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The relationship between GTP binding and hydrolysis on the one hand and septin filament assembly and function on the other has yet to be fully elucidated. The crystal structures of SEPT3 subgroup members bound to either GDP or to a GTP analogue shed light on this question by indicating how the squeezing of one of the inter-subunit interfaces may lead to the exposure of a polybasic region important for membrane association., Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 A, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 A2 and 1070 A2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 A between the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness — which is limited to the parasite's adult form — and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

Conjugated linoleic acid (CLA), a fatty acid found in ruminant food products, has been associated with anticarcinogenic activity. However, its effect on cancer metabolism is unclear. In this paper we evaluated the effects of cis-9, trans-11 CLA on the metabolic profile of MCF-7 and MDA-MB-231 breast cancer cells using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra showed that phosphocholine level, a cancer malignance biomarker, was reduced in both cells treated with CLA, but the reduction was more pronounced in MCF-7 cells. The NMR spectra also showed that CLA has opposite effect on MCF-7 and MDA-MB-231 acetone metabolism. Acetone signal has been observed in the spectra of MDA-MB-231 control cells, but not in the spectra of the cells treated with 50 and 100 µM CLA. Conversely, the acetone signal is very small or not observed in the NMR spectra of MCF-7 control cells and in cells treated with 50 µM of CLA, but is very strong in the spectra of the cells treated with 100 µM of CLA. Therefore, this CLA concentration is causing a ketosis in MCF-7 cells by inducing the use of fatty acids as an energy source or by reducing acetone catabolism. These results indicate that CLA interfere in the metabolism of both cells. However, the strongest effect has been observed on the metabolism of MCF-7 cells cultivated in the presence of 100 µM CLA. Therefore, CLA could be a potential anticarcinogenic drug, especially for cells with positive estrogen receptor, such as MCF-7.

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function., This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (grants #2016/04658-9 to DAL, #2013/04433-9 and #2018/19992-7 to IAC, #2015/00062-1 to FAS, #2018/20209-5 to DCM, #2019/22000-9 to HVDR, #2017/07709-6 to PSK, #2014/15546-1 to RCG and APUA and EMU/FAPESP grants #2015/16811-3 and #2015/16812-0 for the acquisition of the Gryphon crystallization robot and the SEC-MALS apparatus, respectively).

Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 A and 1.68 A, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

Septins are able to polymerize into long apolar filaments and have long been considered to be a component of the cytoskeleton alongside intermediate filaments (which are also apolar in nature), microtubules and actin filaments (which are not). Their central guanosine triphosphate (GTP)-binding domain, which is essential for stabilizing the filament itself, is flanked by N- and C-terminal domains for which no direct structural information is yet available. In most cases, physiological filaments are built from a number of different septin monomers, and in the case of mammalian septins this is most commonly either three or four. Comprehending the structural basis for the spontaneous assembly of such filaments requires a deeper understanding of the interfaces between individual GTP-binding domains than is currently available. Nevertheless, in this review we will summarize the considerable progress which has been made over the course of the last 10 years. We will provide a brief description of each structure determined to date and comment on how it has added to the body of knowledge which is rapidly growing. Rather than simply repeat data which have already been described in the literature, as far as is possible we will try to take advantage of the full set of information now available (mostly derived from human septins) and draw the reader's attention to some of the details of the structures themselves and the filaments they form which have not be commented on previously. An additional aim is to clarify some misconceptions.

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107–108 M−1⋅s−1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M−1⋅s−1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host–pathogen interactions.

Two cysteine proteinase inhibitors from cowpea, Vu Cys1 and Vu Cys2, were produced in E . coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100 °C for up to 60 min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after Vu Cys1 and Vu Cys2 were subjected to these treatments. The crystal structure of Vu Cys1 was determined at a resolution of 1.95 A, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in Vu Cys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of Vu Cys1.

The three-dimensional structure of canecystatin-1, a potent inhibitor of cysteine proteases from sugarcane (Saccharum officinarum), has been solved in two different crystal forms. In both cases, it is seen to exist as a domain-swapped dimer, the first such observation for a cystatin of plant origin. Size exclusion chromatography and multidimensional NMR spectroscopy show the dimer to be the dominant species in solution, despite the presence of a measurable quantity of monomer undergoing slow exchange. The latter is believed to be the active species, whereas the domain-swapped dimer is presumably inactive, as its first inhibitory loop has been extended to form part of a long β-strand that forms a double-helical coiled coil with its partner from the other monomer. A similar structure is observed in human cystatin C, but the spatial disposition of the two lobes of the dimer is rather different. Dimerization is presumably a mechanism by which canecystatin-1 can be kept inactive within the plant, avoiding the inhibition of endogenous proteases. The structure described here provides a platform for the rational design of specific cysteine protease inhibitors for biotechnological applications.

Protein-protein interactions play a critical role in promoting the stability of protein quaternary structure and in the assembly of large macromolecular complexes. What drives the stabilization of such assemblies is a central question in biology. A limiting factor in fully understanding such systems is the transient nature of many complexes, making structural studies difficult. Septins comprise a conserved family of guanine nucleotide binding proteins that polymerize in the form of heterofilaments. In structural terms, they have a common organization: a central GTPase domain, an N-terminal domain, and a C-terminal domain; the latter is predicted to form a coiled coil. Currently, even for the best characterized human septin heterocomplex (SEPT2/SEPT6/SEPT7), the role of C-terminal domain is not fully established, and this is partly due to the absence of electron density for the C-terminal domains in the x-ray structure. Here we present results on the homo/heterotypical affinity for the C-terminal domains of human septins belonging to the SEPT6 and SEPT7 groups (SEPT6C/8C/10C/11C and SEPT7C, respectively) and provide clear evidence that this domain determines the preference for heterotypic interactions at one specific interface during the assembly of the heterofilament. This observation has wider implications where macromolecular assemblies are defined by coiled-coil protein interactions.

Septins are filament-forming proteins found in many eukaryotes. Despite being important components of the cytoskeleton, only recently details of their macromolecular assemblies and crystal structures have started to appear in the literature. These are of fundamental importance to the understanding of cytoskeleton dynamics, membrane barrier formation, and bacterial caging, as well as essential cellular processes such as cell division, exocytosis, and vesicle trafficking. However, obtaining this data is frequently hindered by several experimental difficulties common to the majority of septin samples. Here we provide an overview of the current approaches to circumvent or minimize the experimental complications observed in septin crystallography focusing mainly, but not exclusively, on the choice of the septin construct and how to best prepare the sample itself.

Alzheimer’s disease is an ultimately fatal neurodegenerative disease, and BACE-1 has become an attractive validated target for its therapy, with more than a hundred crystal structures deposited in the PDB. In the present study, we present a new methodology that integrates ligand-based methods with structural information derived from the receptor. 128 BACE-1 inhibitors recently disclosed by GlaxoSmithKline R&D were selected specifically because the crystal structures of 9 of these compounds complexed to BACE-1, as well as five closely related analogs, have been made available. A new fragment-guided approach was designed to incorporate this wealth of structural information into a CoMFA study, and the methodology was systematically compared to other popular approaches, such as docking, for generating a molecular alignment. The influence of the partial charges calculation method was also analyzed. Several consistent and predictive models are reported, including one with r 2 = 0.88, q 2 = 0.69 and r pred 2 = 0.72. The models obtained with the new methodology performed consistently better than those obtained by other methodologies, particularly in terms of external predictive power. The visual analyses of the contour maps in the context of the enzyme drew attention to a number of possible opportunities for the development of analogs with improved potency. These results suggest that 3D-QSAR studies may benefit from the additional structural information added by the presented methodology.

2D QSAR studies were carried out for a series of 55 ligands for the Thyroid receptors, TRα and TRβ. Significant cross-validated correlation coefficients ( q 2 = 0.781 (TRα) and 0.693 (TRβ)) were obtained. The models’ predictive abilities were proved more valuable than the classical 2D-QSAR, and were further investigated by means of an external test set of 13 compounds. The predicted values are in good agreement with experimental values, suggesting that the models could be useful in the design of novel, more potent TR ligands. Contribution map analysis identified a number of positions that are promising for the development of receptor isoform specific ligands.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium that causes serious infections worldwide. This pathogen is resistant to all beta lactam antibiotics due the presence of PBP2a, a transpeptidase enzyme that presents very low beta-lactam affinity. Here we report the generation and characterization of mouse monoclonal antibodies to PBP2a of MRSA strains. Two clones were obtained and characterized by immunoassays (ELISA, avidity index determination, and immunoblotting), isotyping, association/dissociation rate constants by surface plasmon resonance (SPR), and flow cytometry. Clone 38, which showed the best avidity and affinity, bound to PBP2a located on the bacterial surface by flow cytometry. Further studies are warranted in order to evaluate if these antibodies may help inhibit bacterial growth and be used to treat infections by MRSA.

The human genome codes for 13 members of a family of filament-forming GTP-binding proteins known as septins. These have been divided into four different subgroups on the basis of sequence similarity. The differences between the subgroups are believed to control their correct assembly into heterofilaments which have specific roles in membrane remodelling events. Many different combinations of the 13 proteins are theoretically possible and it is therefore important to understand the structural basis of specific filament assembly. However, three-dimensional structures are currently available for only three of the four subgroups. In the present study we describe the crystal structure of a construct of human SEPT3 which belongs to the outstanding subgroup. This construct (SEPT3-GC), which includes the GTP-binding and C-terminal domains, purifies as a nucleotide-free monomer, allowing for its characterization in terms of GTP-binding and hydrolysis. In the crystal structure, SEPT3-GC forms foreshortened filaments which employ the same NC and G interfaces observed in the heterotrimeric complex of human septins 2, 6 and 7, reinforcing the notion of ‘promiscuous’ interactions described previously. In the present study we describe these two interfaces and relate the structure to its tendency to form monomers and its efficiency in the hydrolysis of GTP. The relevance of these results is emphasized by the fact that septins from the SEPT3 subgroup may be important determinants of polymerization by occupying the terminal position in octameric units which themselves form the building blocks of at least some heterofilaments.

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K(D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K(D) of 4 μM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Background Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Most physiological effects of thyroid hormones are mediated by the two thyroid hormone receptor subtypes, TRalpha and TRbeta. Several pharmacological effects mediated by TRbeta might be beneficial in important medical conditions such as obesity, hypercholesterolemia and diabetes, and selective TRbeta activation may elicit these effects while maintaining an acceptable safety profile. To understand the molecular determinants of affinity and subtype selectivity of TR ligands, we have successfully employed a ligand- and structure-guided pharmacophore-based approach to obtain the molecular alignment of a large series of thyromimetics. Statistically reliable three-dimensional quantitative structure-activity relationship (3D-QSAR) and three-dimensional quantitative structure-selectivity relationship (3D-QSSR) models were obtained using the comparative molecular field analysis (CoMFA) method, and the visual analyses of the contour maps drew attention to a number of possible opportunities for the development of analogs with improved affinity and selectivity. Furthermore, the 3D-QSSR analysis allowed the identification of a novel and previously unmentioned halogen bond, bringing new insights to the mechanism of activity and selectivity of thyromimetics.

Os receptores dos hormônios da tireóide (TRs) são fatores de transcrição envolvidos na diferenciação celular, metabolismo e funções fisiológicas da maioria dos tecidos. Muitos estudos mostram que diversos efeitos farmacológicos mediados pelos TRs podem ser benéficos na farmacoterapia, especialmente aqueles mediados pelo TR que podem ser úteis em condições médicas importantes como obesidade, hipercolesterolemia e diabetes. Além disso, a descoberta que o TR é a isoforma predominante no coração, mediando a maioria dos efeitos cardiovasculares prejudiciais, estimulou a pesquisa por ligantes seletivos para o TR que poderiam ser utilizados em quadros clínicos importantes com perfil de segurança aceitável. Foi realizado um estudo das relações quantitativas entre a estrutura e atividade (QSAR) de um conjunto de compostos com atividade biológica descrita para TR e TR, que gerou modelos de Holograma QSAR com elevada consistência interna e externa, apresentando bom poder de correlação e predição das propriedades biológicas. Também foi realizado um minucioso estudo de triagem virtual, que propiciou a seleção de 7 compostos que foram adquiridos para terem suas atividades biológicas avaliadas. Ensaios de transfecção e gene repórter foram estabelecidos e utilizados na avaliação da atividade biológica dos compostos selecionados pelo ensaio virtual. Finalmente, um ensaio utilizando ressonância plasmônica de superfície (SPR) foi desenvolvido e utilizado para avaliar a atividade agonista desses compostos, e que pode ser útil para avaliar a atividade de novos ligantes. A técnica de SPR também foi empregada em um cuidadoso estudo da interação do TR com seus correguladores, que incluiu estudos cinéticos e termodinâmicos, propiciando a determinação das taxas cinéticas e parâmetros termodinâmicos para a interação do complexo TR-T3 com peptídeos derivados de dois de seus correguladores. Os resultados obtidos são relevantes e devem ser considerados no planejamento de futuros experimentos utilizando o LBD de TR e agonistas. The thyroid hormone receptors (TRs) are transcriptional factors involved in cell differentiation, development, metabolism and physiological function of most tissues. Many lines of evidence show that several pharmacological actions of TRs might be beneficial in medical therapy, specially those mediated by TR that target important medical conditions like obesity, hypercholesterolemia and diabetes. Additionally, the findings that TR is the predominant isoform in the heart and mediates most of the TRs deleterious cardiovascular effects, stimulated the research for selective TR ligands which could address important medical needs with an acceptable safety profile. In this PhD thesis, studies of the quantitative structure-activity relationships (QSAR) of a dataset of compounds with reported biologic activity for both TR and TR were performed, and statistically significant Hologram QSAR models with good predictive ability for untested compounds were created. In parallel, a careful virtual screening procedure was executed, leading to the selection of 7 compounds which were purchased for the evaluation of their biological activities. Cell transfection and reporter gene assays were developed, validated and used to evaluate the biological activities of these compounds. Finally, a surface plasmon resonance (SPR) assay was developed and used to assess the agonistic activity of these compounds. The SPR technique was also employed in a careful study of the interaction between the ligand binding domain of TR and peptides derived from its coregulators, which included the determination of the kinetic and thermodynamic parameters for this interaction. The results suggest that flexibility plays an important role in the interaction between the receptor and its coregulators, and point out important aspects of experimental design that should be addressed when using TR LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR ligands.

Thyroid hormones exert most of their physiological effects through two thyroid hormone receptor (TR) subtypes, TRalpha and TRbeta, which associate with many transcriptional coregulators to mediate activation or repression of target genes. The search for selective TRbeta ligands has been stimulated by the finding that several pharmacological actions mediated by TRbeta might be beneficial in medical conditions such as obesity, hypercholesterolemia and diabetes. Here, we present a new methodology which employs surface plasmon resonance to investigate the interactions between TRbeta ligand binding domain (LBD) complexes and peptides derived from the nuclear receptor interaction motifs of two of its coregulators, SRC2 and DAX1. The effect of several TRbeta ligands, including the TRbeta selective agonist GC-1 and the TRbeta selective antagonist NH-3, were investigated. We also determined the kinetic rate constants for the interaction of TRbeta-T3 with both coregulators, and accessed the thermodynamic parameters for the interaction with DAX1. Our findings suggest that flexibility plays an important role in the interaction between the receptor and its coregulators, and point out important aspects of experimental design that should be addressed when using TRbeta LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TRbeta ligands.

Septins constitute a family of conserved guanine nucleotide binding proteins that are found in different organisms. The human septins comprise 13 genes which encode proteins involved in important cellular processes, such as cytokinesis, exocytosis, membrane compartmentalization. They can be divided into 4 groups based on sequence similarity. Septins from different groups have showed to interact with each other assembling into heteromeric complexes that can polymerize to form filaments. Currently, only one of these possible heterocomplexes had its structure solved, but it did not include any member of the SEPT3 group. Recent publications suggest SEPT9, a member of the SEPT3 group, can occupy the terminal position in the assembly of octameric complexes. Here, we present the first determination of the crystallographic structure and biochemical properties of the SEPT3. The analysis of this structure shows unique features that can be useful in understanding the assembling of septin octamer units and the polymerization control of the septin filaments. A truncated version of SEPT3 lacking the N-terminal domain (SEPT3-GC) was found to be a monomer in solution, and the dimerization was dependent on salt concentration and the presence of GTPS. Additional site-directed mutagenesis experiments proved that the SEPT3-GC monomeric state is related to a tyrosine residue which is present in all human septins, except in the SEPT3 group. The SEPT3-GC affinity by GTPS was Mg2+ dependent and this septin was also active and able to hydrolyze GTP in vitro. The crystal structure of SEPT3-GC forms foreshortened filaments which employ the same NC and G interfaces observed in the hexameric complex of human septins 2, 6 and 7, reinforcing the notion of ‘promiscuous' interactions described previously.

As septinas são proteínas filogeneticamente classificadas na superclasse das P-loop GTPases e que, juntamente com actina, microtúbulos e filamentos intermediários, são consideradas o quarto componente do citoesqueleto. Para exercer essa função, as septinas tendem a interagir entre si formando heterocomplexos que, posteriormente, polimerizam-se em filamentos. A fim de compreender a arquitetura e dinâmica das septinas, realizou-se a caracterização estrutural e biofísica da septina 7 humana e de seus complexos com as septinas 3 e 9. Devido ao fato da septina 7 ser única em seu grupo, tem-se que a mesma é insubstituível e, portanto, está presente em todos os heterocomplexos descritos. Visando compreender os elementos moleculares responsáveis por tal unicidade, são apresentadas duas estruturas em alta resolução do domínio GTPase da septina 7 ligada a GDP. Pela primeira vez, verificou-se que o cofator Mg2+ está coordenado de uma maneira mais fraca (em relações aos padrões já descritos na literatura) e que o contato entre o switches II na interface-G (ponte-β antiparalela) aparenta estar relacionado ao fenômeno de deslizamento da fita-β3. Na verdade, os resultados indicam que tal fenômeno seria um artifício utilizado pelas septinas a fim de desestimular interfaces promíscuas. Além disso, estudos de termoestabilidade mostraram que as septinas 3 e 9 acabam por diminuir a estabilidade de seus heterodímeros com a septina 7; um resultado que pode estar relacionado com a dinâmica de formação de filamentos. Devido a uma suposta interação mais fraca, tem-se que tais septinas poderiam talvez apresentar uma maior flexibilidade na constituição dos filamentos. Por outro lado, ao realizar a mutação T282Y na septina 3, nota-se que a mesma é responsável por aumentar significativamente a termoestabilidade do heterodímero. A partir da estrutura cristalográfica deste complexo, observou-se que tal tirosina é responsável pelo estabelecimento de interações de hidrogênio com o anel guanina e com a septina 7; promovendo a estabilização da interface-G. Por último, com o intuito de identificar qual interface-NC do heterodímero constituído pelas septinas 7 e 9 é a responsável pela sua oligomerização a um tetrâmero, avaliou-se a dependência de tais septinas com a força iônica. The septins phylogenetically belong to the P-loop GTPase superclass of proteins and, together with actin, microtubules and intermediate filaments, are considered the fourth component of the cytoskeleton. To perform this role, the septins tend to interact with each other to form heterocomplexes, which can further polymerize into filaments. In order to understand the architecture and dynamics of septins, a structural and biophysical characterization of human septin 7 and its complexes with septins 3 and 9 were performed. Since septin 7 is single in its own group, it is irreplaceable and therefore present in all heterocomplexes. Toward understanding the molecular elements responsible for such unicity, two high-resolution structures of septin 7 GTPase domain complexed with GDP are presented. For the first time, it has been found that the Mg2+ cofactor is weakly coordinated (in relation to the patterns already described in the literature) and that the contact between the switches II at the G-interface (antiparallel β-bridge) appears to be related to the phenomenon of β3-stand slippage. In fact, the results indicate this phenomenon could be thought of as a way to discourage promiscuous interfaces. In addition, thermostability assays have shown that septin 3 and 9 end up decreasing the stability of their heterodimers with septin 7; a result that may be related to the filament dynamics. Due to this supposed weaker interaction, it has been speculated that this septins could perhaps have a greater flexibility in the composition of the filaments. On the other hand, the T282Y mutation inserted into septin 3 significantly increases the heterodimer thermostability. In the crystal structure of this complex, it was observed that this particular tyrosine establishes a hydrogen bond with the guanine ring and with septin 7; promoting the stabilization of the G-interface. Finally, in order to identify which NC-interface of the heterodimer constituted by septin 7 and 9 is responsible for its oligomerization to a tetramer, the dependence of this septins with the ionic strength was analyzed.