Plant organellar MSH1 is a displacement loop specific endonuclease.

MSH1 is an organellar targeted protein that obstructs ectopic recombination and the accumulation of mutations in plant organellar genomes. MSH1 also modulates the epigenetic status of nuclear DNA, and its absence induces a variety of phenotypic responses. MSH1 is a member of the MutS family of DNA mismatch repair proteins but harbors an additional GIY-YIG nuclease domain that distinguishes it from the rest of this family. How MSH1 hampers recombination and promotes fidelity in organellar DNA inheritance is unknown. Here, we elucidate its enzymatic activities by recombinantly expressing and purifying full-length MSH1 from Arabidopsis thaliana (AtMSH1). AtMSH1 is a metalloenzyme that shows a strong binding affinity for displacement loops (D-loops). The DNA binding abilities of AtMSH1 reside in its MutS domain and not in its GIY-YIG domain, which is the ancillary nickase of AtMSH1. In the presence of divalent metal ions, AtMSH1 selectively executes multiple incisions at D-loops, but not other DNA structures including Holliday junctions or dsDNA, regardless of the presence or absence of mismatches. The selectivity of AtMSH1 to dismantle D-loops supports the role of this enzyme in preventing recombination between short repeats. Our results suggest that plant organelles have evolved novel DNA repair routes centered around the anti-recombinogenic activity of MSH1.

The novel EhHSTF7 transcription factor displays an oligomer state and recognizes a heat shock element in the Entamoeba histolytica parasite.

The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.

Expression, purification, and refolding of the recombinant extracellular domain ß1-subunit of the dog Na+/K+-ATPase of the epithelial cells.

The ß1-subunit of the Na+/K+-ATPase is a cell membrane protein, beyond its classic functions, it is also a cell adhesion molecule. ß1-subunits on the lateral membrane of dog kidney epithelial cells trans-interact with ß1-subunits from another neighboring cells. The ß-ß interaction is essential for the formation and stabilization of intercellular junctions. Previous studies on site-directed mutagenesis and in silico revealed that the interaction interface involves residues 198-207 and 221-229. However, it is necessary to report the interaction interface at the structural level experimentally. Here, we describe the successful cloning, overexpression in E. coli, and purification of the extracellular domain of the ß1-subunit from inclusion bodies. Experimental characterization by size exclusion chromatography and DLS indicated similar hydrodynamic properties of the protein refolded. Structural analysis by circular dichroism and Raman spectroscopy revealed the secondary structures in the folded protein of type ß-sheet, α-helix, random coil, and turn. We also performed ß1-ß1 interaction assays with the recombinant protein, showing dimers' formation (6xHisß1-ß1). Given our results, the recombinant extracellular domain of the ß1-subunit is highly similar to the native protein, therefore the current work in our laboratory aims to characterize at the atomic level the interaction interface between EDß1.

Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site.

The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended ß-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended ß-hairpin is involved in nucleotide incorporation on substrates with 5'-overhangs longer than 2 nt, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3'-5' exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating ß-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels.

Synergistic effect of compounds directed to triosephosphate isomerase, a combination to develop drug against trichomoniasis.

The development of new drugs is continuous in the world; currently, saving resources (both economic ones and time) and preventing secondary effects have become a necessity for drug developers. Trichomoniasis is the most common nonviral sexually transmitted infection affecting more than 270 million people around the world. In our research group, we focussed on developing a selective and more effective drug against Trichomonas vaginalis, and we previously reported on a compound, called A4, which had a trichomonacidal effect. Later, we determined another compound, called D4, which also had a trichomonacidal effect together with favorable toxicity results. Both A4 and D4 are directed at the enzyme triosephosphate isomerase. Thus, we made combinations between the two compounds, in which we determined a synergistic effect against T. vaginalis, determining the IC50 and the toxicity of the best relationship to obtain the trichomonacidal effect. With these results, we can propose a combination of compounds that represents a promising alternative for the development of a new therapeutic strategy against trichomoniasis.

Foaming and Structural Studies on the Acidic Subunit of Amaranth 11S Globulin Modified with Antihypertensive Peptides as a Function of pH and Ionic Strength.

Some studies aimed at revealing the relationship between protein structure and their functional properties. However, the majority of these reports have been carried out using protein isolates. There are limited reports on the possible relationship between the functional properties and the structure of a purified protein. In this work the amaranth 11S globulin acidic subunit (AAC) and five mutations of the same protein that were modified in their variable regions with antihypertensive peptides (VYVYVYVY and RIPP), were analyzed at two ionic strength (2.9 and 17.6 g/L NaCl) and pH (3.0-7.0). Results revealed better solubility for the proteins mutated at the terminal ends (AACM.1 and AACM.4) and lower solubility for the protein inserted with RIPP peptide. Spectroscopy studies revealed an increase of ß-sheet structure at high salt concentration for all proteins. It was also observed that salt concentration acted as a modulator, which allowed a better foam features for all modified proteins limiting movement of side chains and reducing red-shifted displacement of λmax. All proteins showed foam capacity ranging from 76 to 93% although foam stability was twofold better for modified proteins than for AAC at high salt concentration. This study allowed better understanding about the structural changes that influence the foaming properties of engineered proteins.

Effect of the single mutation N9Y on the catalytical properties of xylanase Xyn11A from Cellulomonas uda: a biochemical and molecular dynamic simulation analysis.

Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 °C in the optimum temperature and an upward shift of 1 unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 °C, compared to that displayed by Xyn11A-WT. Molecular dynamics simulations of Xyn11A-WT and Xyn11A-N9Y suggest that the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.

Amoebicidal effect of 5,5'-[(4-nitrophenyl)methylene]bis-6-hydroxy-2-mercapto-3-methyl-4(3H)-pyrimidinone), a new drug against Entamoeba histolytica.

Entamoeba histolytica is a cosmopolitan protozoan parasite that can produce infections in the intestine and some organs (liver, lungs, and brain), with worldwide prevalence. There are treatments against E. histolytica (antiparasitics), but as the drugs used in these treatments have presented some type of resistance and/or side effects, there are cases with complications of this disease. Therefore, it is necessary to develop new drugs aimed at a specific therapeutic target against this parasite. Here, we used the compound 5,5'-[(4-nitrophenyl)methylene]bis(6-hydroxy-2-mercapto-3-methyl-4(3H)-pyrimidinone) in the patenting process (called D4). D4 has a reported specific use against a glycolytic enzyme, the triosephosphate isomerase of Trichomonas vaginalis (TvTIM). We determined that D4 has an amoebicidal effect in in vitro cultures, with an IC50 value of 18.5 µM, and we proposed a specific site of interaction (Lys77, His110, Gln115, and Glu118) in the triosephosphate isomerase of E. histolytica (EhTIM). Furthermore, compound D4 has favorable experimental and theoretical toxicity results. Therefore, D4 should be further investigated as a potential drug against E. histolytica.

Exploring the Conformational Space of Bcl-2 Protein Variants: Dynamic Contributions of the Flexible Loop Domain and Transmembrane Region.

Members of the Bcl-2 protein family regulate apoptosis through interactions with several proteins. A critical intrinsically disordered region (IDR) present in some members of the Bcl-2 family is essential for their function. Also, the structural and conformational plasticity of disordered regions is essential for the regulation of the Bcl-2 protein's activity. Further, some proteins of the family contain transmembrane-helical regions, which anchor them into organelle membranes. Bcl-2, the archetypical member of the family, is characterized by an IDR labeled as a flexible loop domain (FLD) and a transmembrane domain (TMD). Another member of this family is the Bcl-2A1 protein, containing a TMD but lacking the FLD. To our knowledge, this is the first report which characterizes the individual and simultaneous dynamical contributions of FLD and TMD in Bcl-2 and Bcl-2A1 using molecular dynamics simulations (MDS). We examined the conformational spaces of Bcl-2, Bcl-2A1, and two artificial constructs lacking the TMD (Bcl-2ΔTM and Bcl-2A1ΔTM). As the results show, FLD and TMD stabilized each protein independently when they are present. When they coincided, such as in Bcl-2, an additive stabilizing effect is observed. This information is crucial for understanding the structural mechanisms of interaction in the Bcl-2 family.

Mimicking a p53-MDM2 interaction based on a stable immunoglobulin-like domain scaffold.

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide-bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin-like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig-like domain of the human T cell co-receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein-protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino-acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1-p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1-p53(FG)-loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1-p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.

Insertion of antihypertensive peptides in acidic subunit from amaranth 11S induces contrasting effects in stability.

The insertion of peptides is a biotechnology tool widely used to improve the nutraceutical properties of proteins. Because the effect of these insertions in protein stability and function is difficult to predict, it should be determined experimentally. In this study, we created two variants of amarantin acidic subunit and analyzed them along with other four proteins reported previously. We measured their response against two destabilizing agents: temperature and urea. The six proteins presented the insertion of antihypertensive peptides (VYVYVYVY or RIPP) in the variable regions of the protein. We observed that their effect strongly depended on the site of the insertion. The insertion in the variable region I stabilized the protein both thermally and chemically, but it affected the inhibitory activity of the angiotensin-converting enzyme in vitro. In contrast, insertions in other three regions were severely destabilizing, producing molten globules. Our findings reveal that the insertion of bioactive peptides in variable regions of a protein can increase or decrease the protein's thermal and chemical stability and that these conformational changes may also alter its final activity.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei.

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable ß-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein.

Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis.

Exploring the structure and conformational landscape of human leptin. A molecular dynamics approach.

Leptin is a hormone that regulates energy homeostasis, inflammation, hematopoiesis and immune response, among other functions (Houseknecht et al., 1998; Zhang et al., 1995; Paz-Filho et al., 2010). To obtain its crystallographic structure, it was necessary to substitute a tryptophan for a glutamic acid at position 100, thus creating a mutant leptin that has been reported to have biological activity comparable to the activity of the wild type but that crystallizes more readily. Here, we report a comparative study of the conformational space of WT and W100E leptin using molecular dynamics simulations performed at 300, 400, and 500 K. We detected differences between the interactions of the two proteins with local and distal effects, resulting in changes in the conformation, accessible surface area, compactness, electrostatic potential and dynamic behavior. Additionally, the series of unfolding events that occur when leptin is subjected to high temperature differs for the two constructs. We observed that both proteins are mostly unstructured after 20 ns of MD simulation at 500 K. However, WT leptin maintains a significant amount of secondary structure in helix α2, while the most stable region of W100E leptin is helix α3. Furthermore, we found that the region between residues 25 and 42 might adopt interconverting secondary structures ranging from α-helices and random coils to ß-strand structures. Thus, this region can be considered an intrinsically disordered region. This atomistic description supports our understanding of leptin signaling and consequently might facilitate the use of leptin in treatments for the pathophysiologies in which it is implicated.

Structural and thermodynamic folding characterization of triosephosphate isomerases from Trichomonas vaginalis reveals the role of destabilizing mutations following gene duplication.

We report the structures and thermodynamic analysis of the unfolding of two triosephosphate isomerases (TvTIM1 and TvTIM2) from Trichomonas vaginalis. Both isoforms differ by the character of four amino acids: E/Q 18, I/V 24, I/V 45, and P/A 239. Despite the high sequence and structural similarities between both isoforms, they display substantial differences in their stabilities. TvTIM1 (E18, I24, I45, and P239) is more stable and less dissociable than TvTIM2 (Q18, V24, V45, and A239). We postulate that the identities of residues 24 and 45 are responsible for the differences in monomer stability and dimer dissociability, respectively. The structural difference between both amino acids is one methyl group. In TvTIMs, residue 24 is involved in packing α-helix 1 against α-helix 2 of each monomer and residue 45 is located at the center of the dimer interface forming a "ball and socket" interplay with a hydrophobic cavity. The mutation of valine at position 45 for an alanine in TvTIM2 produces a protein that migrates as a monomer by gel filtration. A comparison with known TIM structures indicates that this kind of interplay is a conserved feature that stabilizes dimeric TIM structures. In addition, TvTIMs are located in the cytoplasm and in the membrane. As TvTIM2 is an easily dissociable dimer, the dual localization of TvTIMs may be related to the acquisition of a moonlighting activity of monomeric TvTIM2. To our knowledge, this is the simplest example of how a single amino acid substitution can provide alternative function to a TIM barrel protein.

Identification of a cryptic functional apolipophorin-III domain within the Prominin-1 gene of Litopenaeus vannamei.

The Apolipophorin-III (apoLp-III) is reported as an essential protein element in lipids transport and incorporation in lepidopterans. Structurally, apoLp-III has an α-helix bundle structure composed of five α-helices. Interestingly, classic studies proposed a structural switch triggered by its interaction with lipids, where the α-helix bundle opens. Currently, the study of the apoLp-III has been limited to insects, with no homologs identified in other arthropods. By implementing a structure-based search with the Phyre2 algorithm surveying the shrimp Litopenaeus vannamei's transcriptome, we identified a putative apoLp-III in this farmed penaeid (LvApoLp-III). Unlike canonical apoLp-III, the LvApoLp-III was identified as an internal domain within the transmembrane protein Prominin-1. Structural modeling using the template-based Phyre2 and template-free AlphaFold algorithms rendered two distinct structural topologies: the α-helix bundle and a coiled-coil structure. Notably, the secondary structure composition on both models was alike, with differences in the orientation and distribution of the α-helices and hydrophobic moieties. Both models provide insights into the classical structural switch induced by lipids in apoLp-III. To corroborate structure/function inferences, we cloned the synthetic LvApoLp-III domain, overexpressed, and purified the recombinant protein. Circular dichroism measurements with the recombinant LvApoLp-III agreed with the structural models. In vitro liposome interaction demonstrated that the apoLp-III domain within the PROM1 of L.vannamei associated similarly to exchangeable apolipoproteins. Altogether, this work reports the presence of an apolipophorin-III domain in crustaceans for the first time and opens questions regarding its function and importance in lipid metabolism or the immune system.

TtCel7A: A Native Thermophilic Bifunctional Cellulose/Xylanase Exogluclanase from the Thermophilic Biomass-Degrading Fungus Thielavia terrestris Co3Bag1, and Its Application in Enzymatic Hydrolysis of Agroindustrial Derivatives.

The biomass-degrading thermophilic ascomycete fungus Thielavia terrestris Co3Bag1 produces TtCel7A, a native bifunctional cellulase/xylanase GH7 family. The purified TtCel7A, with an estimated molecular weight of 71 kDa, was biochemically characterized. TtCel7A displayed an optimal pH of 5.5 for both activities and an optimal temperature of 60 and 50 °C for cellulolytic and xylanolytic activities, respectively. The half-lives determined for cellulase activity were 140, 106, and 41 min at 50, 60, and 70 °C, respectively, whereas the half-lives observed for xylanase activity were 24, 10, and 1.4 h at 50, 60, and 70 °C, respectively. The KM and Vmax values were 3.12 mg/mL and 50 U/mg for cellulase activity and 0.17 mg/mL and 42.75 U/mg for xylanase activity. Circular dichroism analysis suggests changes in the secondary structure of TtCel7A in the presence of CMC as the substrate, whereas no modifications were observed with beechwood xylan. TtCel7A displayed the excellent capability to hydrolyze CMC, beechwood xylan, and complex substrates such as oat bran, wheat bran, and sugarcane bagasse, with glucose and cellobiose being the main products released; also, slightly less endo cellulase and xylanase activities were observed. Thus, suggesting TtCel7A has an exo- and endomode of action. Based on the characteristics of the enzyme, it might be considered a good candidate for industrial applications.

Atomistic mechanism of leptin and leptin-receptor association.

The leptin-leptin receptor complex is at the very core of energy homeostasis and immune system regulation, among many other functions. In this work, we built homology models of leptin and the leptin binding domain (LBD) of the receptor from humans and mice. Docking analyses were used to obtain the coordinates of the native leptin-LBD complexes and a mixed heterodimer formed by human leptin and mouse LBD. Molecular dynamics (MD) simulations were performed using all models (monomers and heterodimers) as initial coordinates and the GROMACS program. The overall structural and dynamical behaviors are similar for the three complexes. Upon MD simulations, several new interactions appear. In particular, hydrophobic interactions, with more than 90% persistence, seem to be the most relevant for the stability of the dimers, as well as the pair formed by Asp85Lep and Arg468LBD. This in silico analysis provides structural and dynamical information, at the atomistic level, about the mechanism of leptin-LBD complex formation and leptin receptor activation. This knowledge might be used in the rational drug design of therapeutics to modulate leptin signaling.Communicated by Ramaswamy H. Sarma.