The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 Å resolution

AbstractBackground: The allosteric hexameric enzyme glucosamine-6-phosphate deaminase from Escherichia coli catalyses the regulatory step of N-acetylglucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia. The reversibility of the catalysis and its rapid-equilibrium random kinetic mechanism, among other properties, make this enzyme a good model for studying allosteric processes.Results: Here we present the structure of P6322 crystals, obtained in sodium acetate, of GlcN6P deaminase in its ligand-free T state. These crystals are very sensitive to X-ray radiation and have a high (78%) solvent content. The active-site lid (residues 162–185) is highly disordered in the T conformer; this may contribute significantly to the free-energy change of the whole allosteric transition. Comparison of the structure with the crystallographic coordinates of the R conformer (Brookhaven Protein Data Bank entry 1dea) allows us to describe the geometrical changes associated with the allosteric transition as the movement of two rigid entities within each monomer. The active site, located in a deep cleft between these two rigid entities, presents a more open geometry in the T conformer than in the R conformer.Conclusions: The differences in active-site geometry are related to alterations in the substrate-binding properties associated with the allosteric transition. The rigid nature of the two mobile structural units of each monomer seems to be essential in order to explain the observed kinetics of the deaminase hexamer. The triggers for both the homotropic and heterotropic allosteric transitions are discussed and particular residues are assigned to these functions. A structural basis for an entropic term in the allosteric transition is an interesting new feature that emerges from this study

Two- and Three-Dimensional Quantitative Structure-Activity Relationships Studies on a Series of Liver X Receptor Ligands

Liver X receptor (LXR) is an attractive drug target for the development of novel therapeutic agents for the treatment of dyslipidaemia and cholestasis. In the present work, comparative molecular field analysis (CoMFA) and hologram quantitative structure-activity relationship (HQSAR) studies were conducted on a series of potent LXR ligands. Significant correlation coefficients (CoMFA, r2 = 0.98 and q2 = 0.69; HQSAR, r2 = 0.99 and q2 = 0.85) were obtained, indicating the potential of the models for untested compounds. The models were then used to predict the potency of an external test set, and the predicted values obtained from the 2D and 3D models were in good agreement with the experimental results. The final QSAR models, along with the information obtained from 3D steric and electrostatic contour maps and 2D contribution maps should be useful for the design of novel LXR ligands having improved potency

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.State of Sao Paulo Research Foundation (FAPESP)[1998/14138-2]State of Sao Paulo Research Foundation (FAPESP)[05/59833-5]State of Sao Paulo Research Foundation (FAPESP)[08/58316-5

Effect of cis-9, trans-11 conjugated linoleic acid (CLA) on the metabolism profile of breast cancer cells determined by H HR-MAS NMR spectroscopy

Conjugated linoleic acid (CLA), a fatty acid found in ruminant food products, has been associated with anticarcinogenic activity. However, its effect on cancer metabolism is unclear. In this paper we evaluated the effects of cis-9, trans-11 CLA on the metabolic profile of MCF-7 and MDA-MB-231 breast cancer cells using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra showed that phosphocholine level, a cancer malignance biomarker, was reduced in both cells treated with CLA, but the reduction was more pronounced in MCF-7 cells. The NMR spectra also showed that CLA has opposite effect on MCF-7 and MDA-MB-231 acetone metabolism. Acetone signal has been observed in the spectra of MDA-MB-231 control cells, but not in the spectra of the cells treated with 50 and 100 µM CLA. Conversely, the acetone signal is very small or not observed in the NMR spectra of MCF-7 control cells and in cells treated with 50 µM of CLA, but is very strong in the spectra of the cells treated with 100 µM of CLA. Therefore, this CLA concentration is causing a ketosis in MCF-7 cells by inducing the use of fatty acids as an energy source or by reducing acetone catabolism. These results indicate that CLA interfere in the metabolism of both cells. However, the strongest effect has been observed on the metabolism of MCF-7 cells cultivated in the presence of 100 µM CLA. Therefore, CLA could be a potential anticarcinogenic drug, especially for cells with positive estrogen receptor, such as MCF-7

Molecular recognition and maturation of SOD1 by its evolutionarily destabilised cognate chaperone hCCS

<div><p>Superoxide dismutase-1 (SOD1) maturation comprises a string of posttranslational modifications which transform the nascent peptide into a stable and active enzyme. The successive folding, metal ion binding, and disulphide acquisition steps in this pathway can be catalysed through a direct interaction with the copper chaperone for SOD1 (CCS). This process confers enzymatic activity and reduces access to noncanonical, aggregation-prone states. Here, we present the functional mechanisms of human copper chaperone for SOD1 (hCCS)–catalysed SOD1 activation based on crystal structures of reaction precursors, intermediates, and products. Molecular recognition of immature SOD1 by hCCS is driven by several interface interactions, which provide an extended surface upon which SOD1 folds. Induced-fit complexation is reliant on the structural plasticity of the immature SOD1 disulphide sub-loop, a characteristic which contributes to misfolding and aggregation in neurodegenerative disease. Complexation specifically stabilises the SOD1 disulphide sub-loop, priming it and the active site for copper transfer, while delaying disulphide formation and complex dissociation. Critically, a single destabilising amino acid substitution within the hCCS interface reduces hCCS homodimer affinity, creating a pool of hCCS available to interact with immature SOD1. hCCS substrate specificity, segregation between solvent and biological membranes, and interaction transience are direct results of this substitution. In this way, hCCS-catalysed SOD1 maturation is finessed to minimise copper wastage and reduce production of potentially toxic SOD1 species.</p></div

Structures of substrate- and product-bound forms of a multi-domain copper nitrite reductase shed light on the role of domain tethering in protein complexes

Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitrogen cycle where nitrate is used in place of dioxygen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd 1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering than the intuitive advantage for a partner-protein electron-transfer complex by narrowing the conformational search in such a combined system.</jats:p

Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid

Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date. After incubation for 15 days at 37 °C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation in a biologically relevant environment, opening new avenues of investigation into the molecular mechanisms of LA.CNPqFAPERJCAPE

Protecting an ecosystem service: approaches to understanding and mitigating threats to wild insect pollinators

Insect pollination constitutes an ecosystem service of global importance, providing significant economic and aesthetic benefits as well as cultural value to human society, alongside vital ecological processes in terrestrial ecosystems. It is therefore important to understand how insect pollinator populations and communities respond to rapidly changing environments if we are to maintain healthy and effective pollinator services. This paper considers the importance of conserving pollinator diversity to maintain a suite of functional traits to provide a diverse set of pollinator services. We explore how we can better understand and mitigate the factors that threaten insect pollinator richness, placing our discussion within the context of populations in predominantly agricultural landscapes in addition to urban environments. We highlight a selection of important evidence gaps, with a number of complementary research steps that can be taken to better understand: i) the stability of pollinator communities in different landscapes in order to provide diverse pollinator services; ii) how we can study the drivers of population change to mitigate the effects and support stable sources of pollinator services; and, iii) how we can manage habitats in complex landscapes to support insect pollinators and provide sustainable pollinator services for the future. We advocate a collaborative effort to gain higher quality abundance data to understand the stability of pollinator populations and predict future trends. In addition, for effective mitigation strategies to be adopted, researchers need to conduct rigorous field-testing of outcomes under different landscape settings, acknowledge the needs of end-users when developing research proposals and consider effective methods of knowledge transfer to ensure effective uptake of actions

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations