Clonagem, expressão e purificação de domínios da proteína AtRLI2 (RNase L Inhibitor), um supressor endógeno de silenciamento por RNA de Arabidopis thaliana, visando estudos estruturais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudos estruturais com fosfolipases A2 isoladas de venenos de serpentes dos gêneros Bothrops e Crotalus

Pós-graduação em Ciências Biológicas (Genética) - IBB

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

Structure of the active site of sulfite dehydrogenase from Starkeya novella

In this paper, we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella. Our results provide the first direct structural information on the active site of the oxidized form of this enzyme and confirm the conclusions derived from protein crystallography that the molybdenum coordination is analogous to that of the sulfite oxidases. The molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 angstrom and three thiolate Mo-S ligands at 2.42 angstrom, whereas the reduced enzyme has one oxo at 1.74 angstrom, one long oxygen at 2.19 angstrom (characteristic of Mo-OH2), and three Mo-S ligands at 2.40 angstrom.

Structural studies on antifolate drugs

Single crystal X-ray structure determinations are reported for eleven compounds all of which are either biologically active or potentially biologically important. The compounds fall into two distinct classes:- 1. Substituted diaminopyrimidines 2. Substituted aminopyrimidinones The first class of compounds were all selected on the basis of their common diaminopyrimidine nucleus which has been demonstrated to be a vital requirement for antifolate activity. They may all be described as non-classical or small molecule lipophilic dihydrofolate reductase (DHFR) inhibitors, as opposed to the classical folate analogues, having the ability to cross the blood-brain barrier, enter cells via a rapid passive diffusion process, and achieve high intracellular concentrations. Thus they are an excellent choice in the search for crystallography in the solid state, providing geometrical and distance data not available from any other analytical techniques to date; supporting and enhancing data obtained in the lower resolution studies of protein crystallography. The biological importance of these compounds is discussed and an attempt is made to relate/predict their pharmacological activity to observed structural features in the crystalline environment. Special attention is focussed on hydrogen bonding, confirmational flexibility and hydrophobicity of substituents; each of which appear to make contributions to tight binding in the enzyme active site. Chapter 9 describes the use of data from the literature and the solid state modelling of an observed enzyme-substrate interaction in an attempt to define it more accurately in terms of its geometric flexibility. Of the second class, one compound (ABPP) is reported; studies in two different crystal forms. In demonstrating both antiviral and high interferon inducing activity it is possible that this compound could be useful against cancer and also viral infections.

Glycosylation and Glycodiversification in Polyketide Antibiotics: Unraveling Biosynthetic Steps in Nogalamycin Formation

Soil-dwelling Streptomyces bacteria are known for their ability to produce biologically active compounds such as antimicrobial, immunosuppressant, antifungal and anticancer drugs. S. nogalater is the producer of nogalamycin, a potential anticancer drug exhibiting high cytotoxicity and activity against human topoisomerases I and II. Nogalamycin is an anthracycline polyketide comprising a four-ring aromatic backbone,a neutral deoxy sugar at C7, and an amino sugar attached via an O–C bond at C1 and a C–C bond between C2 and C5´´. This kind of attachment of the amino sugar is unusual thus making the structure of the compound highly interesting. The sugar is also associated with the biological activity of nogalamycin, as it facilitates binding to DNA. Furthermore, the sugar moieties of anthracyclines are often crucial for their biological activity. Together the interesting attachment of the amino sugar and the general reliance of polyketides on the sugar moieties for bioactivity have made the study of the biosynthesis of nogalamycin attractive. The sugar moieties are typically attached by glycosyltransferases, which use two substrates: the donor and the acceptor. The literature review of the thesis is focused on the glycosylation of polyketides and the possibilities to alter their glycosylation patterns. My own thesis work revolves around the biosynthesis of nogalamycin. We have elucidated the individual steps that lead to its rather unique structure. We reconstructed the whole biosynthetic pathway in the heterologous host S. albus using a cosmid and a plasmid. In the process, we were able to isolate new compounds when the cosmid, which contains the majority of the nogalamycin gene cluster, was expressed alone in the heterologous host. The new compounds included true intermediates of the pathway as well as metabolites, which were most likely altered by the endogenous enzymes of the host. The biological activity of the most interesting new products was tested against human topoisomerases I and II, and they were found to exhibit such activities. The heterologous expression system facilitated the generation of mutants with inactivated biosynthetic genes. In that process, we were able to identify the functions of the glycosyltransferases SnogE and SnogD, solve the structure of SnogD, discover a novel C1-hydroxylase system comprising SnoaW and SnoaL2, and establish that the two homologous non-heme α-ketoglutarate and Fe2+ dependent enzymes SnoK and SnoN catalyze atypical reactions on the pathway. We demonstrated that SnoK was responsible for the formation of the additional C–C bond, whereas SnoN is an epimerase. A combination of in vivo and in vitro techniques was utilized to unravel the details of these enzymes. Protein crystallography gave us an important means to understand the mechanisms. Furthermore, the solved structures serve as platforms for future rational design of the enzymes.

Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X-ray crystallography

The oxalate oxidase enzyme expressed in barley roots is a thermostable, protease-resistant enzyme that generates H2O2. It has great medical importance because of its use to assay plasma and urinary oxalate, and it has also been used to generate transgenic, pathogen-resistant crops. This protein has now been purified and three types of crystals grown. X-ray analysis shows that the symmetry present in these crystals is consistent with a hexameric arrangement of subunits, probably a trimer of dimers. This structure may be similar to that found in the related seed storage proteins.

Direct structure analysis in protein electron crystallography: crystallographic phases for halorhodopsin to 6-A resolution.

The crystal structure of halorhodopsin was determined in (centrosymmetric) projection to 6-A resolution by direct methods that use only the amplitudes of the electron diffraction pattern. A multisolution technique was used to generate initial 15-A-resolution basis sets, and after selection of the best phase set (by the closest match of magnitude of Eobs and magnitude of Ecalc), annealing of individual reflections was used to improve its accuracy. The Sayre equation was then used to expand the phase terms to 10 A, followed again by phase annealing. A final expansion with the Sayre equation enlarged this corrected phase set to 6 A. When the condition of density flatness was used to locate the best phase solution after each extension, a final structure could be observed that was quite similar to the one found earlier by analysis of electron micrographs.

A Staggered Decameric Assembly of Human C-Reactive Protein Stabilized by Zinc Ions Revealed by X-ray Crystallography

International audience

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

The MX2 macromolecular crystallography beamline: a wiggler X-ray source at the LNLS

The Brazilian Synchrotron Light Laboratory [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP, Brazil] is the first commissioned synchrotron light source in the southern hemisphere. The first wiggler macromolecular crystallography beamline (MX2) at the LNLS has been recently constructed and brought into operation. Here the technical design, experimental set-up, parameters of the beamline and the first experimental results obtained at MX2 are described. The beamline operates on a 2.0 T hybrid 30-pole wiggler, and its optical layout includes collimating mirror, Si( 111) double-crystal monochromator and toroidal bendable mirror. The measured flux density at the sample position at 8.7 eV reaches 4.8 x 10(11) photons s(-1) mm(-2) (100 mA)(-1). The beamline is equipped with a MarResearch Desktop Beamline Goniostat (MarDTB) and 3 x 3 MarMosaic225 CCD detector, and is controlled by a customized version of the Blu-Ice software. A description of the first X-ray diffraction data sets collected at the MX2 LNLS beamline and used for macromolecular crystal structure solution is also provided.

Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae

The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pill and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 Angstrom) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares similar to 40% sequence identity. Ln addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors. (C) 1997 Academic Press Limited.

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Forkhead-associated (FHA) domains are modular protein–protein interaction domains of ~130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6122 or P6522, with unit-cell parameters a = b = 127.3, c = 386.3 Å; diffraction data have been collected to 3.1 Å resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 Å resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 Å.

Crystallization of PNMT, the adrenaline-synthesizing enzyme, is critically dependent on a high protein concentration

Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 Angstrom. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 Angstrom.