Journal of Structural Biology

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  • Cover 2 - Editorial Board
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3









    Categories: Journal Articles
  • Table of Contents / barcode
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3









    Categories: Journal Articles
  • Alpbach special issue
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): David A.D. Parry







    Categories: Journal Articles
  • Fifty years of fibrous protein research: A personal retrospective
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): David A.D. Parry

    As a result of X-ray fiber diffraction studies on fibrous proteins and crystallographic data on fragments derived from them, new experimental techniques across the biophysical and biochemical spectra, sophisticated computer modeling and refinement procedures, widespread use of bioinformatics and improved specimen preparative procedures the structures of many fibrous proteins have now been determined to at least low resolution. In so doing these structures have yielded insight into the relationship that exists between sequence and conformation and this, in turn, has led to improved methodologies for predicting structure from sequence data alone. In this personal retrospective a selection of progress made during the past 50years is discussed in terms of events to which the author has made some contribution.





    Categories: Journal Articles
  • Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Tim Kükenshöner , Daniel Wohlwend , Christoph Niemöller , Padmarupa Dondapati , Janina Speck , Adebola V. Adeniran , Anita Nieth , Stefan Gerhardt , Oliver Einsle , Kristian M. Müller , Katja M. Arndt

    The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.





    Categories: Journal Articles
  • Axial helix rotation as a mechanism for signal regulation inferred from the crystallographic analysis of the E. coli serine chemoreceptor
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Hedda U. Ferris , Kornelius Zeth , Michael Hulko , Stanislaw Dunin-Horkawicz , Andrei N. Lupas

    Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15–20nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation–demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors.





    Categories: Journal Articles
  • A soluble mutant of the transmembrane receptor Af1503 features strong changes in coiled-coil periodicity
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Marcus D. Hartmann , Stanislaw Dunin-Horkawicz , Michael Hulko , Jörg Martin , Murray Coles , Andrei N. Lupas

    Structures of full-length, membrane-bound proteins are essential for understanding transmembrane signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1 and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure offers the first view of the junction between the transmembrane region and HAMP and explains the conservation of a key sequence motif in HAMP domains.





    Categories: Journal Articles
  • A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Emma S. Gleave , Helgo Schmidt , Andrew P. Carter

    Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our S accharomyces cerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins.





    Categories: Journal Articles
  • Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Hedda U. Ferris , Murray Coles , Andrei N. Lupas , Marcus D. Hartmann

    Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.





    Categories: Journal Articles
  • Your personalized protein structure: Andrei N. Lupas fused to GCN4 adaptors
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Silvia Deiss , Birte Hernandez Alvarez , Kerstin Bär , Carolin P. Ewers , Murray Coles , Reinhard Albrecht , Marcus D. Hartmann

    This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date.





    Categories: Journal Articles
  • TRDistiller: A rapid filter for enrichment of sequence datasets with proteins containing tandem repeats
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): François D. Richard , Andrey V. Kajava

    The dramatic growth of sequencing data evokes an urgent need to improve bioinformatics tools for large-scale proteome analysis. Over the last two decades, the foremost efforts of computer scientists were devoted to proteins with aperiodic sequences having globular 3D structures. However, a large portion of proteins contain periodic sequences representing arrays of repeats that are directly adjacent to each other (so called tandem repeats or TRs). These proteins frequently fold into elongated fibrous structures carrying different fundamental functions. Algorithms specific to the analysis of these regions are urgently required since the conventional approaches developed for globular domains have had limited success when applied to the TR regions. The protein TRs are frequently not perfect, containing a number of mutations, and some of them cannot be easily identified. To detect such “hidden” repeats several algorithms have been developed. However, the most sensitive among them are time-consuming and, therefore, inappropriate for large scale proteome analysis. To speed up the TR detection we developed a rapid filter that is based on the comparison of composition and order of short strings in the adjacent sequence motifs. Tests show that our filter discards up to 22.5% of proteins which are known to be without TRs while keeping almost all (99.2%) TR-containing sequences. Thus, we are able to decrease the size of the initial sequence dataset enriching it with TR-containing proteins which allows a faster subsequent TR detection by other methods. The program is available upon request.





    Categories: Journal Articles
  • The spectrin family of proteins: A unique coiled-coil fold for various molecular surface properties
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Aurélie Nicolas , Olivier Delalande , Jean-François Hubert , Elisabeth Le Rumeur

    The spectrin superfamily is composed of proteins involved in cytolinker functions. Their main structural feature is a large central subdomain with numerous repeats folded in triple helical coiled-coils. Their similarity of sequence was considered to be low without detailed quantification of the intra- and intermolecular levels. Among the superfamily, we considered as essential to propose an overview of the surface properties of all the repeats of the five proteins of the spectrin family, namely α- and β-spectrins, α-actinin, dystrophin and utrophin. Therefore, the aim of this work was to obtain a quantitative comparison of all the repeats at both the primary sequence and the three-dimensional levels. For that purpose, we applied homology modelling methods to obtain structural models for successive and overlapping tandem repeats of the human erythrocyte α- and β-spectrins and utrophin, as previously undertaken for dystrophin, and we used the known structure of α-actinin. The matrix calculation of the pairwise similarities of all the repeat sequences and the electrostatic and hydrophobic surface properties throughout the protein family support the view that spectrins and α-actinin on one hand and utrophin and dystrophin on the other hand share some structural similarities, but a detailed molecular characterisation highlights substantial differences. The repeats within the family are far from identical, which is consistent with their multiple interactions with different cellular partners, including proteins and membrane lipids.





    Categories: Journal Articles
  • Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Tara D. Sutherland , Holly E. Trueman , Andrew A. Walker , Sarah Weisman , Peter M. Campbell , Zhaoming Dong , Mickey G. Huson , Andrea L. Woodhead , Jeffrey S. Church

    The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages—the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises—and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins – which likely produce deviations from canonical coiled-coil structure – likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins.





    Categories: Journal Articles
  • Effect of sequence features on assembly of spider silk block copolymers
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Olena S. Tokareva , Shangchao Lin , Matthew M. Jacobsen , Wenwen Huang , Daniel Rizzo , David Li , Marc Simon , Cristian Staii , Peggy Cebe , Joyce Y. Wong , Markus J. Buehler , David L. Kaplan

    Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.





    Categories: Journal Articles
  • Decoration of silk fibroin by click chemistry for biomedical application
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Hongshi Zhao , Eva Heusler , Gabriel Jones , Linhao Li , Vera Werner , Oliver Germershaus , Jennifer Ritzer , Tessa Luehmann , Lorenz Meinel

    Silkfibroin (SF) has an excellent biocompatibility and its remarkable structure translates into exciting mechanical properties rendering this biomaterial particularly fascinating for biomedical application. To further boost the material’s biological/preclinical impact, SF is decorated with biologics, typically by carbodiimide/N-hydroxysuccinimide coupling (EDC/NHS). For biomedical application, this chemistry challenges the product risk profile due to the formation of covalent aggregates, particularly when decoration is with biologics occurring naturally in humans as these aggregates may prime for autoimmunity. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry) provides the necessary specificity to avoid such intermolecular, covalent aggregates. We present a blueprint outlining the necessary chemistry rendering SF compatible with CuAAC and with a particular focus on structural consequences. For that, the number of SF carboxyl groups (carboxyl-SF; required for EDC/NHS chemistry) or azido groups (azido-SF; required for click chemistry) was tailored by means of diazonium coupling of the SF tyrosine residues. Structural impact on SF and decorated SF was characterized by Fourier transform infrared spectroscopy (FTIR). The click chemistry yielded a better controlled product as compared to the EDC/NHS chemistry with no formation of inter- and intramolecular crosslinks as demonstrated for SF decorated with fluorescent model compounds or a biologic, fibroblast growth factor 2 (FGF2), respectively. In conclusion, SF can readily be translated into a scaffold compatible with click chemistry yielding decorated products with a better risk profile for biomedical application.





    Categories: Journal Articles
  • Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Martin Humenik , Michael Magdeburg , Thomas Scheibel

    Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n ⩾2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-β structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-β-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into β-sheet structures, which is necessary for nucleation and beneficial for fibril growth.





    Categories: Journal Articles
  • A first census of collagen interruptions: Collagen’s own stutters and stammers
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Jordi Bella

    The repetitive Gly-X-Y sequence is the telltale sign of triple helical domains in collagens and collagen-like proteins. Most collagen sequences contain sporadic interruptions of this pattern, which may have functional roles in molecular flexibility, assembly or molecular recognition. However, the structural signatures of the different interruptions are not well defined. Here, a first comprehensive survey of collagen interruptions on collagen sequences from different taxonomic groups is presented. Amino acid preferences at the sites of interruption and the flanking triplets are analysed separately for metazoan and prokaryotic collagens and the concept of commensurateness between interruptions is introduced. Known structural information from model peptides is used to present a common framework for hydrogen bonding topology and variations in superhelical twist for the different types of interruptions. Several collagen interruptions are further classified here as stutters or stammers in analogy to the heptad breaks observed in alpha-helical coiled coils, and the structural consequences of commensurate interruptions in heterotrimeric collagens are briefly discussed. Data presented here will be useful for further investigation on the relation between structure and function of collagen interruptions.





    Categories: Journal Articles
  • Bacterial collagen-like proteins that form triple-helical structures
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Zhuoxin Yu , Bo An , John A.M. Ramshaw , Barbara Brodsky

    A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.





    Categories: Journal Articles
  • Nano-thrombelastography of fibrin during blood plasma clotting
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Tímea Feller , Miklós S.Z. Kellermayer , Balázs Kiss

    Hemostasis is a complex process that relies on the sensitive balance between the formation and breakdown of the thrombus, a three-dimensional polymer network of the fibrous protein fibrin. Neither the details of the fibrinogen–fibrin transition, nor the exact mechanisms of fibrin degradation are fully understood at the molecular level. In the present work we investigated the nanoscale-changes in the viscoelasticity of the 3D-fibrin network during fibrinogenesis and streptokinase (STK)-induced fibrinolysis by using a novel application of force spectroscopy, named nano-thrombelastography. In this method the changes in the bending of an oscillating atomic-force-microscope (AFM) cantilever in human blood–plasma droplet were followed as a function of time. Whereas the global features of the time-dependent change in cantilever deflection corresponded well to a macroscopic thrombelastogram, the underlying force spectra revealed large, sample-dependent oscillations in the range of 3–50nN and allowed the separation of elastic and viscous components of fibrin behavior. Upon STK treatment the nano-thrombelastogram signal decayed gradually. The decay was driven by a decrease in thrombus elasticity, whereas thrombus viscosity decayed with a time delay. In scanning AFM images mature fibrin appeared as 17-nm-high and 12–196-nm-wide filaments. STK-treatment resulted in the decrease of filament height and the appearance of a surface roughness with 23.7nm discrete steps that corresponds well to the length of a fibrinogen monomer. Thus, the initial decay of thrombus elasticity during fibrinolysis may be caused by the axial rupture of fibrin fibers.





    Categories: Journal Articles
  • Stretching desmin filaments with receding meniscus reveals large axial tensile strength
    [Jun 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): Balázs Kiss , Miklós S.Z. Kellermayer

    Desmin forms the intermediate filament system of muscle cells where it plays important role in maintaining mechanical integrity and elasticity. Although the importance of intermediate-filament elasticity in cellular mechanics is being increasingly recognized, the molecular basis of desmin’s elasticity is not fully understood. We explored desmin elasticity by molecular combing with forces calculated to be as large as 4nN. Average filament contour length increased 1.55-fold axial on average. Molecular combing together with EGTA-treatment caused the fragmentation of the filament into short, 60 to 120-nm-long and 4-nm-wide structures. The fragments display a surface periodicity of 38nm, suggesting that they are composed of laterally attached desmin dimers. The axis of the fragments may deviate significantly from that of the overstretched filament, indicating that they have a large orientational freedom in spite of being axially interconnected. The emergence of protofibril fragments thus suggests that the interconnecting head or tail domains of coiled-coil desmin dimers are load-bearing elements during axial stretch.





    Categories: Journal Articles