Journal of Structural Biology

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Structural insights on the catalytic site protection of human carbonyl reductase 1 by glutathione

Wed, 09/30/2015 - 03:56
Publication date: Available online 14 September 2015
Source:Journal of Structural Biology

Author(s): Qingnan Liang, Rui Liu, Shuqi Du, Yu Ding

The NADPH-dependent human carbonyl reductase 1 (hCBR1), a member of the short-chain dehydrogenase/reductase protein family, plays an important role in the ubiquitous metabolism of endogenous and xenobiotic carbonyl containing compounds. Glutathione (GSH) is also a cofactor of hCBR1, however, its role in the carbonyl reductase function of the enzyme is still unclear. In this study, we presented the crystal structure of hCBR1 in complex with GSH, in the absence of its substrates or inhibitors. Interestingly, we found that the GSH molecule presents in a configuration quite different from that was previously reported when substrate is binding to hCBR1. Our structure indicates that GSH contributes to the substrate selectivity of hCBR1 and protects the catalytic center of hCBR1 through a switch-like mechanism. The isothermal titration calorimetry and enzymology data shows that GSH directly binding with hCBR1 when there’s no substrate exist. The enzymology data also shows GSH protects NADPH being attacked by oxidative small molecules. This is the first time that GSH is found to demonstrate such functions as a co-enzyme. Our crystal structure succeeds in providing critical insights into the substrate selectivity of hCBR1 and the interaction between hCBR1 and GSH.





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Asynchronous data acquisition and on-the-fly analysis of dose fractionated cryoEM images by UCSFImage

Wed, 09/30/2015 - 03:56
Publication date: Available online 11 September 2015
Source:Journal of Structural Biology

Author(s): Xueming Li, Shawn Zheng, David A. Agard, Yifan Cheng

Newly developed direct electron detection cameras have a high image output frame rate that enables recording dose fractionated image stacks of frozen hydrated biological samples by electron cryomicroscopy (cryoEM). Such novel image acquisition schemes provide opportunities to analyze cryoEM data in ways that were previously impossible. The file size of a dose fractionated image stack is 20–60 times larger than that of a single image. Thus, efficient data acquisition and on-the-fly analysis of a large number of dose-fractionated image stacks become a serious challenge to any cryoEM data acquisition system. We have developed a computer-assisted system, named UCSFImage4, for semi-automated cryo-EM image acquisition that implements an asynchronous data acquisition scheme. This facilitates efficient acquisition, on-the-fly motion correction, and CTF analysis of dose fractionated image stacks with a total time of ∼60s/exposure. Here we report the technical details and configuration of this system.





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The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-di-GMP receptor and flagella gene regulator

Wed, 09/30/2015 - 03:56
Publication date: Available online 8 September 2015
Source:Journal of Structural Biology

Author(s): Tiantian Su, Shiheng Liu, Kang Wang, Kaikai Chi, Deyu Zhu, Tiandi Wei, Yan Huang, Liming Guo, Wei Hu, Sujuan Xu, Zong Lin, Lichuan Gu

FleQ is an AAA+ ATPase enhancer-binding protein that regulates both flagella and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. FleQ belongs to the NtrC subfamily of response regulators, but lacks the corresponding aspartic acid for phosphorylation in the REC domain (FleQR, also named FleQ domain). Here, we show that the atypical REC domain of FleQ is essential for the function of FleQ. Crystal structure of FleQR at 2.3Å reveals that the structure of FleQR is significantly different from the REC domain of NtrC1 which regulates gene expression in a phosphorylation dependent manner. FleQR forms a novel active dimer (transverse dimer), and mediates the dimerization of full-length FleQ in an unusual manner. Point mutations that affect the dimerization of FleQ lead to loss of function of the protein. Moreover, a c-di-GMP binding site deviating from the previous reported one is identified through structure analysis and point mutations.





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Investigating interactions of the Bacillus subtilis spore coat proteins CotY and CotZ using single molecule force spectroscopy

Wed, 09/30/2015 - 03:56
Publication date: Available online 2 September 2015
Source:Journal of Structural Biology

Author(s): Huiqing Liu, Daniela Krajcikova, Zhe Zhang, Hongda Wang, Imrich Barak, Jilin Tang

Spores formed by Bacillus subtilis are surrounded by a protective and multilayered shell, termed the coat, which grants the spores resistance to various environmental stresses and facilitates spore germination. The spore coat consists of more than seventy different proteins, arranged into at least four distinct structural layers: the undercoat, inner coat, outer coat and crust. However, how these proteins, especially the morphogenetic proteins, interact to establish the organized, functional coat layers remains poorly understood. CotY and CotZ as the components of the crust, play a morphogenetic role in the crust assembly around the spore. In this study, the single molecule force spectroscopy was used to investigate the interaction and dynamics between CotY and CotZ at the single-molecule level. The results show that homotypic interactions of CotY and CotZ and the heterotypic interaction between CotY and CotZ exist. Furthermore, the dissociation kinetics of the complexes were studied by monitoring the relationship between the unbinding forces and the loading rates at different pulling velocities. In this way, a series of kinetic parameters regarding the three different complexes were obtained. It revealed the strong interactions between CotY and CotZ, CotY and CotY, and a relatively weak interaction of CotZ and CotZ.





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Cover 2 - Editorial Board

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3









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Table of Contents / barcode

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3









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Austromegabalanus psittacus barnacle shell structure and proteoglycan localization and functionality

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): M.S. Fernández, J.I. Arias, A. Neira-Carrillo, J.L. Arias

Comparative analyzes of biomineralization models have being crucial for the understanding of the functional properties of biominerals and the elucidation of the processes through which biomacromolecules control the synthesis and structural organization of inorganic mineral-based biomaterials. Among calcium carbonate-containing bioceramics, egg, mollusk and echinoderm shells, and crustacean carapaces, have being fairly well characterized. However, Thoraceca barnacles, although being crustacea, showing molting cycle, build a quite stable and heavily mineralized shell that completely surround the animal, which is for life firmly cemented to the substratum. This makes barnacles an interesting model for studying processes of biomineralization. Here we studied the main microstructural and ultrastructural features of Austromegabalanus psittacus barnacle shell, characterize the occurrence of specific proteoglycans (keratan-, dermatan- and chondroitin-6-sulfate proteoglycans) in different soluble and insoluble organic fractions extracted from the shell, and tested them for their ability to crystallize calcium carbonate in vitro. Our results indicate that, in the barnacle model, proteoglycans are good candidates for the modification of the calcite crystal morphology, although the cooperative effect of some additional proteins in the shell could not be excluded.





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Exploring the ‘aggregation-prone’ core of human Cystatin C: A structural study

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Paraskevi L. Tsiolaki, Nikolaos N. Louros, Stavros J. Hamodrakas, Vassiliki A. Iconomidou

Amyloidogenic proteins like human Cystatin C (hCC) have been shown to form dimers and oligomers by exchange of subdomains of the monomeric proteins. Normally, the hCC monomer, a low molecular type 2 Cystatin, consists of 120 amino acid residues and functions as an inhibitor of cysteine proteases. The oligomerization of hCC is involved in the pathophysiology of a rare form of amyloidosis namely Icelandic hereditary cerebral amyloid angiopathy, in which an L68Q mutant is deposited as amyloid in brain arteries of young adults. In order to find the shortest stretch responsible to drive the fibril formation of hCC, we have previously demonstrated that the LQVVR peptide forms amyloid fibrils, in vitro (Tsiolaki et al., 2015). Predictions by AMYLPRED, an amyloidogenic determinant prediction algorithm developed in our lab, led us to synthesize and experimentally study two additional predicted peptides derived from hCC. Along with our previous findings, in this work, we reveal that these peptides self-assemble, in a similar way, into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies have shown. Further to our experimental results, all three peptides seem to have a fundamental contribution in forming the “aggregation-prone” core of human Cystatin C.





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The Ku–Mar zinc finger: A segment-swapped zinc ribbon in MarR-like transcription regulators related to the Ku bridge

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Gurmeet Kaur, Srikrishna Subramanian

Two putative oxidative-stress sensor proteins from Pseudomonas aeruginosa, PA1607 and PA1374, belong to the MarR family of transcription regulators and possess a unique mode of dimerization. In these proteins, in addition to the α-helices involved in dimerization, inter-subunit contacts are strengthened by additional C-terminal β-strands. Using sequence and structure analysis we show that these β-strands constitute a novel segment-swapped zinc ribbon domain. We detect the presence of the zinc ribbon domain in MarR proteins from many bacterial homologs. While the metal-chelating residues of the zinc ribbons are absent in most members of this family, we could however identify several species of Proteobacteria, Actinobacteria and Firmicutes that possess intact zinc-chelating sites. Conservation pattern of metal-chelating residues together with the extensive structural resemblance to zinc ribbons, in particular to the bridge-region of the dsDNA break repair protein Ku, suggests that the C-terminal β-rich region of these proteins is a zinc ribbon. Sequence analysis also supports a distant evolutionary connection between the zinc ribbons of the MarR and Ku families. However, unlike Ku where the segment-swapped zinc ribbons play a role in DNA-binding and obligate dimerization, their primary role in MarR appears to be in dimerization and strengthening of inter-subunit contacts.





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The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Jochen Wuerges, Lorenzo Caputi, Michele Cianci, Stephane Boivin, Rob Meijers, Stefano Benini

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site.





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Effect of fringe-artifact correction on sub-tomogram averaging from Zernike phase-plate cryo-TEM

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Gregory P. Kishchenko, Radostin Danev, Rebecca Fisher, Jie He, Chyongere Hsieh, Michael Marko, Haixin Sui

Zernike phase-plate (ZPP) imaging greatly increases contrast in cryo-electron microscopy, however fringe artifacts appear in the images. A computational de-fringing method has been proposed, but it has not been widely employed, perhaps because the importance of de-fringing has not been clearly demonstrated. For testing purposes, we employed Zernike phase-plate imaging in a cryo-electron tomographic study of radial-spoke complexes attached to microtubule doublets. We found that the contrast enhancement by ZPP imaging made nonlinear denoising insensitive to the filtering parameters, such that simple low-frequency band-pass filtering made the same improvement in map quality. We employed sub-tomogram averaging, which compensates for the effect of the “missing wedge” and considerably improves map quality. We found that fringes (caused by the abrupt cut-on of the central hole in the phase plate) can lead to incorrect representation of a structure that is well-known from the literature. The expected structure was restored by amplitude scaling, as proposed in the literature. Our results show that de-fringing is an important part of image-processing for cryo-electron tomography of macromolecular complexes with ZPP imaging.





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Complementarity and congruence between exact NOEs and traditional NMR probes for spatial decoding of protein dynamics

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Beat Vögeli, Simon Olsson, Roland Riek, Peter Güntert

The study of the spatial sampling of biomolecules is essential to understanding the structure–dynamics–function relationship. We have established a protocol for the determination of multiple-state ensembles based on exact measurements of the nuclear Overhauser effect (eNOE). The protocol is practical since it does not require any additional data, while all other NMR data sets must be supplemented by NOE restraints. The question arises as to how much structural and dynamics information is shared between the eNOEs and other NMR probes. We compile one of the largest and most diverse NMR data sets of a protein to date consisting of eNOEs, RDCs and J couplings for GB3. We show that the eNOEs improve the back-prediction of RDCs and J couplings, either upon use of more than one state, or in comparison to conventional NOEs. Our findings indicate that the eNOE data is self-consistent, consistent with other data, and that the structural representation with multiple states is warranted.





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A Bayesian approach for suppression of limited angular sampling artifacts in single particle 3D reconstruction

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Toshio Moriya, Erman Acar, R. Holland Cheng, Ulla Ruotsalainen

In the single particle reconstruction, the initial 3D structure often suffers from the limited angular sampling artifact. Selecting 2D class averages of particle images generally improves the accuracy and efficiency of the reference-free 3D angle estimation, but causes an insufficient angular sampling to fill the information of the target object in the 3D frequency space. Similarly, the initial 3D structure by the random-conical tilt reconstruction has the well-known “missing cone” artifact. Here, we attempted to solve the limited angular sampling problem by sequentially applying maximum a posteriori estimate with expectation maximization algorithm (sMAP-EM). Using both simulated and experimental cryo-electron microscope images, the sMAP-EM was compared to the direct Fourier method on the basis of reconstruction error and resolution. To establish selection criteria of the final regularization weight for the sMAP-EM, the effects of noise level and sampling sparseness on the reconstructions were examined with evenly distributed sampling simulations. The frequency information filled in the missing cone of the conical tilt sampling simulations was assessed by developing new quantitative measurements. All the results of visual and numerical evaluations showed the sMAP-EM performed better than the direct Fourier method, regardless of the sampling method, noise level, and sampling sparseness. Furthermore, the frequency domain analysis demonstrated that the sMAP-EM can fill the meaningful information in the unmeasured angular space without detailed a priori knowledge of the objects. The current research demonstrated that the sMAP-EM has a high potential to facilitate the determination of 3D protein structures at near atomic-resolution.





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Different binding and recognition modes of GL479, a dual agonist of Peroxisome Proliferator-Activated Receptor α/γ

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Jademilson Celestino dos Santos, Amanda Bernardes, Letizia Giampietro, Alessandra Ammazzalorso, Barbara De Filippis, Rosa Amoroso, Igor Polikarpov

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-dependent transcription factors that control various functions in human organism, including the control of glucose and lipid metabolism. PPARγ is a target of TZD agonists, clinically used to improve insulin sensitivity whereas fibrates, PPARα ligands, lower serum triglyceride levels. We report here the structural studies of GL479, a synthetic dual PPARα/γ agonist, designed by a combination of clofibric acid skeleton and a phenyldiazenyl moiety, as bioisosteric replacement of stilbene group, in complex with both PPARα and PPARγ receptors. GL479 was previously reported as a partial agonist of PPARγ and a full agonist of PPARα with high affinity for both PPARs. Our structural studies reveal different binding modes of GL479 to PPARα and PPARγ, which may explain the distinct activation behaviors observed for each receptor. In both cases the ligand interacts with a Tyr located at helix 12 (H12), resulting in the receptor active conformation. In the complex with PPARα, GL479 occupies the same region of the ligand-binding pocket (LBP) observed for other full agonists, whereas GL479 bound to PPARγ displays a new binding mode. Our results indicate a novel region of PPARs LBP that may be explored for the design of partial agonists as well dual PPARα/γ agonists that combine, simultaneously, the therapeutic effects of the treatment of insulin resistance and dyslipidemia.





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The proteomics of wool fibre morphogenesis

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Jeffrey E. Plowman, Duane P. Harland, Sivasangary Ganeshan, Joy L. Woods, Bede van Shaijik, Santanu Deb-Choudhury, Ancy Thomas, Stefan Clerens, David R. Scobie

Gel and gel-free proteomic techniques have been used for the first time to directly study the proteins present in whole wool follicles and dissected portions of follicles that correlated with morphological changes in the developing fibre as determined by transmission electron microscopy. Individual wool follicles were dissected into four portions designated as the bulb, elongation, keratogenous and keratinisation portions. Gel-free proteomic analysis of dissected portions from 30 follicles showed that the first keratins to appear were K31, K35 and K85, in the bulb portion. The first epithelial KAP, trichohyalin, was detected in the bulb portion and the first cortical KAP, KAP11.1 was found in the elongation portion. Other major trichocyte keratins and cortical KAPs began to appear further up the follicle in the keratogenous and keratinisation zones. These results were consistent with what has been observed from gene expression studies and correlated well with the morphological changes observed in the follicle. Other proteins detected by this approach included the keratin anchor protein desmoplakin, as well as vimentin and epithelial keratins, histones, ribosomal proteins and collagens. Two-dimensional electrophoretic (2DE) analysis of dissected portions of 50 follicles revealed substantial changes in the position, number and intensity of the spots of the trichocyte keratins as they progressed through the follicle zones, suggesting that they are subject to modification as a result of the keratinisation process. Also present in the 2DE maps were a number of epithelial keratins, presumably from the inner and outer root sheaths, and the dermal components.





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Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): David F. Flannelly, Thalia G. Aoki, Ludmilla Aristilde

The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs.





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High resolution structures of Plasmodium falciparum GST complexes provide novel insights into the dimer–tetramer transition and a novel ligand-binding site

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Markus Perbandt, Raphael Eberle, Lena Fischer-Riepe, Huaixing Cang, Eva Liebau, Christian Betzel

Protection from oxidative stress and efficient redox regulation are essential for malarial parasites which have to grow and multiply rapidly in pro-oxidant rich environments. Therefore, redox active proteins currently belong to the most attractive antimalarial drug targets. The glutathione S-transferase from Plasmodium falciparum (PfGST) is a redox active protein displaying a peculiar dimer–tetramer transition that causes full enzyme-inactivation. This distinct structural feature is absent in mammalian GST isoenzyme counterparts. A flexible loop between residues 113–119 has been reported to be necessary for this tetramerization process. However, here we present structural data of a modified PfGST lacking loop 113–119 at 1.9Å resolution. Our results clearly show that this loop is not essential for the formation of stable tetramers. Moreover we present for the first time the structures of both, the inactive and tetrameric state at 1.7Å and the active dimeric state in complex with reduced glutathione at 2.4Å resolution. Surprisingly, the structure of the inactive tetrameric state reveals a novel non-substrate binding-site occupied by a 2-(N-morpholino) ethane sulfonic acid (MES) molecule in each monomer. Although it is known that the PfGST has the ability to bind lipophilic anionic ligands, the location of the PfGST ligand-binding site remained unclear up to now.





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2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): David Kalbermatter, Jean-Marc Jeckelmann, Po-Lin Chiu, Zöhre Ucurum, Thomas Walz, Dimitrios Fotiadis

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IICglc) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IICglc was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IICglc into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IICglc, which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IICglc lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.





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Crystal structure of the first WW domain of human YAP2 isoform

Wed, 09/30/2015 - 03:56
Publication date: September 2015
Source:Journal of Structural Biology, Volume 191, Issue 3

Author(s): Sergio Martinez-Rodriguez, Julio Bacarizo, Irene Luque, Ana Camara-Artigas

The WW domains are the smallest modular domains known. The study of the structural basis of their stability is important to understand their physiological role. These domains are intrinsically flexible, which makes them difficult to crystallize. The first WW domain of the human Yes tyrosine kinase Associated Protein (YAP) has been crystallized and its structure has been solved by X-ray diffraction at 1.6Å resolution. Crystals belong to the orthorhombic space group P21212 with unit cell parameters a =42.67, b =43.10 and c =21.30. The addition of proline and other small-molecule additives improves drastically the quality of the crystals. The interactions that stabilize this minimal modular domain have been analysed. This crystal structure reveals that, besides the stabilization of the hydrophobic core of the protein by the aromatic cluster formed by Trp177–Phe189–Pro202, some salt-bridges interactions might affect the stability of the domain.





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Structure of neurotropic adeno-associated virus AAVrh.8

Wed, 09/30/2015 - 03:56
Publication date: Available online 31 August 2015
Source:Journal of Structural Biology

Author(s): Sujata Halder, Kim Van Vliet, J. Kennon Smith, Thao Thi Phuong Duong, Robert McKenna, James M. Wilson, Mavis Agbandje-McKenna

Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ∼84%, ∼91%, and ∼87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies and human donor serum directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment.





Categories: Journal Articles