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Hepatic steatosis is strongly associated with chronic liver disease and systemic metabolic disorder. Adipose lipolysis is a recognized principal source of intrahepatic fat in various metabolic disorders, including non-alcoholic fatty liver disease. We hypothesized that, in the premorbid state, hepatic de novo lipogenesis (DNL) driven by excess carbohydrates abundance might play a more significant role. We employed a novel nutritional model in sheep of two distinct carbohydrates abundances. During 4 months of the dietary treatment, lambs were monitored for metabolic and terminal liver parameters. Lambs grown on the high-calorie (HC) diet were consistently more hyperglycemic and hyperinsulinemic than lambs grown on the lower-calorie (LC) diet (P P P P P CCL2 (P TNF-alpha (P IL1B trended higher (P P P

Gene augmentation therapy based on subretinal delivery of adeno-associated viral (AAV) vectors is proving to be highly efficient in treating several inherited retinal degenerations. However, due to potential complications and drawbacks posed by subretinal injections, there is a great impetus to find alternative methods of delivering the desired genetic inserts to the retina. One such method is an intravitreal delivery of the vector. Our aim was to evaluate the efficacy of two capsid-modified vectors that are less susceptible to cellular degradation, AAV8 (doubleY-F) and AAV2 (quadY-F+T-V), as well as a third, chimeric vector AAV[max], to transduce photoreceptor cells following intravitreal injection in sheep. We further tested whether saturation of inner limiting membrane (ILM) viral binding sites using a nonmodified vector, before the intravitreal injection, would enhance the efficacy of photoreceptor transduction. Only AAV[max] resulted in moderate photoreceptor transduction following intravitreal injection. Intravitreal injection of the two other vectors did not result in photoreceptor transduction nor did the saturation of the ILM before the intravitreal injection. However, two of the vectors efficiently transduced photoreceptor cells following subretinal injection in positive control eyes. Previous trials with the same vectors in both murine and canine models resulted in robust and moderate transduction efficacy, respectively, of photoreceptors following intravitreal delivery, demonstrating the importance of utilizing as many animal models as possible when evaluating new strategies for retinal gene therapy. The successful photoreceptor transduction of AAV[max] injected intravitreally makes it a potential candidate for intravitreal delivery, but further trials are warranted to determine whether the transduction efficacy is sufficient for a clinical outcome.

Fatty liver is an abnormal metabolic condition of excess intrahepatic fat. This condition, referred to as hepatic steatosis, is tightly associated with chronic liver disease and systemic metabolic morbidity. The most prevalent form in humans, i.e. non-alcoholic fatty liver, generally develops due to overnutrition and sedentary lifestyle, and has as yet no approved drug therapy. Previously, we have developed a relevant large-animal model in which overnourished sheep raised on a high-calorie carbohydrate-rich diet develop hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. Here, we tested the hypothesis that treatment with thiamine (vitamin B1) can counter the development of hepatic steatosis driven by overnutrition. Remarkably, the thiamine-treated animals presented with completely normal levels of intrahepatic fat, despite consuming the same amount of liver-fattening diet. Thiamine treatment also decreased hyperglycemia and increased the glycogen content of the liver, but it did not improve insulin sensitivity, suggesting that steatosis can be addressed independently of targeting insulin resistance. Thiamine increased the catalytic capacity for hepatic oxidation of carbohydrates and fatty acids. However, at gene-expression levels, more-pronounced effects were observed on lipid-droplet formation and lipidation of very-low-density lipoprotein, suggesting that thiamine affects lipid metabolism not only through its known classic coenzyme roles. This discovery of the potent anti-steatotic effect of thiamine may prove clinically useful in managing fatty liver-related disorders. This article has an associated First Person interview with the joint first authors of the paper., Editor's choice: Experiments using overnourished sheep raised on a high-calorie liver-fattening diet, treated with and without thiamine, revealed that vitamin B1 protects against the development of fatty liver driven by overnutrition.

Fatty liver represents a significant metabolic pathology of excess intrahepatic fat in domestic animals and humans. Quantification of hepatic-fat content is therefore essential for diagnosis and investigation of liver and metabolic disease. However, the reproducibility of hepatic steatosis analysis is often low due to subjective and technical factors. We hypothesized that improvement in tissue-lipids extraction efficiency would contribute to the accuracy and precision of liver-fat determination. To test it, we investigated the effect of standardized tissue sonication on liver-fat quantification by the Folch method in sheep. Liver samples from grownup lambs of lean (n = 16) and fatty (n = 15) livers, and from pregnant ewes (n = 6) who died from pregnancy toxemia (PT), were used for hepatic-fat content determination with or without tissue sonication. In the grown lambs, an average hepatic-fat content of 6.6% was determined in sonicated compared to 5.1% in non-sonicated specimens (P = 0.0002). Similarly, in ewes with PT, an average of 12.5% was determined with sonication compared to 10.8% without it (P = 0.0006), and the reproducibility was higher with sonication (CV of 3.1 vs. 6.1%, respectively). Thus, tissue sonication improved the efficiency of liver-lipids extraction and was significant to the accuracy and precision of hepatic-fat determination. Enzymatic quantification of triglycerides was moderately correlated with the results obtained gravimetrically (r = 0.632, P < 0.005). The reported data provide reliable reference values for pregnancy toxemic sheep. The significant improvement in liver-fat quantification observed with the reported revised protocol is likely applicable to most mammals and humans.

Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p

The necrotrophic fungus Botrytis cinerea, is considered a major cause of postharvest losses in a wide range of crops. The common fungal extracellular membrane protein (CFEM), containing a conserved eight-cysteine pattern, was found exclusively in fungi. Previous studies in phytopathogenic fungi have demonstrated the role of membrane-bound and secreted CFEM-containing proteins in different aspects of fungal virulence. However, non-G protein-coupled receptor (non-GPCR) membrane CFEM proteins have not been studied yet in phytopathogenic fungi. In the present study, we have identified a non-GPCR membrane-bound CFEM-containing protein, Bcin07g03260, in the B. cinerea genome, and generated deletion mutants, &Delta, CFEM-Bcin07g03260, to study its potential role in physiology and virulence. Three independent &Delta, CFEM-Bcin07g03260 mutants showed significantly reduced progression of a necrotic lesion on tomato (Solanum lycopersicum) leaves. Further analysis of the mutants revealed significant reduction (approximately 20&ndash, 30%) in conidial germination and consequent germ tube elongation compared with the WT. Our data complements a previous study of secreted &Delta, CFEM1 mutants of B. cinerea that showed reduced progression of necrotic lesions on leaves, without effect on germination. Considering various functions identified for CFEM proteins in fungal virulence, our work illustrates a potential new role for a non-GPCR membrane CFEM in pathogenic fungi to control virulence in the fungus B. cinerea.

As a precursor for a universal metabolic coenzyme, vitamin B1, also known as thiamine, is a vital nutrient in all living organisms. We previously found that high-dose thiamine therapy prevents overnutrition-induced hepatic steatosis in sheep by enhancing oxidative catabolism. Based on this capacity, we hypothesized that thiamine might also reduce whole-body fat and weight. To test it, we investigated the effects of high-dose thiamine treatment in sheep under overnutrition and calorie-restricted undernutrition to respectively induce positive energy balance (PEB) and negative energy balance (NEB). Eighteen mature ewes were randomly assigned to three treatment groups (n = 6 each). The control group (CG) was administered daily with subcutaneous saline, whereas the T5 and T10 groups were administered daily with equivoque of saline containing 5 mg/kg and 10 mg/kg of thiamine, respectively. Bodyweight and blood biochemistry were measured twice a week for a period of 22 days under PEB and for a consecutive 30 days under NEB. Surprisingly, despite the strong effect of thiamine on liver fat, no effect on body weight or blood glucose was detectable. Thiamine did, however, increase plasma concentration of non-esterified fatty acids (NEFA) during NEB (575.5 ± 26.7, 657.6 ± 29.9 and 704.9 ± 26.1 µEqL−1 for CG, T5, and T10, respectively: p &lt, 0.05), thereby favoring utilization of fatty acids versus carbohydrates as a source of energy. Thiamine increased serum creatinine concentrations (p &lt, 0.05), which paralleled a trending increase in urea (p = 0.09). This may indicate an increase in muscle metabolism by thiamine. Reduction of fat content by thiamine appears more specific to the liver than to adipose tissue. Additional studies are needed to evaluate the potential implications of high-dose vitamin B1 therapy in muscle metabolism.

Xylans are polymeric sugars constituting a significant part of the plant cell wall. They are usually substituted with acetyl side groups attached at positions 2 or 3 of the xylose backbone units. Acetylxylan esterases are part of the hemicellulolytic system of many microorganisms which utilize plant biomass for growth. These enzymes hydrolyze the ester linkages of the xylan acetyl groups and thus improve the accessibility of main-chain-hydrolyzing enzymes and their ability to break down the sugar backbone units. The acetylxylan esterases are therefore critically important for those microorganisms and as such could be used for a wide range of biotechnological applications. The structure of an acetylxylan esterase (Axe2) isolated from the thermophilic bacteriumGeobacillus stearothermophilusT6 has been determined, and it has been demonstrated that the wild-type enzyme is present as a unique torus-shaped octamer in the crystal and in solution. In order to understand the functional origin of this unique oligomeric structure, a series of rational noncatalytic, site-specific mutations have been made on Axe2. Some of these mutations led to a different dimeric form of the protein, which showed a significant reduction in catalytic activity. One of these double mutants, Axe2-Y184F-W190P, has recently been overexpressed, purified and crystallized. The best crystals obtained belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 71.1,b= 106.0,c= 378.6 Å. A full diffraction data set to 2.3 Å resolution has been collected from a flash-cooled crystal of this type at 100 K using synchrotron radiation. This data set is currently being used for the three-dimensional structure analysis of the Axe2-Y184F-W190P mutant in its dimeric form.

Pathogenic microorganisms must cope with extremely low free-iron concentrations in the host's tissues. Some fungal pathogens rely on secreted haemophores that belong to the Common in Fungal Extracellular Membrane (CFEM) protein family, to extract haem from haemoglobin and to transfer it to the cell's interior, where it can serve as a source of iron. Here we report the first three-dimensional structure of a CFEM protein, the haemophore Csa2 secreted by Candida albicans. The CFEM domain adopts a novel helical-basket fold that consists of six α-helices, and is uniquely stabilized by four disulfide bonds formed by its eight signature cysteines. The planar haem molecule is bound between a flat hydrophobic platform located on top of the helical basket and a peripheral N-terminal ‘handle’ extension. Exceptionally, an aspartic residue serves as the CFEM axial ligand, and so confers coordination of Fe3+ haem, but not of Fe2+ haem. Histidine substitution mutants of this conserved Asp acquired Fe2+ haem binding and retained the capacity to extract haem from haemoglobin. However, His-substituted CFEM proteins were not functional in vivo and showed disturbed haem exchange in vitro, which suggests a role for the oxidation-state-specific Asp coordination in haem acquisition by CFEM proteins. The first crystal structure of a fungal secreted haemophore—Csa2 from Candida albicans—provides insight into how pathogenic fungi extract haem from haemoglobin and transfer it to the cytoplasm, where it serves as a source of iron.

Hypercholesterolemia, high serum cholesterol in the form of LDL, is a major risk factor for atherosclerosis. LDL is mostly degraded in the liver after its cellular internalization with the LDL receptor (LDLR). This clathrin-mediated endocytosis depends on the protein autosomal recessive hypercholesterolemia (ARH), which binds the LDLR cytoplasmic tail. Mutations in either the LDLR tail or in ARH lead to hypercholesterolemia and premature atherosclerosis. Despite the significance of this interaction for cholesterol homeostasis, no structure of either ARH or the LDLR tail is available to determine its molecular basis. We report the crystal structure at 1.37-Å resolution of the phosphotyrosine-binding (PTB) domain of ARH in complex with an LDLR tail peptide containing the FxNPxY 0 internalization signal. Surprisingly, ARH interacts with a longer portion of the tail than previously recognized, which extends to I -7 xF -5 xNPxY 0 QK +2 . The LDLR tail assumes a unique “Hook”-like structure with a double β-turn conformation, which is accommodated in distinctive ARH structural determinants (i.e., an extended backbone hydrogen-bonding platform, three hydrophobic helical grooves, and a hydrophobic pocket for Y 0 ). This unique complementarity differs significantly in related PTB proteins and may account for the unique physiological role of these partners in the hepatic uptake of cholesterol LDL. Moreover, the unusual hydrophobic pocket for Y 0 explains the distinctive ability of ARH to internalize proteins containing either FxNPxY 0 or FxNPxF 0 sequences. Biophysical measurements reveal how mutations associated with hypercholesterolemia destabilize ARH and its complex with LDLR and illuminate LDL internalization defects seen in patients.

By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer’s disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge.

CD1 proteins present self- and foreign lipid Ags to activate specific T cells in the mammalian immune system. These T cells play an important role in controlling autoimmune diseases, suppression of tumor growth, and host defense against invading pathogens. Humans use five CD1 isoforms, whereas only two exist in birds. Unlike mammals’ CD1, the structure of chicken CD1-2 showed a primitive lipid-binding groove, suggesting that chicken may only recognize single-chain lipids. In contrast, the crystal structure of the second chicken CD1 isoform, chCD1-1, reported in this study at 2.2 Å resolution, reveals an elaborated binding groove with a dual-pocket, dual-cleft architecture. The A’ and F’ deep pockets are separated from each other, but each is connected to a hydrophobic surface cleft, which may participate in lipid binding. The long endogenous ligand found inside the binding groove of chCD1-1, together with binding data on various glycolipids and mycolic acid, strongly suggest that the unique avian CD1 family could bind long dual- and possibly triacyl-chain lipids.

Mistic represents a family of unique membrane-associating proteins originally found in Bacillus subtilis (M110). As a fusion partner, it has been shown to assist overexpression of foreign integral membrane proteins in E. coli. We have expressed shorter Mistic homologs from other Bacillus species and surprisingly, unlike M110, found them abundant in the cytoplasm. These Mistic homologs including the corresponding shorter sequence (amino acids 27 through 110 of M110) exist as multimeric assemblies in solution in the absence of detergent. Crystals of Mistic from B. leicheniformis (M2) diffracted to 3.2 A resolution, indicating that it exists as a multimer in the crystalline state as well. Moreover, we show that although M2 is mostly α-helical, it tends to polymerize and form fibrils. Such oligomerization could potentially mask the charged surface of the monomeric Mistic to assist membrane integration.

3-D Structure of Serum Paraoxonase 1 Sheds Light on Its Activity, Stability, Solubility and CrystallizabilitySerum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the "good cholesterol"), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1's active site and suggests possible mechanisms for its catalytic activity on certain substrates.

Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-beta-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the approximately 200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 A resolution. The catalytic domain consists of a (beta/alpha)(8) TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest alpha-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.

Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 A resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE approximately 6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds approximately 3- and approximately 2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.

Quantum chemical DFT-B3LYP/6-31G* method and IR spectrometry have been used to investigate the natural and binding structures of Huperzine B (HupB) in order to better understand the interaction nature between acetylcholinesterase (AChE) and its inhibitor, with the view of designing new AChE inhibitors. The predicted and experimental results reveal that both the natural state and binding form of HupB adopt the chair conformation. Furthermore, the B3LYP/6-31G* results suggest that structure S1 should be the dominant form of the two possible chair structures (S1 and S2, Fig. 2). The calculated results also show that the condensed ring structure composing of rings A, B and C is very rigid. Therefore, its flexibility does not need to be considered when we try to dock this structure to its target. Indeed, this supposition is confirmed by the excellent alignment of the binding structure produced from our recent/break X-ray crystallographic structure of the HupB-AChE complex with the B3LYP/6-31G* predicted geometry. Among all the 111 predicted vibrational bands, the mode 110, which is resulted from the stretching of the bond N2–H and having the second highest frequency, is essential for the geometrical identification. The difference between our predicted strongest absorption band and experimental IR spectrum suggests that a strong intermolecular interaction, which could be a hydrogen bond, exists in HupB crystal. The electrostatic potential surface of HupB derived from our B3LYP/6-31G* CHelpG atomic charge suggests a mechanism of how HupB would interact with its target. In addition, the good agreement between predicted vibrational bands (scaled by a factor of 0.96) and experimental result shows that B3LYP/6-31G* is a good tool for studying such kind of natural compound.

Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 A resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.

Geobacillus stearothermophilusT6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombicP212121space group, with average unit-cell parametersa = 97.7,b= 119.1,c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

Geobacillus stearothermophilus T1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems in G. stearothermophilus T1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure–function study. The best crystals obtained for this enzyme belonged to the triclinic space group P1, with average crystallographic unit-cell parameters of a = 67.0, b = 78.1, c = 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.

RCK is a cytoplasmic regulatory domain of calcium-gated potassium channels. Binding of Ca(2+) by RCK leads to channel activation through a series of yet unknown conformational changes. Structures of the K(+) channel, MthK, and its cytoplasmic RCK domain revealed two binding sites for Ca(2+) per dimer. We determined the crystal structure of RCK in complex with Cd(2+) at 2.2A resolution. Cd(2+) activates MthK more efficiently, and binds at the same binding sites for Ca(2+) but with reduced coordination number. Two additional binding sites for Cd(2+) are found per dimer; one on the main Rossman-fold lobe, and the other on the small lobe of RCK. Using patch-clamp experiments, we demonstrate that Cd(2+) binding to these novel sites enhances activation by Cd(2+) and not by Ca(2+). The structure reveals a large negatively charged surface patch in the proximity of the Ca(2+)/Cd(2+) binding sites, charge neutralization of which appears to promote the channel open state.

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein-gated inwardly rectifying K(+) channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 engineered to strongly bind PIP(2). Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K(+) channels suggests a model for activation of GIRK channels using this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

Serum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the “good cholesterol”), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1’s active site and suggests possible mechanisms for its catalytic activity on certain substrates., Serumske paraoksonaze (PONs) imaju široki raspon fiziološki važnih hidrolitičkih aktivnosti uključujući metabolizam lijekova i detoksikaciju nervnih plinova. PON1 i PON3 smještene su na lipoproteinima visoke gustoće (engl. high-density lipoprotein; HDL - “dobri kolesterol”) i uključene su u ublažavanje ateroskleroze. Članovi skupine PON identificirani su ne samo u sisavaca i drugih kralježnjaka već i kod beskralješnjaka. Prije smo opisali prvu kristalnu strukturu člana PON skupine, direktno razrađenu varijantu PON1 pri rezoluciji 2,2 Å. PON1 je beta-propeler sa šest lopatica s jedinstvenim poklopcem aktivnog mjesta, koji je tako|er uključen u vezanje na HDL. 3-D struktura, gledana zajedno s direktnim razvojnim istraživanjima, omogućila je analizu mutacija koje povećavaju stabilnost, topljivost i kristalizaciju te PON1 varijante. Struktura dopušta detaljan opis aktivnog mjesta PON1 i sugerira moguće mehanizme za njezinu katalitičku aktivnost prema odre|enim supstratima.

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.

The structure of Torpedo californica acetylcholinesterase is examined in complex with several inhibitors that are either in use or under development for treating Alzheimer's disease. The noncovalent inhibitors vary greatly in their structures and bind to different sites of the enzyme, offering many different starting points for future drug design.

In addition to G proteins, ethanol can activate G protein-gated inwardly rectifying K (GIRK) channels. The mechanism underlying GIRK channel activation by alcohol is not well understood. Based on a crystal structure of a related IRK1 channel which contains the alcohol (2-methyl,2-4-pentanediol- MPD) bound to a cytoplasmic hydrophobic pocket, we used structure-based mutagenesis and patch-clamp electrophysiology to investigate the role of the homologous alcohol pocket in GIRK2 channels. In HEK293T cells transfected with GIRK2 cDNA, both ethanol and MPD activated GIRK2 channels. Replacing a conserved Leucine (L257) in this pocket with a bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation of GIRK2 channels, suggesting these larger hydrophobic side-chains filled the pocket. Based on structure and functional evidence, we conclude that this hydrophobic pocket is the site for alcohol activation of GIRK channels. We hypothesized that tethering a hydrophobic group near the pocket might mimic alcohol mediated activation of the channel. To test this idea, we introduced a S246C mutation in a Cysteine-less GIRK2 channel and examined the effect of bath applied MTS-Benzene. Application of 10 micromolar MTS-Benzene dramatically increased the size of basal GIRK currents by 336+66% n=5. This rapid activation was reversed by application of reducing agent DTT (10 mM), indicating a disulfide bond had formed. In addition to the change in basal current, MTS modification of S246C channel altered the rank order for alcohol activation -with significantly less activation by the larger alcohol MPD. These results suggest that attachment of a bulky hydrophobic amino acid near the hydrophobic alcohol-binding pocket can produce sustained activation of the channel by associating with the activation site. These experiments provide a launching point to study molecular events at this hydrophobic pocket that lead to activation of GIRK channels.

When these same mutations are incorporated into the recombinant PON1 variant G2E6, the structure of which was determined and reported in the article, and into another recombinant PON1 variant (G3C9; see Table 1 of the article), we obtained similar activity data as above (not shown). These results indicate that His115 and His134 are responsible for the aryl-esterase activity but not the paraoxonase activity of PON1. Thus, the two hydrolytic activities may be catalyzed by different active site residues, and the model postulated in the article may be valid only for the aryl-esterase activity. We apologize for any inconvenience this may have caused.

Alcohols can activate G protein-gated inwardly rectifying K (GIRK) channels but the molecular mechanism is not well understood. To investigate the possibility of a physical alcohol pocket located in the cytoplasmic domain of GIRK channels, we used a crystal structure of related IRK1 channel containing a bound alcohol (2-methyl, 2-4-pentanediol - MPD) and structure-based mutagenesis of GIRK2 and GIRK4 channels combined with patch-clamp electrophysiology. In transiently transfected HEK293 cells, both wild-type GIRK2 and GIRK4 channels were activated by 100 mM ethanol, MPD and 1-Propanol. Replacing a conserved Leucine (GIRK2-L257 / GIRK4-L252) in the betaD-betaE region of the cytoplasmic domain with bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation for both GIRK2 and GIRK4 channels. Constitutively open channels, such as IRK1 and GIRK2-PIP2 (engineered to bind PIP2 with high affinity), on the other hand, were inhibited by ethanol, 1-propanol, 1-butanol and MPD. Mutating the homologous Leucine in IRK1 (L257) or in GIRK2-PIP2 channels did not alter the sensitivity to inhibition by these alcohols, suggesting a second site in the channel is involved in inhibition by alcohols. Consistent with this, mutagenesis of the extracellular pore-helix of GIRK4 and GIRK2-PIP2 channels reduced the sensitivity to alcohol-mediated inhibition. Interestingly, mutation of the conserved Leucine (L257/L252) in the betaD-betaE domain also disrupted G protein-dependent activation, suggesting a common mechanism of activation by alcohols and G-proteins. Using our data and an analysis of high-resolution structures of inwardly rectifying K channels, we propose a novel model for alcohol activation of GIRK channels that is mediated by the cytoplasmic hydrophobic alcohol pocket.