Amanda Bernardes - Academia.edu (original) (raw)

Papers by Amanda Bernardes

Physical Review B, 1994

A fiber-bundle model in (1+1) dimensions for the breaking of fibrous composite matrix is introduc... more A fiber-bundle model in (1+1) dimensions for the breaking of fibrous composite matrix is introduced. The model consists of N parallel fibers fixed in two plates. When one of the plates is pulled in the direction parallel to the fibers, these can be broken with a probability that depends on their elastic energy. The mechanism of rupture is simulated by the breaking of neighboring fibers that can generate random cracks spreading up through the system. Due to the simplicity of the model we have virtually no computational limitation. The model is sensitive to external conditions such as temperature and traction velocity. The energy versus temperature behavior, the diagrams of stress versus strain, and the histograms of the frequency versus the size of cracks are obtained.

O trabalho foi conduzido em povoamentos de eucalipto de uma empresa florestal do Estado de Goiás,... more O trabalho foi conduzido em povoamentos de eucalipto de uma empresa florestal do Estado de Goiás, com o objetivo de avaliar técnica e economicamente um sistema de colheita florestal de árvores inteiras composto de "feller-buncher", "skidder" e garra traçadora. Foi realizado um estudo de tempos e movimentos e determinados os custos operacionais e de produção das máquinas. Para avaliar esses valores de produtividade do "feller-buncher" e da garra traçadora, foi empregado um delineamento estatístico inteiramente casualizado com seis repetições, em esquema de parcelas subdivididas. Adotaram-se como parcelas as operações que compõem o ciclo operacional das máquinas e como subparcelas, os níveis de produtividade de floresta de 100, 200 e 300 m³ ha -1 . Os valores foram submetidos à análise de variância e ao teste de Tukey a 5% de probabilidade. Para avaliar o efeito dos tempos consumidos nas operações do ciclo operacional do "skidder" nas produtividades de floresta de 100, 200 e 300 m³ ha -1 e nas distâncias de arraste de 100, 200 e 300 m, assim como avaliar suas interações quando significativas, utilizou-se um delineamento estatístico de blocos casualizados, em esquema fatorial 6x3x3, sendo seis operações, três produtividades e três distâncias de arraste, com quatro repetições. Os valores foram submetidos à análise de variância e ao teste de Tukey a 5% de probabilidade. Concluiu-se que o "feller-buncher" registrou o maior custo de produção dentro do sistema, obtendo melhor capacidade efetiva de trabalho nas áreas de maior produtividade. O "skidder" também teve seu melhor desempenho no talhão de 300 m³ ha -1 na distância até 200 m. A garra traçadora apresentou custo de 0.97, 0.75 e 0.63 US$ m -3 para as produtividades de 100, 200 e 300 m³ ha -1 , respectivamente.

Computer Physics Communications, 2009

Biochimie, 2015

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops ... more Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site.

Journal of Molecular Biology, 2013

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear trans... more Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

Acta crystallographica. Section F, Structural biology and crystallization communications, 2013

BMC structural biology, 2007

Background: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron tran... more Background: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.

PLoS ONE, 2014

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reac... more The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Biochemistry, 2007

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding dom... more High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.

PLoS ONE, 2012

Peroxisome proliferator activated receptors (PPARs d, a and c) are closely related transcription ... more Peroxisome proliferator activated receptors (PPARs d, a and c) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs crossreact with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARd more than 300-fold more tightly than PPARa or PPARc but the structural basis of PPARd:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARd:GW0742 complex. Comparisons of the PPARd:GW0742 complex with published structures of PPARs in complex with a and c selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPARd selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARa and c ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARd ligand design. Citation: Batista FAH, Trivella DBB, Bernardes A, Gratieri J, Oliveira PSL, et al. (2012) Structural Insights into Human Peroxisome Proliferator Activated Receptor Delta (PPAR-Delta) Selective Ligand Binding. PLoS ONE 7(5): e33643.

PLoS ONE, 2012

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carb... more The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARc and RXRa is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARc alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARc remains in the monomeric form by itself but forms heterodimers with hRXRa. The lowresolution models of hPPARc/RXRa complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the fulllength receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex. Citation: Bernardes A, Batista FAH, de Oliveira Neto M, Figueira ACM, Webb P, et al. (2012) Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution. PLoS ONE 7(2): e31852.

BMC Structural Biology - BMC STRUCT BIOL, 2007

Background Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron trans... more Background Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. Results The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. Conclusion LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

Journal of Molecular Biology, 2013

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear trans... more Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

Molecular pharmacology, 2012

The peroxisome proliferator-activated receptor ␥ (PPAR␥) is a target for treatment of type II dia... more The peroxisome proliferator-activated receptor ␥ (PPAR␥) is a target for treatment of type II diabetes and other conditions. PPAR␥ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR␥ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR␥ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/ antagonist activity in transfections, inhibits several PPAR␥ target genes in 3T3-L1 cells (LPL, ORL1, and CEBP␣) and PPAR␥dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR␥ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR␥ LBD conformer and occupies a space near the ␤-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR␥, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the ⍀-loop among H2Ј, H3, and the ␤-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR␥-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR␥ modulators. This work was supported by Fundaçã o de Amparo a Pesquisa do Estado de Sã o Paulo (FAPESP) [Grant 2007/58443-4 and 2010/08680-2] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Biochemistry, 2007

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding dom... more High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.

Physical Review B, 1994

A fiber-bundle model in (1+1) dimensions for the breaking of fibrous composite matrix is introduc... more A fiber-bundle model in (1+1) dimensions for the breaking of fibrous composite matrix is introduced. The model consists of N parallel fibers fixed in two plates. When one of the plates is pulled in the direction parallel to the fibers, these can be broken with a probability that depends on their elastic energy. The mechanism of rupture is simulated by the breaking of neighboring fibers that can generate random cracks spreading up through the system. Due to the simplicity of the model we have virtually no computational limitation. The model is sensitive to external conditions such as temperature and traction velocity. The energy versus temperature behavior, the diagrams of stress versus strain, and the histograms of the frequency versus the size of cracks are obtained.

O trabalho foi conduzido em povoamentos de eucalipto de uma empresa florestal do Estado de Goiás,... more O trabalho foi conduzido em povoamentos de eucalipto de uma empresa florestal do Estado de Goiás, com o objetivo de avaliar técnica e economicamente um sistema de colheita florestal de árvores inteiras composto de "feller-buncher", "skidder" e garra traçadora. Foi realizado um estudo de tempos e movimentos e determinados os custos operacionais e de produção das máquinas. Para avaliar esses valores de produtividade do "feller-buncher" e da garra traçadora, foi empregado um delineamento estatístico inteiramente casualizado com seis repetições, em esquema de parcelas subdivididas. Adotaram-se como parcelas as operações que compõem o ciclo operacional das máquinas e como subparcelas, os níveis de produtividade de floresta de 100, 200 e 300 m³ ha -1 . Os valores foram submetidos à análise de variância e ao teste de Tukey a 5% de probabilidade. Para avaliar o efeito dos tempos consumidos nas operações do ciclo operacional do "skidder" nas produtividades de floresta de 100, 200 e 300 m³ ha -1 e nas distâncias de arraste de 100, 200 e 300 m, assim como avaliar suas interações quando significativas, utilizou-se um delineamento estatístico de blocos casualizados, em esquema fatorial 6x3x3, sendo seis operações, três produtividades e três distâncias de arraste, com quatro repetições. Os valores foram submetidos à análise de variância e ao teste de Tukey a 5% de probabilidade. Concluiu-se que o "feller-buncher" registrou o maior custo de produção dentro do sistema, obtendo melhor capacidade efetiva de trabalho nas áreas de maior produtividade. O "skidder" também teve seu melhor desempenho no talhão de 300 m³ ha -1 na distância até 200 m. A garra traçadora apresentou custo de 0.97, 0.75 e 0.63 US$ m -3 para as produtividades de 100, 200 e 300 m³ ha -1 , respectivamente.

Computer Physics Communications, 2009

Biochimie, 2015

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops ... more Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site.

Journal of Molecular Biology, 2013

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear trans... more Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

Acta crystallographica. Section F, Structural biology and crystallization communications, 2013

BMC structural biology, 2007

Background: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron tran... more Background: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.

PLoS ONE, 2014

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reac... more The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Biochemistry, 2007

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding dom... more High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.

PLoS ONE, 2012

Peroxisome proliferator activated receptors (PPARs d, a and c) are closely related transcription ... more Peroxisome proliferator activated receptors (PPARs d, a and c) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs crossreact with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARd more than 300-fold more tightly than PPARa or PPARc but the structural basis of PPARd:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARd:GW0742 complex. Comparisons of the PPARd:GW0742 complex with published structures of PPARs in complex with a and c selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPARd selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARa and c ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARd ligand design. Citation: Batista FAH, Trivella DBB, Bernardes A, Gratieri J, Oliveira PSL, et al. (2012) Structural Insights into Human Peroxisome Proliferator Activated Receptor Delta (PPAR-Delta) Selective Ligand Binding. PLoS ONE 7(5): e33643.

PLoS ONE, 2012

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carb... more The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARc and RXRa is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARc alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARc remains in the monomeric form by itself but forms heterodimers with hRXRa. The lowresolution models of hPPARc/RXRa complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the fulllength receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex. Citation: Bernardes A, Batista FAH, de Oliveira Neto M, Figueira ACM, Webb P, et al. (2012) Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution. PLoS ONE 7(2): e31852.

BMC Structural Biology - BMC STRUCT BIOL, 2007

Background Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron trans... more Background Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. Results The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. Conclusion LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

Journal of Molecular Biology, 2013

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear trans... more Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

Molecular pharmacology, 2012

The peroxisome proliferator-activated receptor ␥ (PPAR␥) is a target for treatment of type II dia... more The peroxisome proliferator-activated receptor ␥ (PPAR␥) is a target for treatment of type II diabetes and other conditions. PPAR␥ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR␥ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR␥ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/ antagonist activity in transfections, inhibits several PPAR␥ target genes in 3T3-L1 cells (LPL, ORL1, and CEBP␣) and PPAR␥dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR␥ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR␥ LBD conformer and occupies a space near the ␤-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR␥, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the ⍀-loop among H2Ј, H3, and the ␤-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR␥-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR␥ modulators. This work was supported by Fundaçã o de Amparo a Pesquisa do Estado de Sã o Paulo (FAPESP) [Grant 2007/58443-4 and 2010/08680-2] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Biochemistry, 2007

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding dom... more High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.