An alternative conformation of ERβ bound to estradiol reveals H12 in a stable antagonist position

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThe natural ligand 17β-estradiol (E2) is so far believed to induce a unique agonist-bound active conformation in the ligand binding domain (LBD) of the estrogen receptors (ERs). Both subtypes, ERα and ERβ, are transcriptionally activated in the presence o7FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2014/22007-02013/08293-72012/24750-6301981/2011-6This work was supported by the São Paulo Research Foundation FAPESP (grants 2014/22007-0, 2013/08293-7, 2012/24750-6) and by CNPq (grant 301981/2011-6

An alternative conformation of ERβ bound to estradiol reveals H12 in a stable antagonist position

The natural ligand 17β-estradiol (E2) is so far believed to induce a unique agonist-bound active conformation in the ligand binding domain (LBD) of the estrogen receptors (ERs). Both subtypes, ERα and ERβ, are transcriptionally activated in the presence of E2 with ERβ being somewhat less active than ERα under similar conditions. The molecular bases for this intriguing behavior are mainly attributed to subtype differences in the amino-terminal domain of these receptors. However, structural details that confer differences in the molecular response of ER LBDs to E2 still remain elusive. In this study, we present a new crystallographic structure of the ERβ LBD bound to E2 in which H12 assumes an alternative conformation that resembles antagonist ERs structures. Structural observations and molecular dynamics simulations jointly provide evidence that alternative ERβ H12 position could correspond to a stable conformation of the receptor under physiological pH conditions. Our findings shed light on the unexpected role of LBD in the lower functional response of ERβ subtype

Sets of Covariant Residues Modulate the Activity and Thermal Stability of GH1 β-Glucosidases

<div><p>The statistical coupling analysis of 768 β-glucosidases from the GH1 family revealed 23 positions in which the amino acid frequencies are coupled. The roles of these covariant positions in terms of the properties of β-glucosidases were investigated by alanine-screening mutagenesis using the fall armyworm <i>Spodoptera frugiperda</i> β-glycosidase (Sfβgly) as a model. The effects of the mutations on the Sfβgly kinetic parameters (<i>k</i>_cat/<i>K</i>_m) for the hydrolysis of three different <i>p</i>-nitrophenyl β-glycosides and structural comparisons of several β-glucosidases showed that eleven covariant positions (54, 98, 143, 188, 195, 196, 203, 398, 451, 452 and 460 in Sfβgly numbering) form a layer surrounding the active site of the β-glucosidases, which modulates their catalytic activity and substrate specificity via direct contact with the active site residues. Moreover, the influence of the mutations on the transition temperature (<i>T</i>_m) of Sfβgly indicated that nine of the coupled positions (49, 62, 143, 188, 223, 278, 309, 452 and 460 in Sfβgly numbering) are related to thermal stability. In addition to being preferentially occupied by prolines, structural comparisons indicated that these positions are concentrated at loop segments of the β-glucosidases. Therefore, due to these common biochemical and structural properties, these nine covariant positions, even without physical contacts among them, seem to jointly modulate the thermal stability of β-glucosidases.</p></div

Distribution of sector S positions on the secondary structure of β-glucosidases.

<p>β-glucosidase from <i>Spodoptera frugiperda</i> Sfβgly; β-glucosidase from <i>Thermus thermophilus</i> (1UG6); β-glucosidase A from <i>Paenibacillus polymyxa</i> (1E4I); β-glucosidase Zmglu from <i>Zea mays</i> (1E56); β-glucosidase SbDhr from <i>Sorghum bicolor</i> (1V03); myrosinase from <i>Sinapis alba</i> (1E6S); β-glucosidase from <i>Trichoderma reesei</i> (3AHY); Human cytosolic β-glucosidase (2ZOX); β-glucosidase from <i>Pyrococcus horikoshii</i> (1VFF). α-Helices are represented by cylinders, β-strands by arrows and loops by lines. Sector S positions are shown as circles, whereas non-sector S positions are shown as stars. The symbols (circles or stars) in black indicate positions placed at loops, whereas white symbols mark positions at helices or strands.</p

Structural comparison of β-glucosidases showing the active site residues (red) and sector A positions (blue).

<p>Myrosinase from <i>Sinapis alba</i> (1E6S); β-glucosidase A from <i>Paenibacillus polymyxa</i> (1EI4); β-glucosidase from <i>Trichoderma reesei</i> (3AHY); β-glucosidase Zmglu from <i>Zea mays</i> (1E56); β-glucosidase from <i>Thermus thermophilus</i> (1UG6); Human cytosolic β-glucosidase (2ZOX); SbDhr from <i>Sorghum bicolor</i> (1V03); β-glucosidase from <i>Pyrococcus horikoshii</i> (1VFF); β-glucosidase from <i>Spodoptera frugiperda</i> Sfβgly. The distances between sector A and the active site residues are shorter than 4.5 Å. The structures were visualized using PyMOL software.</p

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[08/56255-9]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2007/08706-9]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2009/05349-6]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (Capes)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) via INCT do Bioetanol[471834/2009-2

Transition temperatures (<i>T</i>_m) for thermal denaturation of the wild-type and mutant Sfβgly proteins.

<p>Standard deviations were lower than 0.5 K.</p

Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa

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. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage

Palm peroxidades: the most robust enzymes

Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications. Over the last decade, several studies have shown that peroxidases isolated from the leaves of different kinds of palm trees such as the royal palm (Roystonea regia), the date palm (Phoenix dactylifera), the African oil palm (Elaeis guineensis), the ruffle palm (Aiphanes cariotifolia) and the windmill palms (Trachycarpus fortunei and Chamaerops excelsa) exhibit higher activity and stability than commercially available peroxidases isolated from, for example, horseradish roots (Armoracia rusticana) and soybean (Glycine max). Here, the structure, thermal denaturation, and the catalytic cycle of peroxidases from palm trees are reviewed and compared with those of other plant peroxidases. In addition, we report the biotechnological potential of palm peroxidases and their implications in cellular aging and diseases, such as Refsum’s and Alzheimer’s diseases. This paper summarizes the main characteristics of the palm peroxidases studied.Peer reviewe

Joint X-ray crystallographic and molecular dynamics study of cellobiohydrolase I from Trichoderma harzianum: deciphering the structural features of cellobiohydrolase catalytic activity

Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side-chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose-binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher k cat and K m values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.FAPESP (08/56255-9, 09/54035-4, 10/08680-2)CNPq (490022/2009-0, 471834/2009-2, 550985/2010-7, 151951/2008-0)CAPE