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

The natural ligand 17ß-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 ERß, are transcriptionally activated in the presence of E2 with ERß 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 ERß 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 ERß 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 ERß subtype.

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.

Sets of covariant residues modulate the activity and thermal stability of GH1 ß-glucosidases.

The statistical coupling analysis of 768 ß-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 ß-glucosidases were investigated by alanine-screening mutagenesis using the fall armyworm Spodoptera frugiperda ß-glycosidase (Sfßgly) as a model. The effects of the mutations on the Sfßgly kinetic parameters (kcat/Km) for the hydrolysis of three different p-nitrophenyl ß-glycosides and structural comparisons of several ß-glucosidases showed that eleven covariant positions (54, 98, 143, 188, 195, 196, 203, 398, 451, 452 and 460 in Sfßgly numbering) form a layer surrounding the active site of the ß-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 (Tm) of Sfßgly indicated that nine of the coupled positions (49, 62, 143, 188, 223, 278, 309, 452 and 460 in Sfßgly 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 ß-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 ß-glucosidases.

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.

Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa.

Plant peroxidases are presently used extensively in a wide range of biotechnological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å.

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.

Regulation of the transcription factor Ets-1 by DNA-mediated homo-dimerization.

The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.

Expression, purification, crystallization and preliminary crystallographic analysis of the mouse transcription factor MafB in complex with its DNA-recognition motif Cmare.

The MafB transcription factor (residues 211-305) has been overexpressed in and purified from Escherichia coli. A protein-DNA complex between the MafB homodimer and the 21 bp Maf-recognition sequence known as Cmare has been successfully reconstituted in vitro and subsequently crystallized. The diffraction properties of the protein-DNA complex crystals were improved using a combination of protein-construct boundary optimization and targeted mutagenesis to promote crystal lattice stability. Both native and mercury-derivatized crystals have been prepared using these optimized conditions. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 94.8, c = 197.9 A. An anomalous difference Patterson map computed using data collected from crystals grown in the presence of HgCl(2) reveals four peaks. This corresponds to two copies of the protein-DNA complex in the asymmetric unit, with a solvent content of 62% and a Matthews coefficient of 3.22 A(3) Da(-1).