway from different sources. The database contains extensive information on each enzyme, including detailed descriptions about sequence, references, and structural and functional studies. All files (primary sequence, atomic coordinates and quality scores) are available for downloading. The modeled structures can be viewed using the Jmol program.Conclusions: The SKPDB provides a large number of structural models to be used in docking simulations, virtual screening initiatives and drug design. It is freely accessible at http://lsbzix.rc.unesp.br/skpdb/. © 2010 Arcuri et al; licensee BioMed Central Ltd. CEIS/Departamento de Biologia Instituto de Biociências UNESP, Rio Claro, São Paulo Departamento de Ciência da Computação e Estatística UNESP/IBILCE, São Jose do Rio Preto,São Paulo Departamento de Ciências Exatas UNIFAL, Alfenas Minas Gerais Faculdade de Biociências PUCRS, Porto Alegre, Rio Grande do Sul CEIS/Departamento de Biologia Instituto de Biociências UNESP, Rio Claro, São Paulo Departamento de Ciência da Computação e Estatística UNESP/IBILCE, São Jose do Rio Preto,São Paulo

Molecular docking is a simulation process where the binding of a small molecule is identified in the structure of a protein target. There are several different computational approaches to solve this problem. Here it will be described recent developments in application of evolutionary algorithms to molecular docking simulations. Evolutionary algorithms are classified as a group of computational techniques based on the concepts of Darwin's theory of evolution that are designed to the best possible find solution to optimisation problems. A successfully implementation of this algorithm can be found in the program MolDock. The main features of MolDock are reviewed here we also describe application of MolDock to purine nucleoside phosphorylase, shikimate kinase and cyclin-dependent kinase 2.

Toxoplasmosis is a chronic infection that affects approximately 30% of the human population and is caused by Toxoplasma gondii. Determination of the three dimensional structure of PNP from T. gondii could provide new insights into the purine binding site and sub-strate binding, and could be used for future rational design of new drugs against toxoplasmosis. This work describes the molecular model for three dimensional structure of PNP from T.gondii using, as a template, PNP from Plasmodium falciparum. Molecular dynamics showed that this model is stable during a trajectory of 3 ns.

Drug development has become the Holy Grail of many structural bioinformatics groups. The explosion of information about protein structures, ligand-binding affinity, parasite genome projects, and biological activity of millions of molecules opened the possibility to correlate this scattered information in order to generate reliable computational models to predict the likelihood of being able to modulate a target with a small-molecule drug. Computational methods have shown their potential in drug discovery and development allied with in vitro and in vivo methodologies. The present review discusses the main bioinformatics tools available for drug discovery and development.

Molecular recognition process describes the interaction involving two molecules. In the case of biomolecules, these pairs of molecules could be protein-protein, protein-ligand or protein-nucleic acid. The first model to capture the essential features, behind the molecular recognition problem, was the lock-and-key paradigm. The overall analysis protein-protein, protein-nucleic acid and protein-ligand interaction based on the three-dimensional structures and physicochemical parameters, such as binding affinity, opened the possibility to provide further insights in this basic phenomenon. The main ideas behind the molecular recognition are discussed in the present review.

Purine Nucleoside Phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-glycosidic bonds of purine nucleosides and deoxynucleosides, except for adenosine, to generate ribose 1-phosphate and the purine base. PNP has been submitted to intensive structural studies. This work describes for the first time a structural model of PNP from Streptococcus pyogenes (SpPNP). We modeled the complexes of SpPNP with six different ligands in order to determine the structural basis for specificity of these ligands against SpPNP. Molecular dynamics (MD) simulations were performed in order to evaluate the overall stability of SpPNP model. The analysis of the MD simulation was assessed mainly by principal component analysis (PCA) to explore the trimeric structure behavior. Structural comparison, between SpPNP and human PNP, was able to identify the main features responsible for differences in ligand-binding affinities, such as mutation in the purine-binding site and in the second phosphate-binding site. The PCA analysis suggests a different behavior for each subunit in the trimer structure.

Bacillus anthracis has been employed as an agent of bioterrorism, with high mortality, despite anti-microbial treatment, which strongly indicates the need of new drugs to treat anthrax. Shikimate pathway is a seven step biosynthetic route which generates chorismic acid from phosphoenol pyruvate and erythrose-4-phosphate. Chorismic acid is the major branch point in the synthesis of aromatic amino acids, ubiquinone, and secondary metabolites. The shikimate pathway is essential for many pathological organisms, whereas it is absent in mammals. Therefore, these enzymes are potential targets for the development of nontoxic antimicrobial agents and herbicides and have been submitted to intensive structural studies. The forth enzyme of this pathway is responsible for the conversion of dehydroshikimate to shikimate in the presence of NADP. In order to pave the way for structural and functional efforts toward development of new antimicrobials we describe the molecular modeling of shikimate dehydrogenase from Bacillus anthracis complexed with the cofactor NADP. This study was able to identify the main residues of the NADP binding site responsible for ligand affinities. This structural study can be used in the design of more specific drugs against infectious diseases.

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. coli and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies.

Assessing protein–ligand interaction is of great importance for virtual screening initiatives in order to discover new drugs. The present work describes a set of empirical scoring functions to assess the binding affinity, involving terms for intermolecular hydrogen bonds and contact surface. The results show that our methodology works better to predict protein–ligand affinity when compared with XSCORE, a popular empirical scoring function.

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50°C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of ∼63–64°C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents. Proteins 2008. © 2008 Wiley-Liss, Inc.

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:00:06Z No. of bitstreams: 1 WOS000249301900001.pdf: 1962990 bytes, checksum: 0c65e4a3836e97a4f347eb45bda09f6a (MD5) Made available in DSpace on 2014-02-26T17:00:07Z (GMT). No. of bitstreams: 1 WOS000249301900001.pdf: 1962990 bytes, checksum: 0c65e4a3836e97a4f347eb45bda09f6a (MD5) Previous issue date: 2007-08-02 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:20:31Z No. of bitstreams: 1 WOS000249301900001.pdf: 1962990 bytes, checksum: 0c65e4a3836e97a4f347eb45bda09f6a (MD5) Made available in DSpace on 2014-05-20T15:20:31Z (GMT). No. of bitstreams: 1 WOS000249301900001.pdf: 1962990 bytes, checksum: 0c65e4a3836e97a4f347eb45bda09f6a (MD5) Previous issue date: 2007-08-02 Background: Lectins are mainly described as simple carbohydrate- binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds ( CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA- like lectins; a site where a non- protein amino- acid, aaminobutyric acid ( Abu), is bound.Results: the overall structure of native CGL and complexed with alpha- methyl- mannoside and Abu have been refined at 2.3 angstrom and 2.31 angstrom resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.Conclusion: the presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants. Univ Fed Ceara, Dept Bioquim & Biol Mol, Ceara, Brazil Univ Reg Cariri, Dept Biol, Ceara, Brazil Univ Fed Paraiba, Dept Biol, Paraiba, Brazil Univ Estadual Paulista, IBILCE, Dept Fis, São Paulo, Brazil Pontificia Univ Catolica Rio Grande do Sul, Fac Biociencias, BR-90619900 Porto Alegre, RS, Brazil Univ Estadual Paulista, IBILCE, Dept Fis, São Paulo, Brazil

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata , respectively) in the following complexes: ConM/CGL:Man(α1-2)Man(α1- O )Me, ConM/CGL:Man(α1-3)Man(α1- O )Me and ConM/CGL:Man(α1-4)Man(α1- O )Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R factor and R free . Comparisons between ConM, CGL and ConA ( Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.

The complex CDK3-cyclin is involved in the control of the progression of G0. While the mechanisms governing early and late G1 progression are well understood, very little is known about the G0-G1 transition. Human CDK3 is closely related to cyclin-dependent kinase 2 (CDK2). Since there is no crystallographic structure of human CDK3, this work describes for the first time a molecular model of human CDK3 complexed with several inhibitors. Comparison of the binary complexes with different inhibitors strongly indicates that those inhibitors should inhibit CDK3 as well as CDK2.

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of betaalphabetaalphabetabeta-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future.

Parasitic protozoa infecting humans have a great impact on public health, especially in the developing countries. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in Protein Kinase (PK) inhibitors as potential drugs against a variety of diseases, the possibility that PKs may represent targets for novel anti-parasitic agents is being explored. Research into parasite PKs has benefited greatly from genome and EST sequencing projects, with the genomes from a few species fully sequenced (notably that from the malaria parasite Plasmodium falciparum) and several more under way, the structural features that are important to design specific inhibitors against these PKs will be reviewed in the present work.

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

In a routine screening of our small-molecule compound collection we recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of ATP antagonists with moderate potency against CDK2-cyclin E. A preliminary SAR study based on 35 analogues suggests ways in which the pharmacophore could be further optimized, for example, via substitutions in the 4-aryl ring. Enzyme kinetics studies with the lead compound and X-ray crystallography of an inhibitor-CDK2 complex demonstrated that its mode of inhibition is competitive. Functional kinase assays confirmed the selectivity toward CDKs, with a preference for CDK9-cyclin T1. The most potent inhibitor, 4-[(3,5-diamino-1H-pyrazol-4-yl)diazenyl]phenol 31b (CAN508), reduced the frequency of S-phase cells of the cancer cell line HT-29 in antiproliferation assays. Further observed cellular effects included decreased phosphorylation of the retinoblastoma protein and the C-terminal domain of RNA polymerase II, inhibition of mRNA synthesis, and induction of the tumor suppressor protein p53, all of which are consistent with inhibition of CDK9.

Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(alpha1-2)Man(alpha1)OMe, Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe in two crystal forms [the complexes with Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe crystallized in space group P3(2) and those with Man(alpha1-2)Man(alpha1)OMe crystallized in space group I222], which differed from those of the native proteins (P2(1)2(1)2 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(alpha1-4)Man(alpha1)OMe are reported here for the first time.

Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.

The present work analyzed the tetrameric stability of the hemoglobins from the rattlesnake C. durissus terrificus using analytical gel filtration chromatography, SAXS and osmotic stress. We show that the dissociation mechanism proposed for L. miliaris hemoglobin does not apply for these hemoglobins, which constitute stable tetramers even at low concentrations.

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstroms resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstroms, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.

Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 angstroms, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 angstroms resolution using a synchrotron-radiation source.

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 angstrom resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event. (c) 2005 Elsevier Inc. All rights reserved.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:23:58Z No. of bitstreams: 1 WOS000234256300001.pdf: 1197276 bytes, checksum: cc2ee7126ce555d8e5202993cb882d27 (MD5) Made available in DSpace on 2014-02-26T17:23:58Z (GMT). No. of bitstreams: 1 WOS000234256300001.pdf: 1197276 bytes, checksum: cc2ee7126ce555d8e5202993cb882d27 (MD5) Previous issue date: 2005-01-21 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:00:20Z No. of bitstreams: 1 WOS000234256300001.pdf: 1197276 bytes, checksum: cc2ee7126ce555d8e5202993cb882d27 (MD5) Made available in DSpace on 2014-05-20T14:00:20Z (GMT). No. of bitstreams: 1 WOS000234256300001.pdf: 1197276 bytes, checksum: cc2ee7126ce555d8e5202993cb882d27 (MD5) Previous issue date: 2005-01-21 Background: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.Results: the atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.Conclusions: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis. UNESP, Dept Phys, IBILCE, Sao Jose do Rio Preto, SP, Brazil USP, Dept Microbiol, Inst Biomed Sci, BR-09500900 São Paulo, Brazil Adolfo Lutz Inst, São Paulo, Brazil UNESP, Dept Biol, IBILCE, Sao Jose do Rio Preto, SP, Brazil Instituto Butantan, Ctr Appl Toxicol, São Paulo, Brazil UNESP, Dept Phys, IBILCE, Sao Jose do Rio Preto, SP, Brazil UNESP, Dept Biol, IBILCE, Sao Jose do Rio Preto, SP, Brazil