X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin.

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of ß-Galactosidase.

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from ß-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected ß-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

The structure of unliganded sterol carrier protein 2 from Yarrowia lipolytica unveils a mechanism for binding site occlusion.

Isolated or as a part of multidomain proteins, Sterol Carrier Protein 2 (SCP2) exhibits high affinity and broad specificity for different lipidic and hydrophobic compounds. A wealth of structural information on SCP2 domains in all forms of life is currently available; however, many aspects of its ligand binding activity are poorly understood. ylSCP2 is a well-characterized single domain SCP2 from the yeast Yarrowia lipolytica. Herein, we report the X-ray structure of unliganded ylSCP2 refined to 2.0 Å resolution. Comparison with the previously solved liganded ylSCP2 structure unveiled a novel mechanism for binding site occlusion. The liganded ylSCP2 binding site is a large cavity with a volume of more than 800 Å3. In unliganded ylSCP2 the binding site is reduced to about 140 Å3. The obliteration is caused by a swing movement of the C-terminal α helix 5 and a subtle compaction of helices 2-4. Previous pairwise comparisons were between homologous SCP2 domains with a uncertain binding status. The reported unliganded ylSCP2 structure allows for the first time a fully controlled comparative analysis of the conformational effects of ligand occupation dispelling several doubts regarding the architecture of SCP2 binding site.

High resolution crystal structure of NaTrxh from Nicotiana alata and its interaction with the S-RNase.

Thioredoxins are regulatory proteins that reduce disulfide bonds on target proteins. NaTrxh, which belongs to the plant thioredoxin family h subgroup 2, interacts and reduces the S-RNase enhancing its ribonuclease activity seven-fold, resulting an essential protein for pollen rejection inNicotiana.Here, the crystal structure of NaTrxh at 1.7 Å by X-ray diffraction is reported. NaTrxh conserves the typical fold observed in other thioredoxins from prokaryotes and eukaryotes, but it contains extensions towards both N- and C-termini.The NaTrxh N-terminal extension participates in the reduction of S-RNase, and in the structure reported here, this is orientated towards the reactive site. The interaction between SF11-RNase and the NaTrxh N-terminal was simulated and the short-lived complex observed lasted for a tenth of ns. Moreover, we identified certain amino acids as SF11-RNase-E155 and NaTrxh-M104 as good candidates to contribute to the stability of the complex. Furthermore, we simulated the reduction of the C153-C186 SF11-RNase disulfide bond and observed subtle changes that affect the entire core, which might explain the increase in the ribonuclease activity of S-RNase when it is reduced by NaTrxh.

Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.

Benzodiazepine exposure induces transcriptional down-regulation of GABAA receptor α1 subunit gene via L-type voltage-gated calcium channel activation in rat cerebrocortical neurons.

GABAA receptors are targets of different pharmacologically relevant drugs, such as barbiturates, benzodiazepines, and anesthetics. In particular, benzodiazepines are prescribed for the treatment of anxiety, sleep disorders, and seizure disorders. Benzodiazepines potentiate GABA responses by binding to GABAA receptors, which are mainly composed of α (1-3, 5), ß2, and γ2 subunits. Prolonged activation of GABAA receptors by endogenous and exogenous modulators induces adaptive changes that lead to tolerance. For example, chronic administration of benzodiazepines produces tolerance to most of their pharmacological actions, limiting their usefulness. The mechanism of benzodiazepine tolerance is still unknown. To investigate the molecular basis of tolerance, we studied the effect of sustained exposure of rat cerebral cortical neurons to diazepam on the GABAA receptor. Flunitrazepam binding experiments showed that diazepam treatment induced uncoupling between GABA and benzodiazepine sites, which was blocked by co-incubation with flumazenil, picrotoxin, or nifedipine. Diazepam also produced selective transcriptional down-regulation of GABAA receptor α1 subunit gene through a mechanism dependent on the activation of L-type voltage-gated calcium channels. These findings suggest benzodiazepine-induced stimulation of calcium influx through L-type voltage-gated calcium channels triggers the activation of a signaling pathway that leads to uncoupling and an alteration of receptor subunit expression. Insights into the mechanism of benzodiazepine tolerance will contribute to the design of new drugs that can maintain their efficacies after long-term treatments.

A combined Far-FTIR, FTIR Spectromicroscopy, and DFT Study of the Effect of DNA Binding on the [4Fe4S] Cluster Site in EndoIII.

Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe-S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm-1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe-S (sulfide) and Fe-S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe-S bond lengths upon DNA binding, both of the Fe-S (sulfide) and Fe-S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix.

Toluidine blue staining for cell and tissue biology applications.

Toluidine blue (TB) staining either alone or in association with other methodologies has the potential to answer a variety of biological questions regarding the human, animal and plant tissues or cells. In this brief review, we not only report the primary use of TB to detect the anionic substrates and availability of their binding sites, but also unveil the resulting applications of TB staining in biological research. Among these applications, the uses of TB staining to identify the changes in chromatin DNA-protein complexes, nucleolus location, and extracellular matrix proteoglycan complexes associated with different physiological and pathological events are described. The usefulness of TB staining to monitor the effects elicited by environmental insults on chromatin and intercalation of drugs into the DNA is also included.

Synthesis and antinociceptive evaluation of bioisosteres and hybrids of naproxen, ibuprofen and paracetamol.

The aim of this work was to design, synthesize and characterize the potential anti-nociceptive and anti-inflammatory activities of a new series of bioisosteres and hybrids from known non-steroidal anti-inflammatory drugs (NSAIDs). The compounds 4-(acetylamino)phenyl (2S)-2-(6-methoxy-2-naphthyl)propanoate (GUF-1) and 4-(acetylamino)phenyl 2-(R,S)-(4-isobutylphenyl)propanoate (GUF-2) were synthesized as hybrids (also known as heterodimers); whereas those named 2-(R,S)-(4-isobutylphenyl)-N-1H-tetrazol-5-ylpropanamide (GUF-3), (2S)-2-(6-methoxy-2-naphthyl)-N-1H-tetrazol-5-ylpropanamide (GUF-4), [2-(R,S)-N-hydroxy-2-[4-(2-methylpropyl)phenyl]propanamide] (GUF-5), and (2S)-N-hydroxy-2-(6-methoxy-2-naphthyl)propanamide (GUF-6) were synthesized as bioisosteres of the NSAIDs paracetamol, ibuprofen, and naproxen, respectively. All these compounds were characterized by spectroscopic and spectrometric analysis. Antinociceptive activity of GUF-1 to GUF-6 was evaluated using the formalin test in rats. Pharmacological responses of GUF-1, GUF-2 (hybrids), and GUF-5 (bioisostere) demonstrated significant antinociceptive effects; thus these compounds were assayed in an inflammation test like carrageenan-induced paw oedema in rats. Complete molecular docking of cyclooxygenase and the GUF-1 and GUF-2 hybrids showed high docking scores, compared to the reference drugs. Our data demonstrate that compounds GUF-1, GUF-2, and GUF-5 possesses antinociceptive and antiinflammatory activities resembling and improving those known for the traditional NSAIDs, paracetamol, naproxen and ibuprofen.

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α.

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations.

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.

Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.

TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/ß region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase's active site, providing a structural framework for the function of TIPRL in PP2A inhibition.

Early expression of the receptor for advanced glycation end products in a toxic model produced by 6-hydroxydopamine in the rat striatum.

The receptor for advanced glycation end products (RAGE) is commonly involved in different neurodegenerative and inflammatory disorders. The cellular signaling associated to RAGE activation may occur upon binding to different ligands. In this study we investigated whether the toxic model produced by 6-hydroxydopamine (6-OHDA) in rats comprises early noxious responses related to RAGE-mediated signaling cascades. In order to explore a possible interaction between 6-OHDA and RAGE, affinity parameters of RAGE with 6-OHDA were estimated by different means. The possible binding sites of 6-OHDA with the VC1 homodimer for both rat and human RAGE were also modeled. Our results show that the striatal infusion of 6-OHDA recruits RAGE upregulation, as evidenced by an early expression of the receptor. 6-OHDA was also found to bind the VC1 homodimer, although its affinity was moderate when compared to other ligands. This work contributes to the understanding of the role of RAGE activation for 6-OHDA-induced neurotoxicity.

Neurotransmitter GABA activates muscle but not α7 nicotinic receptors.

Cys-loop receptors are neurotransmitter-activated ion channels involved in synaptic and extrasynaptic transmission in the brain and are also present in non-neuronal cells. As GABAA and nicotinic receptors (nAChR) belong to this family, we explored by macroscopic and single-channel recordings whether the inhibitory neurotransmitter GABA has the ability to activate excitatory nAChRs. GABA differentially activates nAChR subtypes. It activates muscle nAChRs, with maximal peak currents of about 10% of those elicited by acetylcholine (ACh) and 15-fold higher EC50 with respect to ACh. At the single-channel level, the weak agonism is revealed by the requirement of 20-fold higher concentration of GABA for detectable channel openings, a major population of brief openings, and absence of clusters of openings when compared with ACh. Mutations at key residues of the principal binding-site face of muscle nAChRs (αY190 and αG153) affect GABA activation similarly as ACh activation, whereas a mutation at the complementary face (εG57) shows a selective effect for GABA. Studies with subunit-lacking receptors show that GABA can activate muscle nAChRs through the α/δ interface. Interestingly, single-channel activity elicited by GABA is similar to that elicited by ACh in gain-of-function nAChR mutants associated to congenital myasthenic syndromes, which could be important in the progression of the disorders due to steady exposure to serum GABA. In contrast, GABA cannot elicit single-channel or macroscopic currents of α7 or the chimeric α7-serotonin-type 3 receptor, a feature important for preserving an adequate excitatory/inhibitory balance in the brain as well as for avoiding activation of non-neuronal receptors by serum GABA.

Expression, purification, crystallization and X-ray crystallographic studies of different redox states of the active site of thioredoxin 1 from the whiteleg shrimp Litopenaeus vannamei.

Thioredoxin (Trx) is a 12 kDa cellular redox protein that belongs to a family of small redox proteins which undergo reversible oxidation to produce a cystine disulfide bond through the transfer of reducing equivalents from the catalytic site cysteine residues (Cys32 and Cys35) to a disulfide substrate. In this study, crystals of thioredoxin 1 from the Pacific whiteleg shrimp Litopenaeus vannamei (LvTrx) were successfully obtained. One data set was collected from each of four crystals at 100 K and the three-dimensional structures of the catalytic cysteines in different redox states were determined: reduced and oxidized forms at 2.00 Šresolution using data collected at a synchrotron-radiation source and two partially reduced structures at 1.54 and 1.88 Šresolution using data collected using an in-house source. All of the crystals belonged to space group P3212, with unit-cell parameters a = 57.5 (4), b = 57.5 (4), c = 118.1 (8) Å. The asymmetric unit contains two subunits of LvTrx, with a Matthews coefficient (VM) of 2.31 Å(3) Da(-1) and a solvent content of 46%. Initial phases were determined by molecular replacement using the crystallographic model of Trx from Drosophila melanogaster as a template. In the present work, LvTrx was overexpressed in Escherichia coli, purified and crystallized. Structural analysis of the different redox states at the Trx active site highlights its reactivity and corroborates the existence of a dimer in the crystal. In the crystallographic structures the dimer is stabilized by several interactions, including a disulfide bridge between Cys73 of each LvTrx monomer, a hydrogen bond between the side chain of Asp60 of each monomer and several hydrophobic interactions, with a noncrystallographic twofold axis.

The disordered N-terminal region of dengue virus capsid protein contains a lipid-droplet-binding motif.

Dengue is the major arthropod-borne human viral disease, for which no vaccine or specific treatment is available. We used NMR, zeta potential measurements and atomic force microscopy to study the structural features of the interaction between dengue virus C (capsid) protein and LDs (lipid droplets), organelles crucial for infectious particle formation. C protein-binding sites to LD were mapped, revealing a new function for a conserved segment in the N-terminal disordered region and indicating that conformational selection is involved in recognition. The results suggest that the positively charged N-terminal region of C protein prompts the interaction with negatively charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to the LD surface. Taken together, the results allowed the design of a peptide with inhibitory activity of C protein-LD binding, paving the way for new drug development approaches against dengue.

N,N'-dicyclohexylsulfamide and N,N'-diphenethylsulfamide are anticonvulsant sulfamides with affinity for the benzodiazepine binding site of the GABA(A) receptor and anxiolytic activity in mice.

A set of sulfamides designed, synthesized and evaluated against maximal electroshock seizure (MES) and pentilenetetrazol (PTZ) tests with promising results, were tested for their affinity for the benzodiazepine binding site of the GABA(A) receptor. The most active compounds, N,N'-dicyclohexylsulfamide (7) and N,N'-diphenethylsulfamide (10), competitively inhibited the binding of [(3)H]-flunitrazepam to the benzodiazepine binding site with K(i)±SEM values of 27.7±4.5µM (n=3) and 6.0±1.2µM (n=3), respectively. The behavioral actions of these sulfamides, i.p. administered in mice, were examined in the plus-maze, hole-board and locomotor activity assays. Compound 7 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the hole-board test (at 1 and 3mg/kg) and the plus-maze assay (at 1 and 3mg/kg). Compound 10 evidenced anxiolytic activity in the plus-maze and the hole-board tests at 1mg/kg. Locomotor activity of mice was not modified by compound 7 or 10 at the doses tested. Flumazenil, a non selective benzodiazepine binding site antagonist, was able to completely reverse the anxiolytic-like effects of these sulfamides, proving that the GABA(A) receptor is implicated in this action. Anxiety represents a major problem for people with epilepsy. The use of anxiolytic and anticonvulsant sulfamides would be beneficial to individuals who suffer from both disorders.

Functional relationships between agonist binding sites and coupling regions of homomeric Cys-loop receptors.

Each subunit in a homopentameric Cys-loop receptor contains a specialized coupling region positioned between the agonist binding domain and the ion conductive channel. To determine the contribution of each coupling region to the stability of the open channel, we constructed a receptor subunit (α7-5-HT(3A)) with both a disabled coupling region and a reporter mutation that alters unitary conductance, and coexpressed normal and mutant subunits. The resulting receptors show single-channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of the number of intact coupling regions with mean open time. We find that each coupling region contributes an equal increment to the stability of the open channel. However, by altering the numbers and locations of active coupling regions and binding sites, we find that a coupling region in a subunit flanked by inactive binding sites can still stabilize the open channel. We also determine minimal requirements for channel opening regardless of stability and find that channel opening can occur in a receptor with one active coupling region flanked by functional binding sites or with one active binding site flanked by functional coupling regions. The overall findings show that, whereas the agonist binding sites contribute interdependently and asymmetrically to open-channel stability, the coupling regions contribute independently and symmetrically.

RNA recognition motifs involved in nuclear import of RNA-binding proteins.

In eukaryotic cells, a regulated import and export of factors is required to fulfill the requirements of precise gene expression. Post-transcriptional regulation of gene expression has proven to provide ubiquitous control, as well as a quick response to environmental changes when required. RNA-binding proteins (RBP) are involved in the several steps at which mRNA biogenesis, stability, translation and decay is exerted. The most characterized RBPs contain single or multiple copies of an RNA Recognition Motif (RRM). Here, we concentrate on RRMs mediating protein nuclear import by virtue of its ability to interact with proteins, besides interacting with nucleic acids. The consensus on how RRM-protein interactions take place is non-existent, and so is the involvement of the RRM as a nuclear localization signal (NLS). Within the cases examined, the single RRM from a trypanosome RBP behaves as a structural NLS, alternating nuclear import and RNA-binding.

The ArcB sensor kinase of Escherichia coli autophosphorylates by an intramolecular reaction.

The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to the respiratory conditions of growth. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here we show that ArcB autophosphorylates through an intramolecular reaction which diverges from the usually envisaged intermolecular autophosphorylation of homodimeric histidine kinases.