M. Cristina Nonato

Investigation of the relationship between the structure and function of Ts2, a neurotoxin from Tityus serrulatus venom

Scorpion toxins targeting voltage‐gated sodium (NaV) channels are peptides that comprise 60–76 amino acid residues cross‐linked by four disulfide bridges. These toxins can be divided in two groups (α and β toxins), according to their binding properties and mode of action. The scorpion α‐toxin Ts2, previously described as a β‐toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian NaV channel isoforms (rNaV1.2, rNaV1.3, rNaV1.4, hNaV1.5, mNaV1.6, rNaV1.7 and rNaV1.8) and one insect NaV channel isoform (DmNaV1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of NaV1.2, NaV1.3, NaV1.5, NaV1.6 and NaV1.7, but does not affect NaV1.4, NaV1.8 or DmNaV1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of NaV1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three β‐strands and one α‐helix, and is arranged in a triangular shape forming a cysteine‐stabilized α‐helix/β‐sheet (CSαβ) motif.

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli.

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18°C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase.

Target sites for the design of anti-trypanosomatid drugs based on the structure of dihydroorotate dehydrogenase.

Trypanosomatids consist of a large group of flagellated parasitic protozoa, including parasites from the genera Leishmania and Trypanosoma, responsible for causing infections in millions of humans worldwide and for which currently no appropriate therapy is available. The significance of pyrimidines in cellular metabolism makes their de novo and salvage pathways ideal druggable targets for pharmacological intervention and open an opportunity for pharmaceutical innovation. In the current review, we discuss the merits in targeting the enzyme dihydroorotate dehydrogenase (DHODH), a flavin-dependent enzyme that catalyzes the fourth and only redox step in pyrimidine de novo biosynthesis, as a strategy for the development of efficient therapeutic strategies for trypanosomatid-related diseases.We also describe the advances and perspectives from the structural biology point of view in order to unravel the structure-function relationship of trypanosomatid DHODHs, and to identify and validate target sites for drug development.

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzymes N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzymes active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.

Crystallization and preliminary X-ray diffraction analysis of Leishmania major dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes that catalyze the oxidation of L-dihydroorotate to orotate, the fourth step in the de novo pyrimidine nucleotide synthesis pathway. In this study, DHODH from Leishmania major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitating agent. The crystals belong to space group P6(1), with unit-cell parameters a = 143.7, c = 69.8 A. X-ray diffraction data were collected to 2.0 A resolution using an in-house rotating-anode generator. Analysis of the solvent content and the self-rotation function indicate the presence of two molecules in the asymmetric unit. The structure has been solved by the molecular-replacement technique.

The dihydroorotate dehydrogenases: Past and present ☆

The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs. It also attempts to describe the latest advances in medicinal chemistry for therapeutic development based on the selective inhibition of DHODH, including an overview of the experimental techniques used for ligand screening during the process of drug discovery.

Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold.

Significance Leishmaniases, Chagas disease, and sleeping sickness are parasitic diseases classified as neglected tropical diseases, affecting approximately one-sixth of the world’s population. Because of the absence of effective medicines to treat these diseases, there is a substantial interest in the identification of new targets for the development of therapeutic strategies to combat neglected tropical diseases. We have determined the crystal structure of a class I fumarate hydratase (FH) from Leishmania major, the parasite responsible for cutaneous leishmaniasis, and find that the structure is distinct from class II human FH. Thus, Leishmania class I FH, which is an essential metabolic enzyme, offers a new perspective for the development of antileishmaniases therapies. Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases.

Cloning, expression, purification, crystallization and preliminary X‐ray diffraction analysis of the N‐terminal carbohydrate‐recognition domain of human galectin‐4

Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2 A resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6(1)22, with unit-cell parameters a = b = 71.25, c = 108.66 A. The asymmetric unit contained one molecule of hGal4-CRD-1, with a V(M) value of 2.34 A(3) Da(-1) and a solvent content of 47.51%.

Crystal structure and molecular dynamics studies of L-amino acid oxidase from Bothrops atrox.

ABSTRACT L‐amino acid oxidases (LAAOs) are dimeric flavoproteins that catalyze the deamination of L‐amino acid to &agr;‐keto acid, producing ammonia and hydrogen peroxide. In this study, we report the crystal structure and molecular dynamics simulations of LAAO from the venom of Bothrops atrox (BatroxLAAO). BatroxLAAO presents several biological and pharmacological properties with promising biomedical applications. BatroxLAAO structure contains the highly conserved structural pattern of LAAOs comprising a FAD‐binding domain, substrate‐binding domain and helical domain, and a dimeric arrangement that can be stabilized by zinc. Also, molecular dynamics results show an asymmetric behavior, and a direct communication between FAD‐ and substrate‐binding domains of counterpart subunits. These findings shed light on the structural role of dimerization to catalytic mechanism of SV‐LAAOs. HIGHLIGHTSThe crystal structure of L‐amino acid oxidase from Bothrops atrox was solved at 2.3 Å resolution.A zinc binding site identified at the dimeric interface may influence the stability of its quaternary structure.Positively charged grooves are found in the majority of SV‐LAAOs in the opposite face of the active site.MD simulations reveal asymmetry between the dynamic behavior of the two monomers.Substrate binding induces a crosstalk between BatroxLAAO FAD‐ and substrate binding domains of counterpart chains.