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Transthyretin was discovered in the 1940s, named after its ability to bind thyroid hormones and retinol. In the genomic era, transthyretins were found to be part of a larger family with homologs of no obvious function, then called transthyretin-related proteins. Thus, it was proposed that the transthyretin gene could be the result of gene duplication of an ancestral of this newly identified homolog, later found out to be an enzyme involved in uric acid degradation, then named HIUase (5-hydroxy-isourate hydrolase). Here, we sought to re-enact the evolutionary history of this protein family by reconstructing, from a phylogeny inferred from 123 vertebrate sequences, three ancestors corresponding to key moments in their evolution-before duplication; the common transthyretin ancestor after gene duplication and the common ancestor of Eutheria transthyretins. Experimental and computational characterization showed the reconstructed ancestor before duplication was unable to bind thyroxine and likely presented the modern HIUase reaction mechanism, while the substitutions after duplication prevented that activity and were enough to provide stable thyroxine binding, as confirmed by calorimetry and x-ray diffraction. The Eutheria transthyretin ancestor was less prone to characterization, but limited data suggested thyroxine binding as expected. Sequence/structure analysis suggests an early ability to bind the Retinol Binding Protein. We solved the X-ray structures from the two first ancestors, the first at 1.46 resolution, the second at 1.55 resolution with well-defined electron density for thyroxine, providing a useful tool for the understanding of structural adaptation from enzyme to hormone distributor.

Protein 3D structure can be remarkably robust to the accumulation of mutations during evolution. On the other hand, sometimes a single amino acid substitution can be sufficient to generate dramatic and completely unpredictable structural consequences. In an attempt to rationally alter the preferences for the metal ion at the active site of a member of the Iron/Manganese superoxide dismutase family, two examples of the latter phenomenon were identified. Site directed mutants of SOD from Trichoderma reesei were generated and studied crystallographically together with the wild type enzyme. Despite being chosen for their potential impact on the redox potential of the metal, two of the mutations (D150G and G73A) in fact resulted in significant alterations to the protein quaternary structure. The D150G mutant presented alternative inter-subunit contacts leading to a loss of symmetry of the wild type tetramer, whereas the G73A mutation transformed the tetramer into an octamer despite not participating directly in any of the inter-subunit interfaces. We conclude that there is considerable intrinsic plasticity in the Fe/MnSOD fold that can be unpredictably affected by single amino acid substitutions. In much the same way as phenotypic defects at the organism level can reveal much about normal function, so too can such mutations teach us much about the subtleties of protein structure.

The Golgi complex is a membranous organelle located in the heart of the eukaryotic secretory pathway. A subfamily of the Golgi matrix proteins, called GRASPs, are key players in the stress-induced unconventional secretion, the Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogs (GRASP55/GRASP65) and two coiled-coil Golgins (GM130/Golgin45), which interact with each otherin vivo. Although essential for the Golgi ribbon formation and the increase in Golgi structural complexity, the molecular details leading to their appearance only in this subphylum are still unknown. Moreover, despite the new functionalities supported by the GRASP paralogy, little is known about the structural and evolutionary differences between these paralogues. In this context, we used ancestor sequence reconstruction and several biophysical/biochemical approaches to assess the evolution of the GRASP structure, flexibility, and how they started anchoring their Golgin partners. Our data showed that the Golgins appeared in evolution and were anchored by the single GRASP ancestor beforegoraspgene duplication and divergence in Metazoans. After thegoraspdivergence, variations inside the GRASP binding pocket determined which paralogue would recruit each Golgin partner (GRASP55 with Golgin45 and GRASP65 with GM130). These interactions are responsible for the protein’s specific Golgi locations and the appearance of the Golgi ribbon. We also suggest that the capacity of GRASPs to form supramolecular structures is a long-standing feature, which likely affects GRASP’s participation as a trigger of the stress-induced secretory pathway.

Coenzyme Q (CoQ) is an essential molecule that consists of a highly substituted benzene ring attached to a polyprenyl tail anchored in the inner mitochondrial membrane. CoQ transfers electrons from NADH dehydrogenase and succinate dehydrogenase complexes toward ubiquinol-cytochrome c reductase, and that allows aerobic growth of cells. In Saccharomyces cerevisiae, the synthesis of CoQ depends on fourteen proteins Coq1p-Co11p, Yah1p, Arh1p, and Hfd1p. Some of these proteins are components of CoQ synthome. Using ab initio molecular modeling and site-directed mutagenesis, we identified the functional residues of the O-methyltransferase Coq3p, which depends on S-adenosylmethionine for catalysis and is necessary for two O-methylation steps required for CoQ maturation. Conserved residues as well as those that coevolved in the protein structure were found to have important roles in respiratory growth, CoQ biosynthesis, and also in the stability of CoQ synthome proteins. Finally, a multiple sequence alignment showed that S. cerevisiae Coq3p has a 45 amino acid residues insertion that is poorly conserved or absent in oleaginous yeast, cells that can store up to 20% of their dry weight as lipids. These results point to the Coq3p structural determinants of its biological and catalytic function and could contribute to the development of lipid-producing yeast for biotechnology.

Transthyretin was discovered in the 1940s, named after its ability to bind thyroid hormones and retinol. In the genomic era, transthyretins were found to be part of a larger family with homologs of no obvious function, then called transthyretin-related proteins. Thus, it was proposed that the transthyretin gene could be the result of gene duplication of an ancestral of this newly identified homolog, later found out to be an enzyme involved in uric acid degradation, then named HIUase (5-hydroxy-isourate hydrolase). Here, we sought to re-enact the evolutionary history of this protein family by reconstructing, from a phylogeny inferred from 123 vertebrate sequences, three ancestors corresponding to key moments in their evolution-before duplication; the common transthyretin ancestor after gene duplication and the common ancestor of Eutheria transthyretins. Experimental and computational characterization showed the reconstructed ancestor before duplication was unable to bind thyroxine and likely presented the modern HIUase reaction mechanism, while the substitutions after duplication prevented that activity and were enough to provide stable thyroxine binding, as confirmed by calorimetry and x-ray diffraction. The Eutheria transthyretin ancestor was less prone to characterization, but limited data suggested thyroxine binding as expected. Sequence/structure analysis suggests an early ability to bind the Retinol Binding Protein. We solved the X-ray structures from the two first ancestors, the first at 1.46 resolution, the second at 1.55 resolution with well-defined electron density for thyroxine, providing a useful tool for the understanding of structural adaptation from enzyme to hormone distributor.

Summary CONAN is a web application developed to detect specificity determinants and function-related sites by amino acids co-variation networks analysis, emphasizing local coevolutionary constraints. The software allows the characterization of structurally and functionally relevant groups of residues and their relationship with subsets of sequences by automatic cross-referencing with GO terms, UniprotKb annotations and INTERPRO. Availability and implementation CONAN is free and open-source, being distributed in the terms of the GPLV3 license. The software is available as a web application and python script versions and can be accessed at http://bioinfo.icb.ufmg.br/conan. We also provide running instructions, the source code and a user guide.

PFstats is a software developed for the extraction of useful information from protein multiple sequence alignments. By analyzing positional conservation and residue coevolution networks, the software allows the identification of structurally and functionally important residue groups and the discovery of probable functional subclasses. Furthermore, it contains tools for the identification of the possible biological significance of these findings. PFstats contains methods for maximizing the significance of alignments through filtering and weighting, residue conservation and coevolution analysis, automatic UniprotKb queries for residue-position annotation and many possible data visualization methods.

Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.

Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed.

Motivation Computational studies of molecular evolution are usually performed from a multiple alignment of homologous sequences, on which sequences resulting from a common ancestor are aligned so that equivalent residues are placed in the same position. Residues frequency patterns of a full alignment or from a subset of its sequences can be highly useful for suggesting positions under selection. Most methods mapping co-evolving or specificity determinant sites are focused on positions, however, they do not consider the case for residues that are specificity determinants in one subclass, but variable in others. In addition, many methods are impractical for very large alignments, such as those obtained from Pfam, or require a priori information of the subclasses to be analyzed. Results In this paper we apply the complex networks theory, widely used to analyze co-affiliation systems in the social and ecological contexts, to map groups of functional related residues. This methodology was initially evaluated in simulated environments and then applied to four different protein families datasets, in which several specificity determinant sets and functional motifs were successfully detected. Availability and implementation The algorithms and datasets used in the development of this project are available on http://www.biocomp.icb.ufmg.br/biocomp/software-and-databases/networkstats/. Supplementary information Supplementary data are available at Bioinformatics online.

Objective Thyroxine-binding globulin (TBG) is the major human thyroid hormone transport protein, encoded by the SERPINA7 gene (Xq22.2). We aim to investigate the molecular basis of partial TBG deficiency (TBG-PD) in a female, by evaluating the X-chromosome inactivation pattern as well as the mutant protein structural modeling. Design and methods Sequencing of the coding region of the SERPINA7 gene was performed in a female with a TBG-PD phenotype and her first-degree relatives. The proband presented with low serum levels of total T3 (TT3) and total T4 (TT4), serum TSH level of 5.4 μUI/mL (normal range, 0.35–5.5), and serum TBG level of 5.5 mg/L (normal range, 13.6–27.2). X-chromosome inactivation pattern was evaluated by methylation analysis of the androgen receptor gene (Xq11.2). Structural analysis of the SERPIN family was performed using Pymol and Areaimol, and PFSTATS for conservation analysis and family-wide investigation of equivalent positions in human homologs. Modeller was used for point mutation structural modeling. Results A novel missense SERPINA7 mutation (p.R35W; c.163C > T) was found in heterozygosity in the proband, and in hemizygosity in her affected siblings. The proband X-chromosome inactivation ratio was 20:80. The substitution of an arginine by a tryptophan is predicted to disrupt the protein surface and main electrostatic interactions. Tryptophans are extremely rare (0.1%) in this position. Conclusions We report a new SERPINA7 variant associated with TBG-PD in three siblings. We named this variant TBG-Brasilia. The X-chromosome inactivation pattern may have accounted for the rare phenotypic expression in a female. The hydrophobic nature of the mutant is predicted to create an apolar patch at the surface, which results in protein aggregation and/or misfolding, potentially responsible for thyroid hormone transport defect.

Phospholipases A2 (PLA2s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA2s family with special emphasis on snake venom PLA2s (svPLA2s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA2s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA2s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for important positions in the family of PLA2s, and potential new objects of investigation.

Bacteria are essential in arsenic cycling. However, few studies have addressed 16S rRNA and arsenic-related functional gene diversity in long-term arsenic-contaminated tropical sediment. Here, using culture-based, metagenomic and computational approaches, we describe the diversity of bacteria, genes and enzymes involved in AsIII and AsV transformation in freshwater sediment and in anaerobic AsIII- and AsV-enrichment cultures (ECs). The taxonomic profile reveals significant differences among the communities. Arcobacter, Dechloromonas, Sedimentibacter and Clostridium thermopalmarium were exclusively found in ECs, whereas Anaerobacillus was restricted to AsV-EC. Novel taxa that are both AsV-reducers and AsIII-oxidizers were identified: Dechloromonas, Acidovorax facilis, A. delafieldii, Aquabacterium, Shewanella, C. thermopalmarium and Macellibacteroides fermentans. Phylogenic discrepancies were revealed among the aioA, arsC and arrA genes and those of other species, indicating horizontal gene transfer. ArsC and AioA have sets of amino acids that can be used to assess their functional and structural integrity and familial subgroups. The positions required for AsV reduction are conserved, suggesting strong selective pressure for maintaining the functionality of ArsC. Altogether, these findings highlight the role of freshwater sediment bacteria in arsenic mobility, and the untapped diversity of dissimilatory arsenate-reducing and arsenate-resistant bacteria, which might contribute to arsenic toxicity in aquatic environments.

In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 Å resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting β-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn2+ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn2+ at pH 4.6–7.5. All four structures reveal three tetra-coordinated Zn2+-binding sites (ZBS 1–3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn2+ binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn2+. Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn2+. HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn2+ binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn2+, which is consistent with the tertiary structural perturbation provoked by Zn2+ binding, tetramer stability is only marginally affected by Zn2+. These data highlight structural and functional roles of Zn2+ in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.

Transthyretin (TTR) is a tetrameric β-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation ( Green et al., 2005 ) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis.

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 A and T. cruzi at 1.9 A resolution, and that from P. falciparum at a higher resolution (2.0 A) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity. Proteins 2009. © 2009 Wiley-Liss, Inc.

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen–deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B–dependent transcription exercised by GR in cells.

A Análise de Acoplamentos Estatísticos é uma técnica computacional capaz de identificar resíduos importantes para a estrutura e função de proteínas em uma família por meio da quantificação de conservação posicional, correlação entre posições e identificação de grupos de resíduos correlacionados entre si. Neste trabalho, a análise de acoplamentos estatísticos foi implementada e aplicada ao estudo das proteínas tirosina fosfatases. Em conjunto com as proteínas tirosina quinases (PTKs), que adicionam um grupo fosforil a um resíduo de tirosina em uma proteína, as proteínas tirosina fosfatases (PTPs), que o removem, são responsáveis por diversos processos de sinalização celular. Elas são um caso de evolução convergente, onde um subgrupo (as proteínas tirosina fosfatases de baixo peso molecular) não apresenta homologia às chamadas PTPs \"clássicas\", capazes de defosforilar apenas resíduos de tirosina, e às fosfatases de especifidicade dupla, capazes de defosforilar também resíduos de serina e treonina, além de substratos não-protéicos. Em comum, as três subfamílias apresentam apenas o motivo CX5R, característico para todas as PTPs. Através do estudo das três subfamílias utilizando a análise de acoplamentos estatísticos, foi possível obter uma descrição detalhada de suas características conservadas e correlacionadas, relacionando-as ao conhecimento acumulado sobre proteínas tirosina fosfatases e a questões em aberto como a regulação por dimerização, a especificidade e mutações relacionadas a patologias. Foi possível também apresentar um método capaz de distinguir proteínas tirosina fosfatases de baixo peso molecular das arsenato redutases, derivadas das primeiras por evolução divergente. Adicionalmente, a técnica foi aplicada ao estudo das hexoquinases, às superóxido dismutases e às peroxidases. A tese descreve também estudos desenvolvidos pelo autor na área de cristalografia de proteínas a determinação das estruturas da Transtirretina humana em complexo com genisteína, da holo-Hexoquinase PI de S. cerevisae, do complexo IL-22/IL-22R1 e da Laminarinase de R. marinus. The statistical coupling analysis is a computational technique which can identify important residues for the structure and function of proteins in a family by quantifying positional conservation, correlation between positions and identifying groups of self-correlating residues. Its implementation in this research group was applied to the study of the protein tyrosine phosphatases. Together with the protein tyrosine kinases (PTKs), which add a phosphoryl group to a tyrosine residue in proteins, the protein tyrosine phosphatases (PTPs), which remove it, are responsible for a variety of cell signaling processes. They are a case of convergent evolution, since one subgroup (the low molecular weight protein tyrosine phosphatases) are not homologous to the classical phosphatases, which can only dephosphorilate tyrosine residues, and the dual-specificity phosphatases, which can also dephosphorilate serine and threonine residues, and also non-proteinaceous substrates. All three sub-families have, in common, the CX5R motif, a characteristic of all PTPs. By applying the statistical coupling analysis to the study of the three sub-families, it was possible to obtain a detailed depiction of their conserved and correlated characteristics, relating them to the accumulated knowledge on protein tyrosine phosphatases and open questions such as protein regulation by dimerization, specificity and disease-related mutations. It was also possible to present a method to distinguish between low molecular weight phosphatases and arsenate reductases, which are derived by the former by divergent evolution. In addition, the technique was applied to the study of hexokinases, superoxide dismutases and peroxidases. The thesis also describe studies developed by the author in the field of protein crystallography the structure determination of human transthyretin in complex with genistein, holo-hexokinase PI from S. cerevisae, the IL-22/IL-22R1 complex and the laminarinase from R. marinus.

Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center.

Made available in DSpace on 2013-08-28T14:09:29Z (GMT). No. of bitstreams: 1 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) Made available in DSpace on 2013-09-30T18:37:20Z (GMT). No. of bitstreams: 2 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) WOS000298666200001.pdf.txt: 56655 bytes, checksum: 10d0e581654ecca834117e332f3f69ca (MD5) Previous issue date: 2011-12-20 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:49:40Z No. of bitstreams: 2 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) WOS000298666200001.pdf.txt: 56655 bytes, checksum: 10d0e581654ecca834117e332f3f69ca (MD5) Made available in DSpace on 2014-05-20T13:49:40Z (GMT). No. of bitstreams: 2 WOS000298666200001.pdf: 402150 bytes, checksum: 3723ce8567931f389f45074a49611911 (MD5) WOS000298666200001.pdf.txt: 56655 bytes, checksum: 10d0e581654ecca834117e332f3f69ca (MD5) Previous issue date: 2011-12-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Correlated mutation analysis has a long history of interesting applications, mostly in the detection of contact pairs in protein structures. Based on previous observations that, if properly assessed, amino acid correlation data can also provide insights about functional sub-classes in a protein family, we provide a complete framework devoted to this purpose. An amino acid specific correlation measure is proposed, which can be used to build networks summarizing all correlation and anti-correlation patterns in a protein family. These networks can be submitted to community structure detection algorithms, resulting in subsets of correlated amino acids which can be further assessed by specific parameters and procedures that provide insight into the relationship between different communities, the individual importance of community members and the adherence of a given amino acid sequence to a given community. By applying this framework to three protein families with contrasting characteristics (the Fe/Mn-superoxide dismutases, the peroxidase-catalase family and the C-type lysozyme/alpha-lactalbumin family), we show how our method and the proposed parameters and procedures are related to biological characteristics observed in these protein families, highlighting their potential use in protein characterization and gene annotation. Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil Univ São Paulo, Inst Fis Sao Carlos, Dept Fis & Informat, Sao Carlos, SP, Brazil Univ Estadual Paulista, Dept Fis & Biofis, Botucatu, SP, Brazil FAPESP: 08/58734-1 FAPESP: 98/14138-2

Diffraction data to 3.2 Å from crystals of the 3.6 MDa erythrocruorin from a Brazilian earthworm represent the highest resolution reported to date for similar complexes. An unambiguous molecular replacement solution shows the particle to belong to the type I class., Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 Å, b = 320.3 Å and c = 332.4 Å. Diffraction data were collected to 3.15 Å using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.

Coq10p is a protein required for coenzyme Q function, but its specific role is still unknown. It is a member of the START domain superfamily that contains a hydrophobic tunnel implicated in the binding of lipophilic molecules. We used site-directed mutagenesis, statistical coupling analysis and molecular modeling to probe structural determinants in the Coq10p putative tunnel. Four point mutations were generated (coq10-K50E, coq10-L96S, coq10-E105K and coq10-K162D) and their biochemical properties analysed, as well as structural consequences. Our results show that all mutations impaired Coq10p function and together with molecular modeling indicate an important role for the Coq10p putative tunnel.

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.

Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75Å resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.Structured summaryMINT-7010533: IL-22 BP (uniprotkb:Q969J5) and IL-22 (uniprotkb:Q9GZX6) bind (MI:0407) by X-ray crystallography (MI:0114)

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9Å crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2.Structured summaryMINT-6693956:Il22 (uniprotkb:Q9GZX6) and IL22R1 (uniprotkb:Q8N6P7) bind (MI:0407) by X-ray crystallography (MI:0114)

Hexokinase is the first enzyme in the glycolytic pathway that catalyzes the transfer of a phosphoryl group from ATP to glucose to form glucose-6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. Here we redetermined the crystal structure of yeast hexokinase PI from Saccharomyces cerevisiae as a complex with its substrate, glucose, and refined it at 2.95 A resolution. Comparison of the holo-PI yeast hexokinase and apo-hexokinase structures shows in detail the rigid body domain closure and specific loop movements as glucose binds and sheds more light on structural basis of the "induced fit" mechanism of reaction in the HK enzymatic action. We also performed statistical coupling analysis of the hexokinase family, which reveals two co-evolved continuous clusters of amino acid residues and shows that the evolutionary coupled amino acid residues are mostly confined to the active site and the hinge region, further supporting the importance of these parts of the protein for the enzymatic catalysis.

Background Thyroid receptors, TRα and TRβ, are involved in important physiological functions such as metabolism, cholesterol level and heart activities. Whereas metabolism increase and cholesterol level lowering could be achieved by TRβ isoform activation, TRα activation affects heart rates. Therefore, β-selective thyromimetics have been developed as promising drug-candidates for treatment of obesity and elevated cholesterol level. GC-1 [3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)-phenoxy acetic acid] has ability to lower LDL cholesterol with 600- to 1400-fold more potency and approximately two- to threefold more efficacy than atorvastatin (Lipitor©) in studies in rats, mice and monkeys. Results To investigate GC-1 specificity, we solved crystal structures and performed molecular dynamics simulations of both isoforms complexed with GC-1. Crystal structures reveal that, in TRα Arg228 is observed in multiple conformations, an effect triggered by the differences in the interactions between GC-1 and Ser277 or the corresponding asparagine (Asn331) of TRβ. The corresponding Arg282 of TRβ is observed in only one single stable conformation, interacting effectively with the ligand. Molecular dynamics support this model: our simulations show that the multiple conformations can be observed for the Arg228 in TRα, in which the ligand interacts either strongly with the ligand or with the Ser277 residue. In contrast, a single stable Arg282 conformation is observed for TRβ, in which it strongly interacts with both GC-1 and the Asn331. Conclusion Our analysis suggests that the key factors for GC-1 selectivity are the presence of an oxyacetic acid ester oxygen and the absence of the amino group relative to T3. These results shed light into the β-selectivity of GC-1 and may assist the development of new compounds with potential as drug candidates to the treatment of hypercholesterolemia and obesity.

Thermophilic endo-1,3(4)-beta glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and contain two protein molecules in the asymmetric unit with a solvent content of 51.75 %. Diffraction data were collected to a resolution of 1.95A and resulted in a dataset with an overall R(merge) of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.

The rat protein tyrosine phosphatase eta, rPTPeta, is a class I "classical" transmembrane RPTP, with an intracellular portion composed of a unique catalytic region. The rPTPeta and the human homolog DEP-1 are downregulated in rat and human neoplastic cells, respectively. However, the malignant phenotype is reverted after exogenous reconstitution of rPTPeta, suggesting that its function restoration could be an important tool for gene therapy of human cancers. Using small-angle x-ray scattering (SAXS) and biophysical techniques, we characterized the intracellular catalytic domain of rat protein tyrosine phosphatase eta (rPTPetaCD) in solution. The protein forms dimers in solution as confirmed by SAXS data analysis. The SAXS data also indicated that rPTPetaCD dimers are elongated and have an average radius of gyration of 2.65 nm and a D(max) of 8.5 nm. To further study the rPTPetaCD conformation in solution, we built rPTPetaCD homology models using as scaffolds the crystallographic structures of RPTPalpha-D1 and RPTPmicro-D1 dimers. These models were, then, superimposed onto ab initio low-resolution SAXS structures. The structural comparisons and sequence alignment analysis of the putative dimerization interfaces provide support to the notion that the rPTPetaCD dimer architecture is more closely related to the crystal structure of autoinhibitory RPTPalpha-D1 dimer than to the dimeric arrangement exemplified by RPTPmicro-D1. Finally, the characterization of rPTPetaCD by fluorescence anisotropy measurements demonstrates that the dimer dissociation is concentration dependent with a dissociation constant of 21.6 +/- 2.0 microM.

Os hormônios tireoidianos estão envolvidos em vários efeitos regulatórios, em órgãos diversos. Suas variações no organismo estão relacionadas a quadros clínicos de grande relevância. A presente dissertação trata do estudo de duas proteínas diretamente relacionadas ao complexo sistema regulatório associado a tais hormônios. A primeira delas é a globulina de ligação à tiroxina (TBG), responsável pelo transporte da grande maioria dos hormônios tireoidianos circulantes, e cujas alterações estão relacionadas a falhas na interpretação de testes de avaliação da função tireoidiana, podendo levar a tratamentos desnecessários. A segunda proteína é o receptor tireoidiano (TR), responsável pela mediação dos efeitos regulatórios do hormônio tireoidiano, tendo sua estimulação de atividade transcricional relacionada à ligação do hormônio em um de seus domínios. A TBG foi estudada através da técnica computacional conhecida como modelagem molecular por homologia, aplicada à proteína em sua forma selvagem e a mutantes observados no Brasil, com o intuito de relacionar a inviabilidade de tais mutantes a aspectos estruturais. Foi proposto que, para dois dos mutantes estudados, a formação das estruturas secundárias como na forma nativa da proteína seria inviável, enquanto que para o terceiro mutante a inviabilidade poderia ser causada por enovelamento incorreto causado por uma possível interação entre um resíduo de cisteína adveniente da mutação e outros resíduos do mesmo aminoácido em posição fisicamente próxima. O estudo do TR teve como base as estruturas cristalográficas das duas isoformas humanas do receptor (hTR α e hTR β) quando ligadas ao tiromimético GC-1. Esse composto tem a propriedade de ligar-se preferencialmente à isoforma β, o que pode ter interessantes aplicações farmacológicas. A análise comparativa da ligação do GC-1 às duas isoformas mostrou que, para tal composto, a seletividade se deve a mudanças consideráveis no modo de ligação ao hTR α e hTR β. Para a isoforma , há dois modos de ligação, envolvendo conformações alternativas de ligante e proteína, onde em uma delas a ligação é mais favorável e semelhante à ligação do composto à isoforma , enquanto no outro modo de ligação há a perda de uma interação direta entre composto e proteína, explicando a mais baixa afinidade do GC-1 à isoforma quando comparado à isoforma . O mecanismo de -seletividade para esse composto está relacionado a um átomo de oxigênio específico que não existe no ligante natural do receptor, o que fornece úteis informações para a criação de novos compostos. The thyroid hormones are involved in various regulatory effects, on diverse organs. Their fluctuations on the body are related to clinical scenarios of great relevance. This work deals with the study of two proteins which are directly related to the regulatory system associated to these hormones. The first one is the thyroxine-binding globulin (TBG), responsible for the transport of a large part of the thyroid hormones in serum, and whose variations are related to misinterpretation of thyroid function tests. The second protein is the thyroid receptor (TR), responsible for the mediation of the thyroid hormone regulatory effects . the transcriptional activity being related to ligand binding to one of the protein domains. The wild-type TBG and three mutants discovered in Brazil were studied by the computational technique known as homology modeling. The purpose of this investigation was to relate protein unviability to structural aspects. It was proposed that, for two mutants, the unviability was related to the impossibility of secondary structure formation as needed to form the native folding, while the third mutant the cause could be the formation of an incorrect folding due to possible interactions involving a cysteine residue created by the mutation and other nearby cysteine residues. The thyroid receptor was studied in the light of the x-ray structures of the two isoforms of the protein (hTR α and hTRβ) bound to GC-1, a synthesized compound which resembles the thyroid hormones. This ligand binds preferably the isoform, a feature that may have interesting pharmacological applications. The comparative analysis of GC-1 binding to the two isoforms allowed the construction of a structural basis of its -selectivity property, which is due to considerable differences in the binding modes for the two isoforms. This involves two different configurations of ligand and protein conformations for the isoform - on one of them, the ligand docks to the molecule the same way it docks to hTRβ, while on the second configuration it loses one direct interaction to the protein, explaining its lower affinity to hTR α when compared to hTRβ. The -selectivity mechanism for this compound is related to a specific oxygen atom which doesn?t exist on the receptor endogenous ligand, providing useful information for the development of new compounds.

Background Nuclear receptors (NRs) are transcription factors which bind small hormones, whose evolutionary history and the presence of different functional surfaces makes them an interesting target for a correlation based analysis. Results Correlation analysis of ligand binding domains shows that correlated residue subsets arise from the differences between functional sites in different nuclear receptor subfamilies. For the DNA binding domain, particularly, the analysis shows that the main source of correlation comes from residues that regulate hormone response element specificity, and one of the conserved residue sub-sets arises due to the presence of an unusual sequence for the DNA binding motif known as P-box in nematodes, suggesting the existence of different DBD-DNA specificities in nuclear receptors. Conclusions We conclude that DNA specificity and functional surface specialization has independently driven nuclear receptor evolution, and suggest possible binding modes for the class of divergent nuclear receptors in nematodes.

The DBWS program, the latest version of which is 9807a (Young et al., 1999), an upgraded version of DBWS-9411, described by Young et al. (1995), is written in Fortran and can carry out Rietveld re®nements using digitized data (from a neutron or X-ray diffractometer) stored in a text ®le. It also requires an Input Control File (ICF), constructed by the user, which stores information about the material and program execution. The ICF is a text ®le that contains crystallographic information about the material and program settings in lines and columns. Given the size of the input ®le needed by DBWS, compiling it can be confusing, dif®cult and time consuming (an ICF for quartz plus alumina, which has only two phases, uses an ICF with more than 60 lines). Most users edit new ICFs by opening previously used ®les in a non-formatted text editor (such as Microsoft Notepad or DOS Edit) and then substituting the old values for new ones. Editing these ®les manually can be confusing because the user needs to know the placement of each parameter in the ICF; furthermore, small errors in the format of the ICF may cause problems when the program runs. After the program has run, a text ®le containing the re®ned data is returned, along with, optionally, a binary ®le that is used to plot the diffraction pattern using a graphics program such as DMPlot (Marciniak, 1997). To solve the aforementioned problems, a graphical interface for DBWS (which works with versions 9411 and 9807 and runs under Windows 95/98/NT) has been developed. Known as DBWS Tools, the program makes DBWS much easier to use and manages each step of the process, from the creation of the ICF to the plotting of the diffraction pattern using the external program DMPlot. In this way, the user need not consult the program manual every time a new sample is analysed (because the meaning of each data ®eld is shown on screen) and the entire process is much faster and user-friendly.

Como ferramenta auxiliar no ensino de Física, é apresentada a utilização do software de computação simbólica Mathematica, através dos seus recursos de programação analítica e multimídia. É possível verificar as relações de freqüências numa escala musical e o efeito do batimento, através de análise matemática e reprodução sonora via computador. Para fins ilustrativos e pedagógicos, são simulados no computador efeitos sonoros comumente utilizados por músicos, como Tremolo, Phaser e Auto-Wah. Estes são modelados matematicamente de forma simplificada, tomando-se como base uma onda sonora padrão, cujos parâmetros associados foram obtidos experimentalmente. Ressalta-se a possibilidade do aprendizado autônomo. As an auxiliary tool in Physics teaching, the utilization of analytical programming and multimedia facilities of the symbolic computing software Mathematica is shown. It is possible to verify the frequency ratios in a musical scale and the beat effect by mathematical analysis and audible reproduction by the computer. For illustrative and pedagogical purposes, sound effects commonly used by musicians, such as Tremolo, Phaser and Auto-Wah are simulated. A simplified mathematical model is defined for them using a standard sound wave whose parameters were experimentally obtained. The possibility of self-teaching is evident.

O processo de descoberta de fármacos consiste em diversas etapas e a utilização de ferramentas computacionais pode ser parte essencial, em especial, durante os primeiros estágios da pesquisa. É possível, por exemplo, o estudo de grande número de moléculas a partir de propriedades fisico-químicas ou mesmo prevendo o possível modo e energia de ligação entre duas ou mais moléculas. O EasyVS é uma ferramenta WEB desenvolvida para simplificar o processo descrito anteriormente a partir da seleção de compostos e bibliotecas de compostos e triagem virtual de ligantes. Com uma interface intuitiva, a ferramenta permite aos usuários ir desde a seleção de uma proteína-alvo com estrutura conhecida, passando pela parametrização do docking até sua execução e visualização de resultados, em poucos clicks. O EasyVS também permite aos usuários a escolha dentre mais de 16 milhões de moléculas através de filtros baseados nas propriedades moleculares, assim como o agrupamento considerando índice de similaridade de Tanimoto. Essa ferramenta foi utilizada, em um estudo de caso, para a triagem virtual de ligantes para as GPCR (receptores acoplados a proteínas G) e apresentou resultados satisfatórios, indicando moléculas já conhecidas como ligantes, separandoas de decoys, assim como novos potenciais ligantes para serem estudados. Esse estudo mostrou-se relevante pois as GPCRs são consideradas a maior família de alvos para fármacos aprovados. Pretende-se aumentar a capacidade de processamento do servidor disponível para o EasyVS a fim de diminuir o tempo de computação das requisições, além de viabilizar novas funcionalidades para o EasyVS. The drug discovery process consists of several steps and the use of computational tools can be an essential part, especially during the early research stages. It is possible, for example, to study large numbers of molecules from various compound libraries from physico-chemical properties or even by predicting the mode and energy of binding between two or more molecules. EasyVS is a web tool developed to simplify the previously described process from the selection of compounds libraries and virtual screening of ligands. With an intuitive interface, this tool allows users to go from the selection of a protein target with known structure, through docking parameterization to its execution and results visualization, in a few clicks. EasyVS also allows users to choose from more than 16 million molecules through filters based on molecular properties, as well as the grouping through Tanimoto’s similarity index. This tool was used, in a case study, for the virtual screening of ligands for GPCR (G protein coupled receptors) and presented satisfactory results, indicating molecules already known as ligands, separating them from decoys, as well as new potential ligands to be studied. This study proved to be relevant since GPCRs are considered the largest family of targets for approved drugs. We intend to increase the processing capacity of the server availableforEasyVSinordertoreducethecomputationtimeoftherequisitions,besides making viable new functionalities for EasyVS.

Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following the overwhelming evidence that NaV channel subtypes NaV1.7- NaV1.9 are involved in nociception (Ahern et al., 2016; Luiz and Wood, 2016). More recently, also NaV1.1, NaV1.3 and NaV1.6 have been identified to be involved in pain pathways. Venom-derived disulphide-rich peptide toxins (1580 amino acids), isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads for pharmaceutical development (Schroeder et al., 2012; Saez et al., 2010). However, few peptide drug leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics, rapid proteolytic cleavage and limited oral bioavailability. This project aims to bridge the gap in the development pipeline between drug leads and drugs candidates by downsizing these larger venom-derived NaV inhibitors into smaller, drug-like molecules. As a first step, we searched the venom of the spider Phoneutria nigriventer for promising NaV channel ligands. A first toxin sparking our interest was the toxin PnTx2-1. Based on the in vivo tests in mice and the sequence identity with PnTx2-5 and PnTx2-6, this peptide is classified as a NaV channel toxin. However, it has never been tested on any NaV channel isoform. Therefore, in this study the NaV channel subtype selectivity and species specificity of PnTx2-1 are investigated. We performed an in-depth functional characterization of the NaV channel modulating properties of PnTx2-1. Furthermore, analysis of the activity of PnTx2-1 on NaV1.5 reveals that this Phoneutria toxin modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current. The obtained results allowed to conclude that PnTx2-1 is an interesting insecticidal peptide but 5 it does not represent a promising lead compound for the development of novel NaV channel ligands with therapeutic potential. Another toxin potentially interesting as a NaV channel modulating ligand is the toxin PnTx1. PnTx1 was found to be the most interesting NaV channel toxin, considering its action in NaVs related to pain (Silva et al., 2012a). Following identification of common sequence motifs from NaV inhibitors PnTx1 (Diniz et al., 2006a) (78 residues, 14 cysteine) and -conotoxin KIIIA (Bulaj et al., 2005) (16 residue, 6 cysteine) from cone snail Conus kinoshitai, we have produced the smallest cyclic peptide-based NaV channel inhibitor known to date (10 residues, 2 cysteines) with demonstrated subtype selectivity across NaV channel subtypes including NaV1.7 and NaV1.9 (Luiz and Wood, 2016). The methodology used in this project, involved a two-pronged approach that incorporates sophisticated peptide engineering carrying out several rounds of small cyclic peptide design to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes. This allowed us to design small, stable and novel NaV probes with high subtype selectivity and potency coupled with improved biopharmaceutical properties, rendering these peptides suitable as analgesic drug candidates.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior β-glicosidases (EC 3.2.1.21) são enzimas chave no processo de produção de biocombustíveis de segunda geração. Elas agem em conjunto com endoglucanases e exoglucanases na conversão de biomassa em açúcares fermentáveis. Entretanto, a maior parte das β-glicosidases conhecidas são altamente inibidas por grandes concentrações de glicose. Assim, a busca por mutações que melhorem a atividade de β-glicosidases não tolerantes a inibição por glicose é de grande importância para a indústria. Nesta tese, é apresentada uma revisão sistemática da literatura para coletar informações sobre β-glicosidases glicose-tolerantes e construir uma base de dados, denominada BETAGDB. Além disso, são caracterizados resíduos importantes no bolsão catalítico para atividade e glicose-tolerância. Por fim, apresenta-se um método baseado na diferença de variação de assinaturas estruturais para propor mutações em enzimas, denominado SSV (Structural Signature Variation). SSV usa modelagem em grafos para criar uma assinatura estrutural identificadora de β-glicosidases glicose-tolerantes. O método SSV foi avaliado em três estudos de caso: (i) 27 mutações descritas na literatura foram manual-mente classificadas como benéficas ou não. A seguir, a classificação foi reproduzida utilizando SSV. O método obteve uma acurácia de 0,74 e uma precisão de 0,89; (ii) 18 mutações benéficas foram propostas para a β-glicosidase não tolerante Bgl1B. Resultados experimen-tais de três mutações corroboram os resultados obtidos pelo segundo estudo de caso e demonstram que SSV é um método eficaz para proposta de mutações em β-glicosidases; e (iii) comparou-se SSV com SVM para verificar se a distância euclidiana, métrica usada por SSV para comparação de assinaturas, era eficaz. Comparou-se ainda com BioGPS, um método que usa fingerprints para proposta de mutações baseadas em estruturas tridimensionais. SSV obteve valores de precisão e especificidade superiores a SVM. Na comparação com BioGPS, SSV foi capaz de predizer corretamente cinco em sete mutações validadas em bancada que inseriam atividade amidase em uma lipase. Os resultados obtidos nesta tese podem ajudar na produção de enzimas β-glicosidases mutantes capazes de aperfeiçoar a produção de biocombustíveis de segunda geração. O método SSV pode ser estendido a outras enzimas e pode ainda ser utilizado em conjunto com outras estratégias e ferramentas para propor mutações mais eficientes. SSV está disponível em: . β-glucosidases (EC 3.2.1.21) are key enzymes in the second-generation biofuel production. They act synergically with endoglucanases and exoglucanases in the conversion of biomass to fermentable sugars. However, most known β-glucosidases are highly inhibited by high glucose concentrations. Hence, the search for mutations that improve the activity of non-tolerant β-glucosidases has great importance to the industry. In this thesis, I present a systematic review of the literature to collect information about glucose-tolerant β-glucosidases and to construct a database, called BETAGDB. In addition, important residues for the activity and glucose-tolerance were characterized in the catalytic pocket. Finally, I proposed a method based on the difference of variation of structural signatures to propose mutations in enzymes, called Structural Signature Variation (SSV). SSV uses graph modeling to create a structural signature that identifies glucose-tolerant β-glucosidases. The SSV method was evaluated in three case studies: (i) 27 mutations described in the literature were manually classified as beneficial or not. The classification was then reproduced using SSV. The method obtained an accuracy of 0.74 and a precision of 0.89; (ii) 18 beneficial mutations were proposed for the non-tolerant β-glucosidase Bgl1B. Experimental results of three mutations corroborate the outcomes obtained by the second case study and demonstrate that SSV is an effective method for the proposal of mutations in β-glucosidases; and (iii) SSV was compared with SVM to verify whether the Euclidean distance, metric used by SSV for comparison of signatures, was effective. It was also compared with BioGPS, a method that uses fingerprints to propose mutations based on three-dimensional structures. SSV obtained values of precision and specificity superior to SVM. In comparison to BioGPS, SSV was able to correctly predict five in seven bench-validated mutations inserted amidase activity into a lipase. The results obtained in this thesis may aid in the production of mutant β-glucosidase enzymes capable of enhancing the production of second-generation biofuels. The SSV method can be extended to other enzymes and can also be used together to other strategies and tools to propose more efficient mutations. SSV is available at .

Molecular Biology is a branch within Science of great importance. Despite the fact it studies microscopic entities, the volume and complexity of information are great. The applications are varied and can be of global interest, such as the spread of antibiotic resistance genes among bacteria and new methods for diagnostic and prognostic of cancer. By understanding biomolecular mechanisms, scientists can define treatments for diseases, support the decisions made by patients, identify the influence of intestinal microbiota over physical and psychological conditions, find cause and source of microbial antibiotic resistance, among many other applications. Computer Science plays key roles in this context, such as enabling complex data analyzes by specialists, creating models that simulate biological structures and processes, and by providing algorithms for extracting information encoded in biological data. During my doctorate, we explored those mechanisms in three main levels: quantification of proteins from cells, analysis of interactions that happen inside cells, and the comparison of genomes and their genetic history. This manuscript reports different projects, four of them already published in scientific journals. They comprise the discovery of candidate proteins for cancer biomarkers, the visual analysis of protein-protein interaction networks and the visual analysis of lateral gene transfer in bacterial phylogenetic trees. Here, we explain these projects and the main findings associated with the use of computational methods. Among the results are the evaluation of stability of ranking and signature methods applied to discovery proteomics data, a new approach to select candidate proteins from discovery to targeted proteomics, lists of candidate biomarkers for oral cancer, and new techniques for the visualization of biological networks and phylogenetic supertrees. A Biologia Molecular é um ramo da Ciência de grande importância. Apesar de estudar entidades microscópicas, o volume e a complexidade das informações são imensos. Suas aplicações são variadas e podem ser de interesse global, como a disseminação de genes de resistência a antibióticos entre bactérias e novos métodos para diagnóstico e prognóstico de câncer. Entendendo os mecanismos biomoleculares, cientistas podem definir tratamentos para doenças, apoiar as decisões tomadas pelos pacientes, identificar a influência da microbiota intestinal sobre as condições físicas e psicológicas, encontrar causas e fontes de resistência microbiana aos antibióticos, entre muitas outras aplicações. A Ciência da Computação desempenha papéis-chave nesse contexto, como permitir análises complexas de dados por especialistas, criar modelos que simulam estruturas e processos biológicos e fornecer algoritmos para extrair informações codificadas em dados biológicos. Durante meu doutorado, exploramos esses mecanismos em três níveis principais: quantificação de proteínas a partir de células, análise de interações que ocorrem dentro das células e comparação de genomas e seus históricos. Este manuscrito relata diferentes projetos, quatro deles já publicados em revistas científicas. Eles compreendem a descoberta de proteínas candidatas a biomarcadores de câncer, a análise visual de redes de interação proteína-proteína e a análise visual da transferência lateral de genes em árvores filogenéticas bacterianas. Aqui, explicamos esses projetos e as principais descobertas associadas ao uso de métodos computacionais. Entre os resultados estão a avaliação da estabilidade dos métodos de ranqueamento e assinaturas aplicados aos dados proteômicos de descoberta, uma nova abordagem para selecionar proteínas candidatas desde a descoberta até proteômica direcionada, listas de candidatos a biomarcadores para câncer oral e novas técnicas para a visualização de redes biológicas e supertrees filogenéticas.

As proteínas evoluem ao longo do tempo, devido ao acúmulo de mutações, inserções e deleções nos genes que as codificam. Posições fortemente conservadas geralmente referem-se a resíduos que são estritamente necessários para a estrutura da proteína ou para sua função. A metodologia DRCN (Decomposition of Residue Coevolution Networks) permite estudar coevolução de resíduos em famílias de proteínas. Para tanto, consiste em cinco etapas básicas: filtragem do alinhamento, cálculo de correlações, decomposição da rede, geração de arquivos de visualização auxiliares e anotação de posições por busca automática no UniProt. As cisteíno proteases são um grande e diverso grupo de enzimas que pertencem ao clã CA e à família C1 (família da papaína) e podem ser encontradas em todos os animais e reinos de plantas bem como em muitos vírus e organismos procariotas. O presente trabalho tem como objetivo analisar e discutir a importância dos resíduos correlacionados e conservados da família das cisteíno proteases a partir do alinhamento múltiplo de sequências. Para tanto utilizamos, o software PFSTATS, que emprega a metodologia DRCN. Foram encontrados 34 resíduos correlacionados dispostos em 5 comunidades de resíduos que coevoluem. A comunidade 1 é a maior comunidade, sendo formada por 22 resíduos que coevoluem tanto por questões estruturais, como por exemplo Cys22 e Cys63 e Cys56 e Cys101, que realizam pontes dissulfeto; quanto por questões funcionais, como Pro2, Phe28, Gly35 e Tyr89, que se localizam em sítios alostéricos preditos da cruzaína. A comunidade 2 é composta por 6 resíduos, dentre eles resíduos da díade catalítica, Cys25 e His162, e resíduos Gly23 e Gly65, que coevoluem por questões estruturais. As comunidades 3, 4 e 5 possuem apenas 2 resíduos cada. O resíduo Cys155 da comunidade 3 possui importância estrutural, bem como os resíduos Val13 e Lys181 (comunidade 4) e Gln51 (comunidade 5). Os resíduos da comunidade 5, Leu48 e Gln51 possuem importância funcional e o resíduo Ser55 (comunidade 3) ainda não apresenta sua função descrita na literatura. Também foram encontrados 10 resíduos que são altamente conservados evolutivamente nessa família. Enquanto alguns resíduos, incluindo os que compõem a díade catalítica (Cys25 e His162), são conservados por questões funcionais, outros o são por questões estruturais (Ser49, Gly168, Gly189 e Gly192). Proteins evolve over time, due to the accumulation of mutations, insertions and deletions in the genes that encode them. Highly conserved positions generally refer to residues that are strictly necessary for the structure of the protein or for its function. The DRCN (Decomposition of Residue Coevolution Networks) methodology allows the study of coevolution of residues in protein families. To do this, it consists of five basic steps: filtering the alignment, calculating correlations, decomposing the network, generating auxiliary visualization files and annotating positions by automatic search in UniProt. Cysteine proteases are a large and diverse group of enzymes belonging to the CA clan and the C1 family (papain family) and can be found in all animals and plant kingdoms as well as in many viruses and prokaryotes. The present work aims to analyze and discuss the importance of correlated and conserved residues of the cysteine protease family from the multiple sequence alignment. For this, we use the PFSTATS software, which employs the DRCN methodology. There were 34 correlated residues found in 5 coevoluted communities. Community 1 is the largest community, consisting of 22 residues coevolved by structural issues, such as Cys22 and Cys63 and Cys56 and Cys101, which carry disulfide bridges; as well as for functional issues, such as Pro2, Phe28, Gly35 and Tyr89, which are located in predicted allosteric sites of cruzain. Community 2 is composed of 6 residues, among them residues of the catalytic dyad, Cys25 and His162, and residues Gly23 and Gly65, which coevolve for structural reasons. Communities 3, 4 and 5 have only 2 residues each. The Cys155 residue of community 3 has structural importance, as well as residues Val13 and Lys181 (community 4) and Gln51 (community 5). The residues of community 5, Leu48 and Gln51 have functional importance and the residue Ser55 (community 3) still does not present its function described in the literature. Also 10 residues were found that are highly conserved evolutionarily in this family. While some residues, including those that make up the catalytic dyad (Cys25 and His162), are conserved for functional reasons, others are for structural reasons (Ser49, Gly168, Gly189 and Gly192).

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico Os Hidrocarbonetos Aromáticos Policíclicos (HAPs), compostos orgânicos formados por anéis benzênicos fusionados, são contaminantes ambientais que ocorrem naturalmente durante a combustão de matéria orgânica como também pelo derramamento de petróleo e outros processos industriais. Existe uma grande diversidade de bactérias capazes de degradar os HAPs e as enzimas presentes na vias metabólicas podem ser utilizadas para remediar esses poluentes. O naftaleno é um dos HAPs mais tóxicos sendo utilizado como modelo no estudo de degradação desses compostos e, em P. putida G7 o metabolismo do naftaleno é um processo bem estudado. O complexo enzimático da naftaleno dioxigenase (NahA) atua na primeira reação da via de degradação desse contaminante e a NahA pertence a uma grande família de Rieske oxigenases que iniciam a degradação de diversos compostos aromáticos. Todavia, a busca por potenciais enzimas envolvidas na degradação de HAPs não está limitada à P. putida G7, sendo que diferentes fontes validadas podem ser incluídas para enriquecer os bancos de dados. Neste trabalho foi realizada a busca por proteínas homólogas a NahA com o intuito de se obter um complexo dioxigenase que atue sobre vários substratos aromáticos. As sequências gênicas das proteínas de Achromobacter xylosoxidans A8, denominadas de Aa, Ab, Ac e Ad foram selecionadas a partir de bases genômicas e metagenômicas de diferentes organismos. As proteínas recombinantes foram expressas separadamente em três componentes (Aa, Ab e AcAd) bem como co-expressas em conjunto e após a expressão, as células foram submetidas a lise celular para a liberação das proteínas e o extrato resultante foi imediatamente utilizado para a purificação por cromatografias de afinidade e de exclusão molecular. O processo de purificação com o sistema contendo o complexo dioxigenase (AaAbAcAd) foi o mais satisfatório e por isso, decidiu-se dar prosseguimento com essa amostra. As proteínas recombinantes foram identificadas por western blotting e por espectrometria de massa. Através dos ensaios de ensaios de espalhamento dinâmico de luz (DLS) as proteínas apresentaram-se, em solução, como monodispersas sendo Aa em forma dimérica e AcAd como hexamérica. A atividade enzimática do complexo dioxigenase foi demonstrada pelo consumo de NADH. A estrutura cristalográfica contendo as proteínas AcAd, foi resolvida a 1,35 Å e o modelo cristalográfico apresentou enovelamento α/β típico das Rieske oxigenases, com os domínios Rieske [2Fe-2S] e catalítico na subunidade alfa bem definidos, com exceção da presença do átomo de manganês mononuclear no sítio ativo, ao invés do ferro, metal conservado para as dioxigenases. Na unidade assimétrica, a molécula foi encontrada na forma de heterodímero αβ e através de operações de simetria cristalográfica observou-se a unidade biológica, na forma de heterohexâmero (α3β3). Os resíduos Cys-104, His-106, Cys-124 e His-127 no domínio Rieske e os resíduos His-232, His-237 e Asp-379 no domínio catalítico são conservados para todas as Rieske oxigenases sendo importantes para a catálise. Os resíduos não conservados presentes no sítio catalítico Leu-325, His-323, Val-375 e Ile-369 se mostraram importantes, podendo contribuir para a especificidade e acomodação de substratos. No modelo cristalográfico contendo o catecol, este apresentou-se ligado ao sítio ativo, interagindo com a Asn-226. Os resultados deste trabalho apresentam pela primeira vez estudos sobre as características estruturais em relação ao componente catalítico do complexo dioxigenase de Achromobacter xylosoxidans A8, os quais contribuem para estudos futuros sobre a identificação de resíduos-chave para catálise sobre diversos substratos. Polycyclic Aromatic Hydrocarbons (PAHs), organic compounds formed by fused benzene rings, are environmental contaminants that occur naturally during the combustion of organic matter as well as by oils spills and other industrial processes. There is a great diversity of bacteria capable of degrading the PAHs and the enzymes present in the metabolic pathways can be used for the bioremediation of these pollutants. Naphthalene is one of the most toxic HAPs being used as a model in the degradation study of these compounds and in P. putida G7 the metabolism of naphthalene is a well-studied process. The enzymatic complex of naphthalene dioxygenase (NahA) acts in the first reaction of the degradation pathway of this contaminant and NahA is a member of a large family of Rieske oxygenases which initiates the degradation of various aromatic compounds. However, the search for potential enzymes involved in the degradation of PAHs is not limited to P. putida G7, and different validated sources can be included to enrich the databases. In this work the search for proteins homologous to NahA was carried out in order to obtain a dioxygenase complex that could act on several aromatic substrates. The gene sequences of the dioxygenase complex proteins Aa, Ab, Ac and Ad from Achromobacter xylosoxidans A8 were selected from genomic and metagenomics databases of different organisms. Recombinants proteins were expressed separately in three components (Aa, Ab and AcAd) as well as co-expressed together and after the expression, cells were lysed for protein release and the resulting extract was immediately subjected to purification by affinity chromatography followed by a size exclusion chromatography. The purification process with the system containing the dioxygenase complex (AaAbAcAd) was the most satisfactory and therefore it was decided to proceed with this sample. Recombinant proteins were identified by western blotting and mass spectrometry. Dynamic light scattering (DLS) assays with the samples showed the proteins presented in solution as monodisperse, with Aa as monomeric and AcAd as hexameric forms.The enzymatic activity of the complex was measured by the NADH consumption. Crystallographic structure of the AcAd proteins was solved at 1.35 Å and the crystallographic model presented an α/β folding typical of the Rieske oxygenases, with the well-defined Rieske [2Fe-2S] and catalytic domains in the alpha subunit, except for the presence of the mononuclear manganese in the active site, rather than iron, a conserved metal of the dioxygenases. In the asymmetric unit, the molecule was found in the form of αβ heterodimer and with crystallographic symmetry operations the biological unit was observed, in the form of heterohexamer (α3β3). Cys-104, His-106, Cys-124 and His-127 residues in the Rieske domain and His-232, His-237 and Asp-379 in the catalytic domain are conserved residues for all Rieske oxygenases being important for catalysis. The non-conserved residues present in the catalytic site Leu-325, His-323, Val-375 and Ile-369 proved to be important and could contribute to the specificity and accommodation of substrates. In the crystallographic model containing catechol, it was bound to the active site, interacting with Asn-226. The results of this work present, for the first time, studies of structural characteristics concerning the catalytic component of the dioxygenase complex from Achromobacter xylosoxidans A8, which can contribute for future studies on the identification of key residues for catalysis on several substrates.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Voltage-gated sodium channels (VGSD) são proteínas responsáveis pela propagação da informação entre neurônios, fibras musculares esqueléticas, fibras musculares cardíacas e outros tipos de células excitáveis. Eles são rapidamente ativados quando a membrana celular é despolarizada, seguido de inativação, o que leva a um influxo de sódio transiente. Até recentemente não existia um modelo estrutural completo do canal de sódio humano. Neste trabalho, realizamos a modelagem comparativa do canal hNav1.4, usando como template o canal de cálcio de coelho (Cav1.1), canal de sódio de barata (NavPaS) e o canal de sódio de enguia elétrica (EeNav1.4), com os programas Rosetta e SWISS-MODEL. Os modelos gerados foram designados hNav1.4/Cav, hNav1.4/PaS e hNav1.4/Ee, conforme o templete usado. Estes modelos foram analisados de duas formas: na fidelidade do seu enovelamento em relação ao seu template, usando a ferramenta TM-Align; e pelos ângulos das ligações peptídicas entre os resíduos, utilizando o gráfico de Ramachandran. Todos os modelos foram considerados bem sucedidos pelos gráficos de Ramachandran e pelo TM-Align. Uma análise mais apurada das estruturas mostrou algumas incongruências no modelo hNav1.4/Cav ao compararmos com o funcionamento dos canais de sódio, tornando o modelo inadequado. O poro do canal se mostra fechado e seus domínios sensíveis a voltagem (VSD) se encontram ativados, o que torna este modelo falho em relação às propriedades funcionais do canal. Os modelos hNav1.4/PaS e hNav1.4/Ee, por outro lado, representam estados conformacionais fechado e aberto, respectivamente, e se mostraram consistentes com os dados biofísicos e farmacológicos conhecidos. Como critério para identificar em qual estado conformacional os canais se encontram, foi realizada a análise dos segmentos S4, e sua posição em relação à constrição hidrofóbica, bem como a verificação das interações dos resíduos carregados com resíduos de aminoácidos dos segmentos S1, S2 e S3. Considerando este critério, foi possível a contagem das cargas elementares necessárias para ativar o canal hNav1.4, chegando-se a um valor aproximado de 6,5. Completando a análise das características dos canais, foi realizado o docking de alfae beta-toxinas de escorpião no canal. A beta-toxina Ts1, se ligou ao sítio 4 do canal hNav1.4/PaS, mostrando alta afinidade com energia livre de ligação (ΔG) de -9.4 kcal/mol, confirmando que o segmento S4 do domínio DII se encontra na posição ativada, embora o canal esteja fechado, conforme análise feita pelo programa MOLEonline. A alfa-toxina AaHII se ligou ao sítio 3 do modelo hNav1.4/PaS e teve sua energia livre de ligação em -10.7 kcal/mol. Um novo docking foi testado com o modelo estado aberto, hNav1.4/Ee, e não obteve sucesso. Este resultado confirma resultados experimentais que comprovam a incapacidade da toxina de se ligar ao canal quando o VSDIV está ativado. Uma análise mais profunda mostrou que o resíduo D1435 do canal, tido como de grande importância para a interação toxina-canal, se encontra em uma posição não favorável para ligação da toxina. Esta mudança é causada pelo movimento do segmento S4 que, ao se mover em direção ao meio externo durante sua ativação, provoca no segmento S3 um movimento para dentro seguido por um giro, colocando o resíduo D1435 na posição desfavorável para a ligação da toxina. Essa observação permite esclarecer uma característica das alfa-toxinas, que é o seu deslocamento do canal quando pulsos altamente despolarizantes são aplicados à membrana. Os resultados apresentados pelo docking das toxinas são compatíveis com os resultados obtidos experimentalmente. Nossos resultados mostram que estes modelos teóricos são capazes de fornecer as bases estruturais para aspectos fisiológicos e farmacológicos desses canais, possibilitando assim uma nova gama de pesquisas teóricas envolvendo o canal hNav1.4 em diferentes estados conformacionais. Voltage-gated sodium channels (VGSD) are proteins responsible for the propagation of information between neurons, skeletal muscle fibers, cardiac muscle fibers and other types of excitable cells. They are rapidly activated when the cell membrane is depolarized, followed by inactivation, which leads to a transient sodium influx. Until recently there was no complete structural model of the human sodium channel. In this work, we performed the comparative modeling of the hNav1.4 channel, using as template the rabbit calcium channel (Cav1.1), cockroach sodium channel (NavPaS) and the electric eel sodium channel (EeNav1.4), with the Rosetta and SWISS-MODEL programs. These models were analyzed in two ways: the fidelity of their folding in relation to their templates using the TM-Align tool, and by angles of the peptide bonds between the amino acid residues, using the Ramachandran plot. All models were considered successful based on these criteria. More accurate analysis of the structures showed that the hNav1.4/Cav model has inconsistencies when compared to the functioning of a sodium channel, making the model inappropriate. The pore of the channel is closed and its voltage sensitive domains (VSD) are activated, which makes this model faulty in relation to the functional properties of the channel. On the other hand, the hNav1.4/PaS and hNav1.4/Ee models were able to represent closed and open conformational states, respectively, and were consistent with known biophysical and pharmacological data. As a criterion to identify the conformational state the channels, the analysis of the segments S4 was performed checking its position in relation to the hydrophobic constriction and the interactions of the charged residues with segments S1, S2 and S3. Considering this criterion, it was possible to count the elementary charges necessary to activate the hNav1.4 channel, reaching an approximate value of 6.5. Completing the analysis of the channel characteristics, the docking of alpha- and beta-scorpion toxins to the canal was performed. Beta-toxin Ts1 bound to channel site 4 with high affinity and binding free energy (ΔG) of -9.4 kcal/mol, thus confirming that the segment S4 of the DII domain is in the activated position, although the pore of the channel is closed state (as shown by the analysis by the MOLEonline program). The alpha toxin AaHII bound to site 3 of the closed-state model hNav1.4/PaS with a free energy of binding of -10.7 kcal/mol. A new docking was tested with the open-state model, hNav1.4/Ee, without success. This result is in agreement with the toxin's ability to bind to the VSDIV of the channel, but not when it is activated. Further analysis showed that the D1435 residue of the channel, considered to be of great importance for the toxin-channel interaction, is at an unfavorable position for toxin binding. This change results from the outward movement of the S4 segment, which causes a rotation of S3, placing the D1435 in unfavorable position. This observation can account for one characteristic of the alpha-toxins, which is their displacement from the channel when strong depolarizing pulses are applied to the membrane. The results presented by the docking of the toxins can explain the results obtained experimentally. Our results show that these theoretical models may provide structural basis to understand the physiological and pharmacological characteristics of these channels, thus enabling a new range of theoretical researches involving the hNav1.4 channel at different conformational states.

Proteínas sinalizadoras membranares são amplamente usadas por bactérias para adequar seu metabolismo a estímulos ambientais. Elas possuem ainda um grande potencial de aplicação, como em técnicas para controle de infecções e o desenvolvimento de ferramentas biotecnológicas, como biossensores. Entretanto, pouco é conhecido sobre os mecanismos estruturais de transdução bacterianos. Em particular, pouco é descrito sobre proteínas sinalizadoras que possuem os domínios GGDEF e/ou EAL, responsáveis por desencadear a síntese, degradação ou captação da molécula c-di-GMP, um dos principais reguladores do metabolismo bacteriano. Por isso, esse estudo objetivou a caracterização de duas proteínas sinalizadoras transmembrana de Pseudomonas aeruginosa, codificadas nos genes PA0847 e PA3343 (HsbD), além de trazer análises por bioinformática de proteínas com domínios GGDEF e EAL consecutivos, comumente associados a membrana celular. É relatado que PA0847 e HsbD são ambas diguanilato ciclases (DGC), que impactam consideravelmente em fenótipos de P. aeruginosa, patógeno modelo de estudo e que infecta pacientes imunocomprometidos. Além disso, um estudo anterior mostrou a interação entre a porção citoplasmática de PA0847 e a região N-terminal de HsbD (HsbD-Nt), pela técnica de duplo híbrido. Nesse estudo, é mostrado que essa interação, associada à presença de um padrão conservado em HsbD-Nt, sugere a presença um sistema de controle específico de c-di-GMP. Ensaios in vitro de pulldown, termoforese e duplo híbrido, utilizando domínios isolados de PA0847, confirmaram a interação com HsbD-Nt e mostraram que a interação é dependente de todos os domínios de PA0847. Análises de massa molecular e de cinética enzimática mostraram que o HAMP eleva a atividade DGC e promove a formação de tetrâmeros. A presença de um transportador de sulfato compartilhando um operon com PA0847, associado a modelos computacionais da região periplasmática de PA0847, suportam o envolvimento de PA0847 na homeostase desse íon. Nesse estudo foram ainda identificados, por meio de análises de sequências, novos potenciais grupos de resíduos que estariam envolvidos na regulação de proteínas GGDEF-EAL. Análises adicionais de sequências e de estruturas indicaram também um mecanismo de dimerização alternativo para EALs, quando a região de dimerização está ausente. Estes resultados são inéditos e fornecem passos para uma melhor compreensão da sinalização bacteriana intermediada pelo c-di-GMP. Membrane signaling protein are widely used by bacteria to adequate their metabolism of environmental cues. They present a great potential of applicability, such as techniques to control bacterial infections and development of biotecnological devices, for example in biosensors. However, little is known regarding the mechanisms of signal transduction in bacteria. In particular, about receptors that posses GGDEF and/or EAL domains, performing synthesis, degradation or recognition of c-di-GMP, which is one of the major bacteria metabolism regulators. For this reason, the present study aimed characterize two important transmembrane signaling proteins from Pseudomonas aeruginosa, coded in genes PA0847 and PA3343 (HsbD) and performed bioinformatic analysis of proteins with GGDEF and EAL in tandem, usually associated with membrane. Previous studies indicated that PA0847 and HsbD are both diguanylate cyclases (DGCs) that significantly impair phenotypes of P. aeruginosa, which is a model organism and common cause of infection in immunocompromised patients. In the present study we have shown that this interaction associated with the presence of a wellconserved pattern from HsbD-Nt, suggests the presence a system used for specific downregulation of c-di-GMP. Furthermore, using isolated domains of PA0847 in thermophoresis, pull down and two-hybrid confirmed the interaction and showed that the full PA0847 cytoplasmic is necessary to interaction with HsbD. Additional amalysis of molecular mass and enzyme kinetic showed that HAMP increases DGC activity, alters the co-purification patern of c-di-GMP and promotes tetramers formation in PA0847 soluble constructs. The presence of sulfate transporter in a common operon with PA0847, associated with computation model of periplasmatic region of PA0847 supports the relation of this protein in sulfate homeostasis. We also identified using new residues groups copled that are potential play roles in GGDEF-EAL proteins. We found by stuctural analisys alternative mechanisms of dimerization in EAL domais that do not posses dimerization site conserved. These results have not been presented elsewhere and provide insights into the bacterial signaling of c-di-GMP pathways.

Corynebacterium diphtheriae (Cd) is a gram-positive human pathogen responsible for diphtheria infection and once regarded for high mortalities worldwide. The fatality gradually decreased with improved living standards and further alleviated when many immunization programs were introduced. However, numerous drug-resistant strains emerged recently that consequently decreased the efficiency of current therapeutics and vaccines, thereby obliging the scientific community to start investigating new therapeutic targets in pathogenic microorganisms. In 2014, our research group introduced the word panmodelome for the first time in the scientific world (Hassan et al., 2014). Inspired by panmodelomics approach, in this study, our group aimed to contribute including the prediction of modelome of thirteen C. diphtheriae strains, using the MHOLline workflow. Considering the quality of the models and using in-house scripts, a set of 465 conserved proteins were selected by combining the results of pangenomics based on core genome and core-modelome analyses. Further, using subtractive proteomics and modelomics approaches for target identification, a set of 23 proteins were selected as essential proteins for bacteria. Considering human as a host, 8 of these proteins (glpX, nusB, rpsH, hisE, smpB, bioB, DIP1084 and DIP0983) were seen as essential and non-host homologs. These proteins were subjected to virtual screening using three different compound libraries (extracted from ZINC database, plant-derived natural compounds, and Di-terpenoid Iso-steviol derivatives). The proposed drug molecules showing favorable interactions, lowered energy values and high complementarity with the predicted targets have also been reported in the present study. Our proposed approach expedites the rapid and efficient selection of C. diphtheriae putative proteins for developing a broad spectrum of novel drugs and vaccines because some of these targets have already been identified and validated in other organisms. Furthermore, we adopted a different approach using same number of genomes of C. diphtheriae to identify drug/vaccine targets based on the druggable pocketome. As a result, we identify 10 targets in which interestingly 3 (hisE-phosphoribosyl-ATP pyrophosphatase, glpX-fructose 1,6-bisphosphatase II, and rpsH 30S ribosomal protein S8) targets were common with our first study. Further, we are working on characterization of hisE-phosphoribosyl-ATP pyrophosphatase, glpX-fructose 1,6-bisphosphatase II, and rpsH 30S ribosomal protein S8 in C. diphtheriae strain NCTC13129. The selection of these proteins (hisE, glpX and rpsH) were made on the bases of their identification through two different computational approaches. Our proposed approaches expedite the selection of C. diphtheriae putative proteins for broad-spectrum development of novel drugs and vaccines, owing to the fact that our study is computational and need experimental validation