Journal of Structural Biology

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  • Robust evaluation of 3D electron cryomicroscopy data using tilt-pairs
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Christopher J. Russo , Lori A. Passmore

    Determining the structure of a protein complex using electron microscopy requires the calculation of a 3D density map from 2D images of single particles. Since the individual images are taken at low electron dose to avoid radiation damage, they are noisy and difficult to align with each other. This can result in incorrect maps, making validation essential. Pairs of electron micrographs taken at known angles to each other (tilt-pairs) can be used to measure the accuracy of assigned projection orientations and verify the soundness of calculated maps. Here we establish a statistical framework for evaluating images and density maps using tilt-pairs. The directional distribution of such angular data is modelled using a Fisher distribution on the unit sphere. This provides a simple, quantitative and easily comparable metric, the concentration parameter κ, for evaluating the quality of datasets and density maps that is independent of the data collection and analysis methods. A large κ is indicative of good agreement between the particle images and the 3D density map. For structure validation, we recommend κ > 10 and a p-value <0.01. The statistical framework herein allows one to objectively answer the question: Is a reconstructed density map correct within a particular confidence interval?





    Categories: Journal Articles
  • Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Dong-Ju You , Nujarin Jongruja , Elias Tannous , Clement Angkawidjaja , Yuichi Koga , Shigenori Kanaya

    RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNase H1) requires ⩾2M NaCl, ⩾10mM MnCl2, or ⩾300mM MgCl2 for folding. To understand the structural basis for this salt-dependent folding of Halo-RNase H1, the crystal structure of Halo-RNase H1was determined in the presence of 10mM MnCl2. The structure of Halo-RNase H1 highly resembles those of metagenome-derived LC11-RNase H1 and Sulfolobus tokodaii RNase H1 (Sto-RNase H1), except that it contains two Mn2+ ions at the active site and has three bi-aspartate sites on its surface. To examine whether negative charge repulsion at these sites are responsible for low-salt denaturation of Halo-RNase H1, a series of the mutant proteins of Halo-RNase H1 at these sites were constructed. The far-UV CD spectra of these mutant proteins measured in the presence of various concentrations of NaCl suggest that these mutant proteins exist in an equilibrium between a partially folded state and a folded state. However, the fraction of the protein in a folded state is nearly 0% for the active site mutant, 40% for the bi-aspartate site mutant, and 70% for the mutant at both sites in the absence of salt. The active site mutant requires relatively low concentration (∼0.5M) of salt for folding. These results suggest that suppression of negative charge repulsion at both active and bi-aspartate sites by salt is necessary to yield a folded protein.





    Categories: Journal Articles
  • Histocompositional organization and toughening mechanisms in antler
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): John G. Skedros , Kendra E. Keenan , David M.L. Cooper , Roy D. Bloebaum

    Mechanical testing studies by Krauss et al. (2009) and Gupta et al. (2013) suggest that the extraordinary toughness of antler bone is primarily achieved by intrinsic/nanostructural mechanisms instead of extrinsic/microstructural mechanisms. However, this conclusion is based on data from extremely small specimens from one antler loaded only in tension, which impedes discernment of the relative importance of intrinsic vs. extrinsic mechanisms. In the present study we conducted analyses into the microstructural features of antler for details of potential additional microscale toughening characteristics, as suggested by recent mechanical testing studies of bulk specimens. The data are also considered in view of the above-mentioned studies concluding that extrinsic/microstructural toughening mechanisms are less important than nanoscale/intrinsic toughening mechanisms in antler. Mule deer antlers were evaluated using: (1) backscattered electron imaging for micro-mineralization, (2) circularly polarized light for osteonal interfacial complexity and collagen fiber orientation (CFO) heterogeneity, and (3) X-ray 3D micro-computed tomography for osteon/vessel orientation, density, and size. Results showed: (1) hyper-mineralized seams of approximately 3–4 microns thickness within relatively hypermineralized “zones” that course circuitously along osteonal interfaces, (2) highly heterogeneous CFO, including increased oblique-to-transverse CFO near/adjacent to osteon peripheries, and (3) osteons are often highly elongated in 2D. 3D reconstructions show that a considerable percentage of the vascular canals course obliquely with respect to the antler long axis. While results show multiple possible extrinsic-level histological characteristics in antler bone, it remains to be determined if microstructural characteristics become subsidiary to nanostructural characteristics in enhancing toughness during the majority of post-yield behavior of antler bone when loaded in a biologically relevant fashion.





    Categories: Journal Articles
  • Synchrotron X-ray micro-tomography imaging and analysis of wood degraded by Physisporinus vitreus and Xylaria longipes
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Marjan Sedighi Gilani , Matthieu N. Boone , Kevin Mader , Francis Willis Mathew Robert Schwarze

    Incubation of Norway spruce with Physisporinus vitreus and sycamore with Xylaria longipes results in reduction in density of these wood species that are traditionally used for the top and bottom plate of a violin, which follows by enhanced acoustic properties. We used Synchrotron X-ray micro-tomography, to study the three-dimensional structure of wood at the micro-scale level and the alterations of the density distribution after incubation with two white-rot fungi. Micro-tomography data from wood treated at different incubation periods are analyzed and compared with untreated (control) specimens to determine the wood density map and changes at the cell-wall level. Differences between the density of early- and latewood, xylem ray and around bordered pits in both Norway spruce and sycamore are studied. Three-dimensional hyphal networks of the P. vitreus and Xylaria longipes hyphae are visualized inside the cell lumina and their significance on the density of the early- and latewood cells after different incubation periods are discussed. The study illustrates the utility of X-ray micro-tomography for both qualitative and quantitative studies of a wide variety of biological systems and due to its high sensitivity, small structural changes can be quantified.





    Categories: Journal Articles
  • Ultrastructure and mineral composition of the cornea cuticle in the compound eyes of a supralittoral and a marine isopod
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Francisca I. Alagboso , Christian Reisecker , Sabine Hild , Andreas Ziegler

    The cuticle of the cornea in Crustacea is an interesting example of a composite material compromising between two distinct functions. As part of the dioptric apparatus of the ommatidia within the complex eye it forms transparent micro-lenses that should as well maintain the mechanical stability of the head capsule. We analyzed the ultrastructure and composition of the isopod cornea cuticle of the terrestrial species Ligia oceanica and the marine Sphaeroma serratum. We used a variety of tissue preparation methods, electron microscopic techniques as well as electron microprobe analysis and Raman spectroscopic imaging. The results reveal various structural adaptations that likely increase light transmission. These are an increase in the thickness of the epicuticle, a reduction of the thickness of the outer layer of calcite, a spatial restriction of pore canals to interommatidial regions, and, for S. serratum only, an increase in calcite crystal size. In both species protein–chitin fibrils within the proximal exocuticle form a peculiar reticular structure that does not occur within the cuticle of the head capsule. In L. oceanica differential mineralization results in a spherically shaped interface between mineralized and unmineralized endocuticle, likely an adaptation to increase the refractive power of the cornea maintaining the mechanical stability of the cuticle between the ommatidia. The results show that the habitat and differences in the general structure of the animal’s cuticle affect the way in which the cornea is adapted to its optical function.





    Categories: Journal Articles
  • X-ray vs. NMR structure of N-terminal domain of δ-subunit of RNA polymerase
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Gabriel Demo , Veronika Papoušková , Jan Komárek , Pavel Kadeřávek , Olga Otrusinová , Pavel Srb , Alžbeta Rabatinová , Libor Krásný , Lukáš Žídek , Vladimír Sklenář , Michaela Wimmerová

    The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important β sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni2+ ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.





    Categories: Journal Articles
  • Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Naoyuki Miyazaki , Masatoshi Esaki , Teru Ogura , Kazuyoshi Murata

    Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae. We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.





    Categories: Journal Articles
  • Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin
    [Aug 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Debiao Zhao , Xuejuan Wang , Junhui Peng , Chongyuan Wang , Fudong Li , Qianqian Sun , Yibo Zhang , Jiahai Zhang , Gang Cai , Xiaobing Zuo , Jihui Wu , Yunyu Shi , Zhiyong Zhang , Qingguo Gong

    c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807SH3a and D881SH3b are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.





    Categories: Journal Articles
  • Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL
    [Aug 2014]

    Publication date: Available online 1 August 2014
    Source:Journal of Structural Biology

    Author(s): Connie Lu , Konstantin V. Korotkov , Wim G.J. Hol

    The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11–15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, “cyto-GspL rods” that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.





    Categories: Journal Articles
  • The structural basis of differential inhibition of human calpain by indole and phenyl α-mercaptoacrylic acids
    [Aug 2014]

    Publication date: Available online 30 July 2014
    Source:Journal of Structural Biology

    Author(s): Sarah E. Adams , Pierre J. Rizkallah , David J. Miller , Emma J. Robinson , Maurice B. Hallett , Rudolf K. Allemann

    Excessive activity of neutrophils has been linked to many pathological conditions, including rheumatoid arthritis, cancer and Alzheimer’s disease. Calpain-I is a Ca2+-dependent protease that plays a key role in the extravasation of neutrophils from the blood stream prior to causing damage within affected tissues. Inhibition of calpain-I with small molecule mercaptoacrylic acid derivatives slows the cell spreading process of live neutrophils and so these compounds represent promising drug leads. Here we present the 2.05 and 2.03Å co-crystal X-ray structures of the pentaEF hand region, PEF(S), from human calpain with (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid and (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid. In both structures, the α-mercaptoacrylic acid derivatives bind between two α-helices in a hydrophobic pocket that is also exploited by a leucine residue of the endogenous regulatory calpain inhibitor calpastatin. Hydrophobic interactions between the aromatic rings of both inhibitors and the aliphatic residues of the pocket are integral for tight binding. In the case of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid, hydrogen bonds form between the mercaptoacrylic acid substituent lying outside the pocket and the protein and the carboxylate group is coplanar with the aromatic ring system. Multiple conformations of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid were found within the pocket. The increased potency of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid relative to (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid may be a consequence of the indole group binding more deeply in the hydrophobic pocket of PEF(S) than the phenyl ring.





    Categories: Journal Articles
  • Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy
    [Aug 2014]

    Publication date: Available online 30 July 2014
    Source:Journal of Structural Biology

    Author(s): Jason Z. Cui , Arash Y. Tehrani , Kimberly A. Jett , Pascal Bernatchez , Cornelis van Breemen , Mitra Esfandiarei

    In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.





    Categories: Journal Articles
  • Iron-rich ferritin in the hypoxia-tolerant rodent Spalax ehrenbergi: A naturally-occurring biomarker confirms the internalization and pathways of intracellular macromolecules
    [Aug 2014]

    Publication date: Available online 19 July 2014
    Source:Journal of Structural Biology

    Author(s): Theodore C. Iancu , Talmon Arad , Imad Shams , Irena Manov

    The discovery of pits/caveolae in the plasmalemma advanced the study of macromolecule internalization. “Transcytosis” describes the transport of macromolecular cargo from one front of a polarized cell to the other within membrane-bounded carrier(s), via endocytosis, intracellular trafficking and exocytosis. Clathrin-mediated transcytosis is used extensively by epithelial cells, while caveolae-mediated transcytosis mostly occurs in endothelial cells. The internalization pathways were monitored by various markers, including radioisotopes, nanoparticles, enzymes, immunostains, and fluorophores. We describe an internalization pathway identified using a naturally-occurring biomarker, in vivo assembled ferritin, containing electron-dense iron cores. Iron, an essential trace metal for most living species and iron homeostasis, is crucial for cellular life. Ferritin is a ubiquitous and highly conserved archeoprotein whose main function is to store a reserve iron supply inside the cytoplasm in a non-toxic form. Ferritin is present in all organisms which have a metabolic requirement for iron and in even in organisms whose taxonomic rank is very low. The newborns of the blind mole, Spalax ehrenbergi, are born and live in a hypoxic environment and have significant iron overload in their liver and heart, but their iron metabolism has not been previously studied. These newborns, which are evolutionarily adapted to fluctuations in the environmental oxygen, have a unique ability to sequester transplacental iron and store it in ferritin without any signs of iron toxicity. Using the ferrihydrite cores of ferritin, we were able to monitor the ferritin internalization from portals of its entry into the cytosol of hepatocytes and cardiomyocytes and into the lysosomes.
    Graphical abstract




    Categories: Journal Articles
  • 3D Raman mapping of the collagen fibril orientation in human osteonal lamellae
    [Aug 2014]

    Publication date: Available online 12 July 2014
    Source:Journal of Structural Biology

    Author(s): Susanne Schrof , Peter Varga , Leonardo Galvis , Kay Raum , Admir Masic

    Chemical composition and fibrillar organization are the major determinants of osteonal bone mechanics. However, prominent methodologies commonly applied to investigate mechanical properties of bone on the micro scale are usually not able to concurrently describe both factors. In this study, we used polarized Raman spectroscopy (PRS) to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment. Specifically, the three-dimensional arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils were derived. Based on the analysis of several osteons, two major fibrillar organization patterns were identified, one with a monotonic and another with a periodically changing twist direction. These results confirm earlier reported twisted and oscillating plywood arrangements, respectively. Furthermore, indicators of the degree of alignment suggested the presence of disordered collagen within the lamellar organization of the osteon. The results show the versatility of the analytical PRS approach and demonstrate its capability in providing not only compositional, but also 3D structural information in a complex hierarchically structured biological material. The concurrent assessment of chemical and structural features may contribute to a comprehensive characterization of the microstructure of bone and other collagen-based tissues.





    Categories: Journal Articles
  • Cover 2 - Editorial Board
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1









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

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1









    Categories: Journal Articles
  • Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Guimei Yu , Frank Vago , Dongsheng Zhang , Jonathan E. Snyder , Rui Yan , Ci Zhang , Christopher Benjamin , Xi Jiang , Richard J. Kuhn , Philip Serwer , David H. Thompson , Wen Jiang

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged E scherichia coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.





    Categories: Journal Articles
  • M-free: Scoring the reference bias in sub-tomogram averaging and template matching
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Zhou Yu , Achilleas S. Frangakis

    Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.





    Categories: Journal Articles
  • Crystal structure of the transport unit of the autotransporter adhesin involved in diffuse adherence from Escherichia coli
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Iris Gawarzewski , Frank DiMaio , Elisa Winterer , Britta Tschapek , Sander H.J. Smits , Joachim Jose , Lutz Schmitt

    Several serious gastrointestinal diseases, which are widespread all over the world, are caused by enteropathogenic Escherichia coli. The monomeric autotransporter AIDA-I (adhesin involved in diffuse adherence) represents an important virulence factor of these strains and is involved in adhesion, biofilm formation, aggregation and invasion into host cells. Here, we present the crystal structure of the transport unit of AIDA-I at 3.0Å resolution, which forms a 12-stranded β-barrel harboring the linker domain in its pore. Mutagenesis studies of the C-terminal amino acid demonstrated the great impact of this terminal residue on membrane integration of AIDA-I and passenger translocation.





    Categories: Journal Articles
  • Heparin induced dimerization of APP is primarily mediated by E1 and regulated by its acidic domain
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Sandra Hoefgen , Ina Coburger , Dirk Roeser , Yvonne Schaub , Sven O. Dahms , Manuel E. Than

    The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimer’s disease (AD). In addition, many physiological functions have been described for APP, including a role in cell–cell- and cell–ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.





    Categories: Journal Articles
  • Molecular dynamics investigation of the active site dynamics of mycobacterial cyclopropane synthase during various stages of the cyclopropanation process
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Chinmayee Choudhury , U. Deva Priyakumar , G. Narahari Sastry

    Mycobacterial cyclopropane synthase 1 (CmaA1) is one of the most important drug targets in anti tuberculosis drug discovery as it is responsible for cis-cyclopropanation at the distal position of unsaturated mycolates, which is an essential step for the pathogenicity, persistence and drug resistance. Five representative models of CmaA1 which correspond to different stages in the cyclopropanation process have been studied using molecular dynamics (MD) simulations. The MD simulations and structural analyses provide a detailed account of the structural changes in the active sites of CmaA1. CmaA1 has two distinct binding sites, i.e., cofactor binding site (CBS) and acyl substrate binding site (ASBS). The apo state of CmaA1 corresponds to a closed conformation where the CBS is inaccessible due to the existence of H-bond between Pro202 of loop10 (L10) and Asn11 of N-terminal α1 helix. However, cofactor binding leads to the breaking of this H-bond and thus the H-bond is absent in the holo form. The hydrophobic side chains orient towards the inner side of the ASBS upon cofactor binding to create a hydrophobic environment for the substrate. The cofactor and substrate tend to come close to each other facilitated by opening of L10 to exchange the methyl group from the cofactor to the substrate. The MD study also revealed that the system tends to regain the apo conformation within 40ns after releasing the product.





    Categories: Journal Articles