Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

PubMed

Wybenga-Groot, Leanne E; McGlade, C Jane

2013-12-01

The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. © 2013.

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

PubMed

Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

2014-05-13

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

PubMed

Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

2004-12-07

Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

Solution structure of the catalytic domain of RICH protein from goldfish.

PubMed

Kozlov, Guennadi; Denisov, Alexey Y; Pomerantseva, Ekaterina; Gravel, Michel; Braun, Peter E; Gehring, Kalle

2007-03-01

Regeneration-induced CNPase homolog (RICH) is an axonal growth-associated protein, which is induced in teleost fish upon optical nerve injury. RICH consists of a highly acidic N-terminal domain, a catalytic domain with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity and a C-terminal isoprenylation site. In vitro RICH and mammalian brain CNPase specifically catalyze the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains unknown. Here, we report the NMR structure of the catalytic domain of goldfish RICH and describe its binding to CNPase inhibitors. The structure consists of a twisted nine-stranded antiparallel beta-sheet surrounded by alpha-helices on both sides. Despite significant local differences mostly arising from a seven-residue insert in the RICH sequence, the active site region is highly similar to that of human CNPase. Likewise, refinement of the catalytic domain of rat CNPase using residual dipolar couplings gave improved agreement with the published crystal structure. NMR titrations of RICH with inhibitors point to a similar catalytic mechanism for RICH and CNPase. The results suggest a functional importance for the evolutionarily conserved phosphodiesterase activity and hint of a link with pre-tRNA splicing.

Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

PubMed

Tao, Y; Strelkov, S V; Mesyanzhinov, V V; Rossmann, M G

1997-06-15

Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal domain and the labile coiled-coil domain may be essential for the fibritin function and for the correct functioning of many other alpha-fibrous proteins.

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Basic Research Program, SAIC-Frederick, Frederick, MD 21702; Gustchina, Alla

2010-08-01

The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix. The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very longmore » C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

Dynamic Identification for Control of Large Space Structures

NASA Technical Reports Server (NTRS)

Ibrahim, S. R.

1985-01-01

This is a compilation of reports by the one author on one subject. It consists of the following five journal articles: (1) A Parametric Study of the Ibrahim Time Domain Modal Identification Algorithm; (2) Large Modal Survey Testing Using the Ibrahim Time Domain Identification Technique; (3) Computation of Normal Modes from Identified Complex Modes; (4) Dynamic Modeling of Structural from Measured Complex Modes; and (5) Time Domain Quasi-Linear Identification of Nonlinear Dynamic Systems.

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

PubMed

Okumura, Masayo; Yamakawa, Hisashi; Ohara, Osamu; Owaribe, Katsushi

2002-02-22

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.

An ambiguity principle for assigning protein structural domains.

PubMed

Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

2017-01-01

Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object-in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our "multipartitioning" approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules.

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn

2012-10-23

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of {approx}456 polypeptide chains contributed by {approx}30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal 'FG' repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain ofmore » Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 {angstrom} resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.« less

Crystal Structure of the Passenger Domain of the Escherichia coli Autotransporter EspP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, Shekeb; Mian, Hira S.; Sandercock, Linda E.

2013-03-07

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal 'passenger domain' responsible for the specific effector functions of the molecule and a C-terminal '{beta}-domain' responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-{angstrom} crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel {beta}-helix precededmore » by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this {beta}-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the {beta}-helix within SPATEs.« less

Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

PubMed

Bussiere, D E; Kong, X; Egan, D A; Walter, K; Holzman, T F; Lindh, F; Robins, T; Giranda, V L

1998-11-01

The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

When a domain isn’t a domain, and why it’s important to properly filter proteins in databases

PubMed Central

Towse, Clare-Louise; Daggett, Valerie

2013-01-01

Summary Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our Consensus Domain Dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1695 folds in the CDD as being non-autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predicted estimates suggest 40% of proteins to have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets. PMID:23108912

Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

PubMed

Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

1994-04-01

We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.

Domain structure of the ribozyme from eubacterial ribonuclease P.

PubMed Central

Loria, A; Pan, T

1996-01-01

Large RNAs can be composed of discrete domains that fold independently. One such "folding domain" has been identified previously in the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA). This domain contains roughly one-third of all residues. Folding of an RNA construct consisting of the remaining two-thirds of B. subtilis P RNA was examined by Fe(II)-EDTA hydroxyl radical protection. This molecule folds into the proper higher-order structure under identical conditions as the full-length P RNA, suggesting the presence of a second folding domain in B. subtilis P RNA. Folding analysis of the Escherichia coli P RNA by hydroxyl radical protection shows that this P RNA is completely folded at 5-6 mM Mg2+. In order to analyze the structural organization of folding domains in E. coli P RNA, constructs were designed based on the domain structure of B. subtilis P RNA. Fe(II)-EDTA protection indicates that E. coli P RNA also contains two folding domains. Despite the significant differences at the secondary structure level, both P RNAs appear to converge structurally at the folding domain level. The pre-tRNA substrate, localized in previous studies, may bind across the folding domains with the acceptor stem/3'CCA contacting the domain including the active site and the T stem-loop contacting the other. Because all eubacterial P RNAs share considerable homology in secondary structure to either B. subtilis or E. coli P RNA, these results suggest that this domain structure may be applicable for most, if not all, eubacterial P RNAs. Identification of folding domains should be valuable in dissecting structure-function relationship of large RNAs. PMID:8718684

Structural analysis of β-prism lectin from Colocasia esculenta (L.) S chott.

PubMed

Vajravijayan, S; Pletnev, S; Pletnev, V Z; Nandhagopal, N; Gunasekaran, K

2016-10-01

The Mannose-binding β-Prism Colocasia esculenta lectin (β-PCL) was purified from tubers using ion exchange chromatography. The purified β-PCL appeared as a single band of ∼12kDa on SDS-PAGE. β-PCL crystallizes in trigonal space group P3121 and diffracted to a resolution of 2.1Å. The structure was solved using Molecular replacement using Crocus vernus lectin (PDB: 3MEZ) as a model. From the final refined model to an R-factor of 16.5% and an Rfree of 20.4%, it has been observed that the biological unit consists of two β-Prism domains augmented through C-terminals swap over to form one of faces for each domain. Cα superposition of individual domains of β-PCL with individual domains of other related structures and superposition of whole protein structures were carried out. The higher RMS deviation for the superposition of whole structures suggest that β-prism domains assume different orientation in each structure. Copyright © 2016 Elsevier B.V. All rights reserved.

Not throwing out the baby with the bathwater: lessons from the Fibromyalgia Impact Questionnaire.

PubMed

van Wilgen, C Paul; Vuijk, Pieter Jelle; van Ittersum, Miriam W; Nijs, Jo

2013-03-01

The Fibromyalgia Impact Questionnaire (FIQ) is the most frequently used questionnaire in patients with fibromyalgia in the last 20 years. Recently, a revised version of the FIQ has been published. In this study, we examined the factor structure of the original version using explorative and confirmative factor analyses in a representative group of about 500 Dutch and Belgian patients with fibromyalgia, in which the work-related item was excluded. The FIQ consisting of a three-factor structure with a functional domain (ten items), physical symptom domain (six items), and mental symptom domain (two items), which is the most accurate. From the data presented, it is concluded that the FIQ is a solid, worldwide-used questionnaire with a history of 20 years consisting of three domains. When constructing a revised FIQ, the results of this study can be incorporated.

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

PubMed Central

Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

2015-01-01

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Bradley R.; Drake, Eric J.; Shi, Ce

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived frommore » understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.« less

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture*

PubMed Central

Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M.

2016-01-01

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. PMID:27597544

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture.

PubMed

Miller, Bradley R; Drake, Eric J; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2016-10-21

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

How Structure Shapes Dynamics: Knowledge Development in Wikipedia - A Network Multilevel Modeling Approach

PubMed Central

Halatchliyski, Iassen; Cress, Ulrike

2014-01-01

Using a longitudinal network analysis approach, we investigate the structural development of the knowledge base of Wikipedia in order to explain the appearance of new knowledge. The data consists of the articles in two adjacent knowledge domains: psychology and education. We analyze the development of networks of knowledge consisting of interlinked articles at seven snapshots from 2006 to 2012 with an interval of one year between them. Longitudinal data on the topological position of each article in the networks is used to model the appearance of new knowledge over time. Thus, the structural dimension of knowledge is related to its dynamics. Using multilevel modeling as well as eigenvector and betweenness measures, we explain the significance of pivotal articles that are either central within one of the knowledge domains or boundary-crossing between the two domains at a given point in time for the future development of new knowledge in the knowledge base. PMID:25365319

Electronic states of domain structure in 1T-TaS2-x Se x observed by STM/STS

NASA Astrophysics Data System (ADS)

Fujii, D.; Iwasaki, T.; Akiyama, K.; Fujisawa, Y.; Demura, S.; Sakata, H.

2018-03-01

We report on a systematic scanning tunneling microscopy and spectroscopy (STM/STS) study on 1T–TaS2-x Se x (x = 0, 0.3, 1.0) at 4.2 K. While the compounds with x = 0 and 0.3, which undergoes the Mott transition, showed the commensurate charge density wave (CDW) with the period of \\sqrt{13}{a}0× \\sqrt{13}{a}0 (a 0 is in-plane lattice constant), the compound with x=1, which shows superconductivity at 3.5 K, exhibits anomalous domain structure: The domain structure consists of regions with regular array of David-stars divided by bright contrasted walls at positive bias voltage. We found the domain wall showed the different electronic state from that of the domain.

An ambiguity principle for assigning protein structural domains

PubMed Central

Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

2017-01-01

Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object—in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our “multipartitioning” approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules. PMID:28097215

Structural and functional properties of hemoglobins from unicellular organisms as revealed by resonance Raman spectroscopy.

PubMed

Egawa, Tsuyoshi; Yeh, Syun-Ru

2005-01-01

Hemoglobins have been discovered in organisms from virtually all kingdoms. Their presence in unicellular organisms suggests that the gene for hemoglobin is very ancient and that the hemoglobins must have functions other than oxygen transport, in view of the fact that O2 delivery is a diffusion-controlled process in these organisms. Based on sequence alignment, three groups of hemoglobins have been characterized in unicellular organisms. The group-one hemoglobins, termed truncated hemoglobins, consist of proteins with 110-140 amino acid residues and a novel two-over-two alpha-helical sandwich motif. The group-two hemoglobins, termed flavohemoglobins, consist of a hemoglobin domain, with a classical three-over-three alpha-helical sandwich motif, and a flavin-containing reductase domain that is covalently attached to it. The group-three hemoglobins consist of myoglobin-like proteins that have high sequence homology and structural similarity to the hemoglobin domain of flavohemoglobins. In this review, recent resonance Raman studies of each group of these proteins are presented. Their implications are discussed in the context of the structural and functional properties of these novel hemoglobins.

Crystal Structures of the Receiver Domain of the Response Regulator PhoP from Escherichia coli in the Absence and Presence of the Phosphoryl Analog Beryllofluoride▿

PubMed Central

Bachhawat, Priti; Stock, Ann M.

2007-01-01

The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg2+. Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation. PMID:17545283

All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

PubMed Central

Mohammadiarani, Hossein; Vashisth, Harish

2016-01-01

The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID:27379020

The PYRIN domain: A member of the death domain-fold superfamily

PubMed Central

Fairbrother, Wayne J.; Gordon, Nathaniel C.; Humke, Eric W.; O'Rourke, Karen M.; Starovasnik, Melissa A.; Yin, Jian-Ping; Dixit, Vishva M.

2001-01-01

PYRIN domains were identified recently as putative protein–protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The ∼95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein–protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction. PMID:11514682

Structural analysis of an oxygen-regulated diguanylate cyclase.

PubMed

Tarnawski, Miroslaw; Barends, Thomas R M; Schlichting, Ilme

2015-11-01

Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

Structure of Urtica dioica agglutinin isolectin I: dimer formation mediated by two zinc ions bound at the sugar-binding site.

PubMed

Harata, K; Schubert, W D; Muraki, M

2001-11-01

Ultica dioica agglutinin, a plant lectin from the stinging nettle, consists of a total of seven individual isolectins. One of these structures, isolectin I, was determined at 1.9 A resolution by the X-ray method. The crystals belong to the space group P2(1) and the asymmetric unit contains two molecules related by local twofold symmetry. The molecule consists of two hevein-like chitin-binding domains lacking distinct secondary structure, but four disulfide bonds in each domain maintain the tertiary structure. The backbone structure of the two independent molecules is essentially identical and this is similarly true of the sugar-binding sites. In the crystal, the C-terminal domains bind Zn(2+) ions at the sugar-binding site. Owing to their location near a pseudo-twofold axis, the two zinc ions link the two independent molecules in a tail-to-tail arrangement: thus, His47 of molecule 1 and His67 of molecule 2 coordinate the first zinc ion, while the second zinc ion links Asp75 of molecule 1 and His47 of molecule 2.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

PubMed

Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

2006-01-24

Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

The crystal structure of pectate lyase peli from soft rot pathogen Erwinia chrysanthemi in complex with its substrate.

PubMed

Creze, Christophe; Castang, Sandra; Derivery, Emmanuel; Haser, Richard; Hugouvieux-Cotte-Pattat, Nicole; Shevchik, Vladimir E; Gouet, Patrice

2008-06-27

The crystallographic structure of the family 3 polysaccharide lyase (PL-3) PelI from Erwinia chrysanthemi has been solved to 1.45 A resolution. It consists of an N-terminal domain harboring a fibronectin type III fold linked to a catalytic domain displaying a parallel beta-helix topology. The N-terminal domain is located away from the active site and is not involved in the catalytic process. After secretion in planta, the two domains are separated by E. chrysanthemi proteases. This event turns on the hypersensitive response of the host. The structure of the single catalytic domain determined to 2.1 A resolution shows that the domain separation unveils a "Velcro"-like motif of asparagines, which might be recognized by a plant receptor. The structure of PelI in complex with its substrate, a tetragalacturonate, has been solved to 2.3 A resolution. The sugar binds from subsites -2 to +2 in one monomer of the asymmetric unit, although it lies on subsites -1 to +3 in the other. These two "Michaelis complexes" have never been observed simultaneously before and are consistent with the dual mode of bond cleavage in this substrate. The bound sugar adopts a mixed 2(1) and 3(1) helical conformation similar to that reported in inactive mutants from families PL-1 and PL-10. However, our study suggests that the catalytic base in PelI is not a conventional arginine but a lysine as proposed in family PL-9.

An investigation of the associations between contingent self-worth and aspirations among Iranian university students.

PubMed

Sabzehara, Milad; Ferguson, Yuna Lee; Sarafraz, Mehdi Reza; Mohammadi, Mostafa

2014-01-01

This study investigated the novel associations between intrinsic and extrinsic aspirations and internal and external domains of contingent self-worth among a sample of 502 Iranian university students. We found a meaningful pattern showing that intrinsic aspirations were positively associated with internal domains, whereas extrinsic aspirations were positively associated with external domains. Our survey data also suggested that the factor structure of the Aspiration Index, as well as the factor structure of the Contingencies of Self-Worth Scale in our Iranian sample were consistent with factor structures of foreign samples. Finally, the types of aspirations and domains of contingencies of self-worth meaningfully predicted variables related to well-being, confirming previous research. We discuss the nature of the associations between the aspirations and the domains of contingent self-worth.

The role of internal duplication in the evolution of multi-domain proteins.

PubMed

Nacher, J C; Hayashida, M; Akutsu, T

2010-08-01

Many proteins consist of several structural domains. These multi-domain proteins have likely been generated by selective genome growth dynamics during evolution to perform new functions as well as to create structures that fold on a biologically feasible time scale. Domain units frequently evolved through a variety of genetic shuffling mechanisms. Here we examine the protein domain statistics of more than 1000 organisms including eukaryotic, archaeal and bacterial species. The analysis extends earlier findings on asymmetric statistical laws for proteome to a wider variety of species. While proteins are composed of a wide range of domains, displaying a power-law decay, the computation of domain families for each protein reveals an exponential distribution, characterizing a protein universe composed of a thin number of unique families. Structural studies in proteomics have shown that domain repeats, or internal duplicated domains, represent a small but significant fraction of genome. In spite of its importance, this observation has been largely overlooked until recently. We model the evolutionary dynamics of proteome and demonstrate that these distinct distributions are in fact rooted in an internal duplication mechanism. This process generates the contemporary protein structural domain universe, determines its reduced thickness, and tames its growth. These findings have important implications, ranging from protein interaction network modeling to evolutionary studies based on fundamental mechanisms governing genome expansion.

A thermodynamic definition of protein domains.

PubMed

Porter, Lauren L; Rose, George D

2012-06-12

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

KIKI-net: cross-domain convolutional neural networks for reconstructing undersampled magnetic resonance images.

PubMed

Eo, Taejoon; Jun, Yohan; Kim, Taeseong; Jang, Jinseong; Lee, Ho-Joon; Hwang, Dosik

2018-04-06

To demonstrate accurate MR image reconstruction from undersampled k-space data using cross-domain convolutional neural networks (CNNs) METHODS: Cross-domain CNNs consist of 3 components: (1) a deep CNN operating on the k-space (KCNN), (2) a deep CNN operating on an image domain (ICNN), and (3) an interleaved data consistency operations. These components are alternately applied, and each CNN is trained to minimize the loss between the reconstructed and corresponding fully sampled k-spaces. The final reconstructed image is obtained by forward-propagating the undersampled k-space data through the entire network. Performances of K-net (KCNN with inverse Fourier transform), I-net (ICNN with interleaved data consistency), and various combinations of the 2 different networks were tested. The test results indicated that K-net and I-net have different advantages/disadvantages in terms of tissue-structure restoration. Consequently, the combination of K-net and I-net is superior to single-domain CNNs. Three MR data sets, the T 2 fluid-attenuated inversion recovery (T 2 FLAIR) set from the Alzheimer's Disease Neuroimaging Initiative and 2 data sets acquired at our local institute (T 2 FLAIR and T 1 weighted), were used to evaluate the performance of 7 conventional reconstruction algorithms and the proposed cross-domain CNNs, which hereafter is referred to as KIKI-net. KIKI-net outperforms conventional algorithms with mean improvements of 2.29 dB in peak SNR and 0.031 in structure similarity. KIKI-net exhibits superior performance over state-of-the-art conventional algorithms in terms of restoring tissue structures and removing aliasing artifacts. The results demonstrate that KIKI-net is applicable up to a reduction factor of 3 to 4 based on variable-density Cartesian undersampling. © 2018 International Society for Magnetic Resonance in Medicine.

Development of quality of life instrument for urban poor in the northeast of Thailand.

PubMed

Surit, Phrutthinun; Laohasiriwong, Wongsa; Sanchaisuriya, Pattara; Schelp, Frank Perter

2008-09-01

Measuring the quality of life is important for evaluation and prediction of life and social care needs. To evaluate Quality of Life (QOL) in an urban poor population in northeast of Thailand, the Urban Poor Quality of Life (UPQOL) instrument was developed To develop an initial instrument to measure urban poor QOL. The development was started with literature review and investigated in urban poor communities. The results were transformed into the items required to build a structured questionnaire. Five hundred twenty three subjects, representatives of urban poor, were selected to test this instrument. Descriptive statistics described feature of items and the samples, exploratory factor analysis conducted the items score, and confirmatory factor analysis conducted the construct validity. The result found that the UPQOL instrument consisted of nine domains (education, income and employment, environment, health, infrastructure, security and safety, shelter and housing, civil society and political, and human rights domains) with egien value rank from 1.5 to 4.2 and 61 items with the factor loading rank from 0.41 to 0.82. The internal consistency was 0.92. The correlation between items to domain ranged from 0.30 to 0.72 and domains to overall QOL ranged from 0.27 to 0.84. Confirmatory factor analysis showed that the structure fit all domains well. Domains and overall structure were good with CFI (> 0.95). The internal consistency value ranged from 0.73-0.93. UPQOL scores were able to discriminate groups of subjects with differences levels of QOL. The UPQOL instrument is conceptually valid. The results support good validity and reliability. It forms the basis for future testing and application in other settings.

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

PubMed Central

Hanaoka, Shingo; Nagadoi, Aritaka; Nishimura, Yoshifumi

2005-01-01

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2. PMID:15608118

Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

PubMed

Trujillo, Uldaeliz; Vázquez-Rosa, Edwin; Oyola-Robles, Delise; Stagg, Loren J; Vassallo, David A; Vega, Irving E; Arold, Stefan T; Baerga-Ortiz, Abel

2013-01-01

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Nanostructure of propylammonium nitrate in the presence of poly(ethylene oxide) and halide salts

NASA Astrophysics Data System (ADS)

Stefanovic, Ryan; Webber, Grant B.; Page, Alister J.

2018-05-01

Nanoscale structure of protic ionic liquids is critical to their utility as molecular electrochemical solvents since it determines the capacity to dissolve salts and polymers such as poly(ethylene oxide) (PEO). Here we use quantum chemical molecular dynamics simulations to investigate the impact of dissolved halide anions on the nanostructure of an archetypal nanostructured protic ionic liquid, propylammonium nitrate (PAN), and how this impacts the solvation of a model PEO polymer. At the molecular level, PAN is nanostructured, consisting of charged/polar and uncharged/nonpolar domains. The charged domain consists of the cation/anion charge groups, and is formed by their electrostatic interaction. This domain solvophobically excludes the propyl chains on the cation, which form a distinct, self-assembled nonpolar domain within the liquid. Our simulations demonstrate that the addition of Cl- and Br- anions to PAN disrupts the structure within the PAN charged domain due to competition between nitrate and halide anions for the ammonium charge centre. This disruption increases with halide concentration (up to 10 mol. %). However, at these concentrations, halide addition has little effect on the structure of the PAN nonpolar domain. Addition of PEO to pure PAN also disrupts the structure within the charged domain of the liquid due to hydrogen bonding between the charge groups and the terminal PEO hydroxyl groups. There is little other association between the PEO structure and the surrounding ionic liquid solvent, with strong PEO self-interaction yielding a compact, coiled polymer morphology. Halide addition results in greater association between the ionic liquid charge centres and the ethylene oxide components of the PEO structure, resulting in reduced conformational flexibility, compared to that observed in pure PAN. Similarly, PEO self-interactions increase in the presence of Cl- and Br- anions, compared to PAN, indicating that the addition of halide salts to PAN decreases its utility as a molecular solvent for polymers such as PEO.

Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

PubMed Central

Trujillo, Uldaeliz; Vázquez-Rosa, Edwin; Oyola-Robles, Delise; Stagg, Loren J.; Vassallo, David A.; Vega, Irving E.; Arold, Stefan T.; Baerga-Ortiz, Abel

2013-01-01

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures. PMID:23469090

Domain walls of linear polarization in isotropic Kerr media

NASA Astrophysics Data System (ADS)

Louis, Y.; Sheppard, A. P.; Haelterman, M.

1997-09-01

We present a new type of domain-wall vector solitary waves in isotropic self-defocusing Kerr media. These domain walls consist of localized structures separating uniform field domains of orthogonal linear polarizations. They result from the interplay between diffraction, self-phase modulation and cross-phase modulation in cases where the nonlinear birefringence coefficient B = {χxyyx(3)}/{χxxxx(3)} is negative. Numerical simulations show that these new vector solitary waves are stable.

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

PubMed Central

Edwards, Thomas E.; Phan, Isabelle; Abendroth, Jan; Dieterich, Shellie H.; Masoudi, Amir; Guo, Wenjin; Hewitt, Stephen N.; Kelley, Angela; Leibly, David; Brittnacher, Mitch J.; Staker, Bart L.; Miller, Samuel I.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

2010-01-01

Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics. PMID:20862217

The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

PubMed

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel

2008-03-14

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.

The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin

2010-01-07

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomersmore » across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.« less

Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly.

PubMed

Chernyatina, Anastasia A; Nicolet, Stefan; Aebi, Ueli; Herrmann, Harald; Strelkov, Sergei V

2012-08-21

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded

PubMed Central

Bourhis, Jean-Marie; Receveur-Bréchot, Véronique; Oglesbee, Michael; Zhang, Xinsheng; Buccellato, Matthew; Darbon, Hervé; Canard, Bruno; Finet, Stéphanie; Longhi, Sonia

2005-01-01

Measles virus is a negative-sense, single-stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in NTAIL, an α-helical molecular recognition element (α-MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and NTAIL, which shows that most of NTAIL remains disordered in the complex despite P-induced folding within the α-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of NTAIL, and of a bulky globular region, corresponding to XD and to the C terminus of NTAIL (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that NTAIL has an additional site (aa 517–525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure. PMID:16046624

Crystal Structure of a Coiled-Coil Domain from Human ROCK I

PubMed Central

Tu, Daqi; Li, Yiqun; Song, Hyun Kyu; Toms, Angela V.; Gould, Christopher J.; Ficarro, Scott B.; Marto, Jarrod A.; Goode, Bruce L.; Eck, Michael J.

2011-01-01

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535–700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation. PMID:21445309

Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions

PubMed Central

Bunney, Tom D.; Cole, Ambrose R.; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W.; Katan, Matilda

2014-01-01

Summary Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition. PMID:25435325

Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

PubMed

Bunney, Tom D; Cole, Ambrose R; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W; Katan, Matilda

2014-12-02

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.

PubMed Central

Liang, H; Olejniczak, E T; Mao, X; Nettesheim, D G; Yu, L; Thompson, C B; Fesik, S W

1994-01-01

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins. Images PMID:7972119

Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.

PubMed

Hu, Wanhui; Wu, Huiwen; Zhang, Hong; Gong, Weibin; Perrett, Sarah

2015-10-01

Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) (1)H, (13)C, (15)N backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following α-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.

X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Domingo Meza-Aguilar, J.; Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F.; Fromme, Petra

Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause ofmore » acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.« less

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Siyang; Gao, Song; Kondabagil, Kiran

2012-04-04

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the centralmore » domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.« less

Structures of EccB 1 and EccD 1 from the core complex of the mycobacterial ESX-1 type VII secretion system

DOE PAGES

Wagner, Jonathan M.; Chan, Sum; Evans, Timothy J.; ...

2016-02-27

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB 1 and EccD 1. The periplasmic domain of EccB 1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. Themore » repeat domains of EccB 1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD 1 has a ubiquitin-like fold and forms a dimer with a negatively charged groove. In conclusion, these structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.« less

Structures of EccB 1 and EccD 1 from the core complex of the mycobacterial ESX-1 type VII secretion system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, Jonathan M.; Chan, Sum; Evans, Timothy J.

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB 1 and EccD 1. The periplasmic domain of EccB 1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. Themore » repeat domains of EccB 1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD 1 has a ubiquitin-like fold and forms a dimer with a negatively charged groove. In conclusion, these structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.« less

Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coli.

PubMed

Tran, An X; Dong, Changjiang; Whitfield, Chris

2010-10-22

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.

Characterizing protein domain associations by Small-molecule ligand binding

PubMed Central

Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

2012-01-01

Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

Polarity Control and Growth of Lateral Polarity Structures in AlN

DTIC Science & Technology

2013-05-10

domains. Transmission electron microscopy shows mixed edge-screw type dislocations with polarity-dependent dislocation bending. Raman 1. REPORT DATE (DD-MM...polarity-dependent dislocation bending. Raman spectroscopy reveals compressively strained Al-polar and relaxed N-polar domains. The near band edge...dislocation bending. Raman spectroscopy reveals compressively strained Al-polar and relaxed N-polar domains. The near band edge luminescence consists of

The Domain of Cognition: An Alternative to Bloom's Cognitive Domain within the Framework of an Information Processing Model.

ERIC Educational Resources Information Center

Stahl, Robert J.; Murphy, Gary T.

Weaknesses in the structure, levels, and sequence of Bloom's taxonomy of cognitive domains emphasize the need for both a new model of how individual learners process information and a new taxonomy of the different levels of memory, thinking, and learning. Both the model and the taxonomy should be consistent with current research findings. The…

1H and 15N NMR resonance assignments and secondary structure of titin type I domains.

PubMed

Muhle-Goll, C; Nilges, M; Pastore, A

1997-01-01

Titin/connectin is a giant muscle protein with a highly modular architecture consisting of multiple repeats of two sequence motifs, named type I and type II. Type I modules have been suggested to be intracellular members of the fibronectin type III (Fn3) domain family. Along the titin sequence they are exclusively present in the region of the molecule located in the sarcomere A-band. This region has been shown to interact with myosin and C-protein. One of the most noticeable features of type I modules is that they are particularly rich in semiconserved prolines, since these residues account for about 8% of their sequence. We have determined the secondary structure of a representative type I domain (A71) by 15N and 1H NMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting of a three- and a four-stranded beta-sheet. When the two sheets are placed on top of each other to form the beta-sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the same side of the molecule and form an exposed hydrophobic patch. This suggests that the semiconserved prolines might be relevant for the function of type I modules, providing a surface for binding to other A-band proteins. The secondary structure of A71 was structurally aligned to other extracellular Fn3 modules of known 3D structure. The alignment shows that titin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.

Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii.

PubMed

Pampa, K J; Madan Kumar, S; Hema, M K; Kumara, Karthik; Naveen, S; Kunishima, Naoki; Lokanath, N K

2017-12-01

Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-L-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subunits, presumably near to the active site(s) of the enzyme. The observed dimeric state might be important for the catalytic function of the enzyme.

Kinetoplast DNA minicircles of phloem-restricted Phytomonas associated with wilt diseases of coconut and oil palms have a two-domain structure.

PubMed

Dollet, M; Sturm, N R; Ahomadegbe, J C; Campbell, D A

2001-11-27

We report the cloning and sequencing of the first minicircle from a phloem-restricted, pathogenic Phytomonas sp. (Hart 1) isolated from a coconut palm with hartrot disease. The minicircle possessed a two-domain structure of two conserved regions, each containing three conserved sequence blocks (CSB). Based on the sequence around CSB 3 from Hart 1, PCR primers were designed to allow specific amplification of Phytomonas minicircles. This primer pair demonstrated specificity for at least six groups of plant trypanosomatids and did not amplify from insect trypanosomatids. The PCR results were consistent with a two-domain structure for other plant trypanosomatids.

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein.

PubMed

Gely, Stéphane; Lowry, David F; Bernard, Cédric; Jensen, Malene R; Blackledge, Martin; Costanzo, Stéphanie; Bourhis, Jean-Marie; Darbon, Hervé; Daughdrill, Gary; Longhi, Sonia

2010-01-01

In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.

Functional and structural characterization of domain truncated violaxanthin de-epoxidase.

PubMed

Hallin, Erik Ingmar; Guo, Kuo; Åkerlund, Hans-Erik

2016-08-01

Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE. © 2016 Scandinavian Plant Physiology Society.

The structure of the exopolyphsophatase (PPX) from Escherchia coli O157:H7 suggests a binding mode for long polyphosphate chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rangarajan,E.; Nadeau, G.; Li, Y.

2006-01-01

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains Imore » and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the 'closed' state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an 'open' state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.« less

Structure and function of POTRA domains of Omp85/TPS superfamily.

PubMed

Simmerman, Richard F; Dave, Ashita M; Bruce, Barry D

2014-01-01

The Omp85/TPS (outer-membrane protein of 85 kDa/two-partner secretion) superfamily is a ubiquitous and major class of β-barrel proteins. This superfamily is restricted to the outer membranes of gram-negative bacteria, mitochondria, and chloroplasts. The common architecture, with an N-terminus consisting of repeats of soluble polypeptide-transport-associated (POTRA) domains and a C-terminal β-barrel pore is highly conserved. The structures of multiple POTRA domains and one full-length TPS protein have been solved, yet discovering roles of individual POTRA domains has been difficult. This review focuses on similarities and differences between POTRA structures, emphasizing POTRA domains in autotrophic organisms including plants and cyanobacteria. Unique roles, specific for certain POTRA domains, are examined in the context of POTRA location with respect to their attachment to the β-barrel pore, and their degree of biological dispensability. Finally, because many POTRA domains may have the ability to interact with thousands of partner proteins, possible modes of these interactions are also explored. © 2014 Elsevier Inc. All rights reserved.

Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

NASA Technical Reports Server (NTRS)

Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

2003-01-01

Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

A Probabilistic Graphical Model to Detect Chromosomal Domains

NASA Astrophysics Data System (ADS)

Heermann, Dieter; Hofmann, Andreas; Weber, Eva

To understand the nature of a cell, one needs to understand the structure of its genome. For this purpose, experimental techniques such as Hi-C detecting chromosomal contacts are used to probe the three-dimensional genomic structure. These experiments yield topological information, consistently showing a hierarchical subdivision of the genome into self-interacting domains across many organisms. Current methods for detecting these domains using the Hi-C contact matrix, i.e. a doubly-stochastic matrix, are mostly based on the assumption that the domains are distinct, thus non-overlapping. For overcoming this simplification and for being able to unravel a possible nested domain structure, we developed a probabilistic graphical model that makes no a priori assumptions on the domain structure. Within this approach, the Hi-C contact matrix is analyzed using an Ising like probabilistic graphical model whose coupling constant is proportional to each lattice point (entry in the contact matrix). The results show clear boundaries between identified domains and the background. These domain boundaries are dependent on the coupling constant, so that one matrix yields several clusters of different sizes, which show the self-interaction of the genome on different scales. This work was supported by a Grant from the International Human Frontier Science Program Organization (RGP0014/2014).

Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose.

PubMed

Tomoo, Koji; Miki, Yasuhiro; Morioka, Hideaki; Seike, Kiho; Ishida, Toshimasa; Ikenishi, Sadao; Miyamoto, Katsushiro; Hasegawa, Tomokazu; Yamano, Akihito; Hamada, Kensaku; Tsujibo, Hiroshi

2017-06-01

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands.

PubMed

Gatta, Alberto T; Sauerwein, Andrea C; Zhuravleva, Anastasia; Levine, Tim P; Matthews, Stephen

2018-01-15

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding. Copyright © 2017 Elsevier Inc. All rights reserved.

Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, A. E., E-mail: schmidt@omrb.pnpi.spb.ru; Shvetsov, A. V.; Kuklin, A. I.

2016-01-15

Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantialmore » contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.« less

Stage Structure of Moral Development: A Comparison of Alternative Models.

ERIC Educational Resources Information Center

Hau, Kit-Tai

This study evaluated the stage structure of several quasi-simplex and non-simplex models of moral development in two domains of moral development in a British and a Chinese sample. Analyses were based on data reported by Sachs (1992): the Chinese sample consisted of 1,005 students from grade 9 to post-college, and the British sample consisted of…

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain.

PubMed

Perederina, Anna; Esakova, Olga; Quan, Chao; Khanova, Elena; Krasilnikov, Andrey S

2010-02-17

Ribonuclease (RNase) P is a site-specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix-loop-helix protein-binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 A. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.

A new crystal structure of the bifunctional antibiotic simocyclinone D8 bound to DNA gyrase gives fresh insight into the mechanism of inhibition.

PubMed

Hearnshaw, Stephen J; Edwards, Marcus J; Stevenson, Clare E; Lawson, David M; Maxwell, Anthony

2014-05-15

Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~1000-fold weaker than the interaction with GyrA. Copyright © 2014. Published by Elsevier Ltd.

A New Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8 Bound to DNA Gyrase Gives Fresh Insight into the Mechanism of Inhibition

PubMed Central

Hearnshaw, Stephen J.; Edwards, Marcus J.; Stevenson, Clare E.; Lawson, David M.; Maxwell, Anthony

2014-01-01

Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~ 1000-fold weaker than the interaction with GyrA. PMID:24594357

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution, Obtained Using Small-Angle X-Ray Scattering

PubMed Central

Taube, Michał; Pieńkowska, Joanna R.; Jarmołowski, Artur; Kozak, Maciej

2014-01-01

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed. PMID:24714665

Comparative analysis of barophily-related amino acid content in protein domains of Pyrococcus abyssi and Pyrococcus furiosus.

PubMed

Yafremava, Liudmila S; Di Giulio, Massimo; Caetano-Anollés, Gustavo

2013-01-01

Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meagher, Martin; Enemark, Eric J.

The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel.

PubMed

Soler-Llavina, Gilberto J; Chang, Tsg-Hui; Swartz, Kenton J

2006-11-22

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

Shear at Twin Domain Boundaries in YBa2Cu3O7-x

NASA Astrophysics Data System (ADS)

Caldwell, W. A.; Tamura, N.; Celestre, R. S.; MacDowell, A. A.; Padmore, H. A.; Geballe, T. H.; Koster, G.; Batterman, B. W.; Patel, J. R.

2004-05-01

The microstructure and strain state of twin domains in YBa2Cu3O7-x are discussed based upon synchrotron white-beam x-ray microdiffraction measurements. Intensity variations of the fourfold twin splitting of Laue diffraction peaks are used to determine the twin domain structure. Strain analysis shows that interfaces between neighboring twin domains are strained in shear, whereas the interior of these domains are regions of low strain. These measurements are consistent with the orientation relationships of twin boundaries within and across domains and show that basal plane shear stresses can exceed 100MPa where twin domains meet. Our results support stress field pinning of magnetic flux vortices by twin domain boundaries.

Structural evolution of the Chugach-Prince William terrane at the hinge of the orocline in Prince William Sound, and implications for ore deposits: A section in Geologic studies in Alaska by the U.S. Geological Survey, 1992

USGS Publications Warehouse

Haeussler, Peter J.; Nelson, Steven W.

1993-01-01

The Chugach-Prince William terrane is a Mesozoic through Tertiary accretionary complex that lies along coastal southern and southeastern Alaska. In Prince William Sound, the regional structural fabric bends about 90°, forming an orocline. Rocks at the hinge of the orocline consist of turbidites, conglomerate, and minor volcanic rocks and limestone. The structural geology in the hinge region defines a number of domains (each >15 km2) consisting of kilometer-scale tight folds. Adjacent domains may have up to a 90° difference in the strike of bedding and trend of fold axes. Four granite to tonalitic or gabbro plutons are dated or inferred to be about 35 Main age, and all were emplaced after regional folding. Base-metal sulfide mineral occurrences, barren quartz veins, and strikeslip late faults locally cut the plutons and generally strike north-south. The mineral occurrences often have a dendritic network of quartz veinlets adjacent to the mineralized zone and brecciated wall rock within the zone. Oroclinal bending, in the style of bending a bar, is consistent with the origin of the complicated domainal geometry of the structures. The Contact fault, thought by some workers to juxtapose two parts of the accretionary prism, truncates one of these 35-Ma plutons with strike-slip offset, but previous reverse motion cannot be ruled out. A magmatic source for the ore-forming fluids is consistent with the structural and mineralogical data. The north-south orientation of mineralized zones suggests that east-west extension occurred possibly during release of fluids from the plutons, which locally hydrofractured the wall rocks and allowed migration of ore-forming fluids along preexisting fractures.

Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore

PubMed Central

Seyedmohammad, Saeed; Fuentealba, Natalia Alveal; Marriott, Robert A.J.; Goetze, Tom A.; Edwardson, J. Michael; Barrera, Nelson P.; Venter, Henrietta

2016-01-01

Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel. PMID:26934982

Molecular weight dependent structure and charge transport in MAPLE-deposited poly(3-hexylthiophene) thin films

DOE PAGES

Dong, Ban Xuan; Smith, Mitchell; Strzalka, Joseph; ...

2018-02-06

In this work, poly(3-hexylthiophene) (P3HT) films prepared using the matrix-assisted pulsed laser evaporation (MAPLE) technique are shown to possess morphological structures that are dependent on molecular weight (MW). Specifically, the structures of low MW samples of MAPLE-deposited film are composed of crystallites/aggregates embedded within highly disordered environments, whereas those of high MW samples are composed of aggregated domains connected by long polymer chains. Additionally, the crystallite size along the side-chain (100) direction decreases, whereas the conjugation length increases with increasing molecular weight. This is qualitatively similar to the structure of spin-cast films, though the MAPLE-deposited films are more disordered. In-planemore » carrier mobilities in the MAPLE-deposited samples increase with MW, consistent with the notion that longer chains bridge adjacent aggregated domains thereby facilitating more effective charge transport. The carrier mobilities in the MAPLE-deposited simples are consistently lower than those in the solvent-cast samples for all molecular weights, consistent with the shorter conjugation length in samples prepared by this deposition technique.« less

Molecular weight dependent structure and charge transport in MAPLE-deposited poly(3-hexylthiophene) thin films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Ban Xuan; Smith, Mitchell; Strzalka, Joseph

In this work, poly(3-hexylthiophene) (P3HT) films prepared using the matrix-assisted pulsed laser evaporation (MAPLE) technique are shown to possess morphological structures that are dependent on molecular weight (MW). Specifically, the structures of low MW samples of MAPLE-deposited film are composed of crystallites/aggregates embedded within highly disordered environments, whereas those of high MW samples are composed of aggregated domains connected by long polymer chains. Additionally, the crystallite size along the side-chain (100) direction decreases, whereas the conjugation length increases with increasing molecular weight. This is qualitatively similar to the structure of spin-cast films, though the MAPLE-deposited films are more disordered. In-planemore » carrier mobilities in the MAPLE-deposited samples increase with MW, consistent with the notion that longer chains bridge adjacent aggregated domains thereby facilitating more effective charge transport. The carrier mobilities in the MAPLE-deposited simples are consistently lower than those in the solvent-cast samples for all molecular weights, consistent with the shorter conjugation length in samples prepared by this deposition technique.« less

X-Ray Crystal Structure of the passenger domain of Plasmid encoded toxin(Pet), an Autotransporter Enterotoxin from enteroaggregative Escherichia coli (EAEC)

PubMed Central

Meza-Aguilar, J. Domingo; Fromme, Petra; Torres-Larios, Alfredo; Mendoza-Hernández, Guillermo; Hernandez-Chiñas, Ulises; Monteros, Roberto A. Arreguin-Espinosa de los; Campos, Carlos A. Eslava; Fromme, Raimund

2014-01-01

Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50 % compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181-190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135-143 compared to the structure of EspP. PMID:24530907

Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant

NASA Astrophysics Data System (ADS)

Yuchi, Zhiguang; Yuen, Siobhan M. Wong King; Lau, Kelvin; Underhill, Ainsley Q.; Cornea, Razvan L.; Fessenden, James D.; van Petegem, Filip

2015-08-01

Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca2+-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2-1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding.

Enhanced sampling of glutamate receptor ligand-binding domains.

PubMed

Lau, Albert Y

2018-04-14

The majority of excitatory synaptic transmission in the central nervous system is mediated by ionotropic glutamate receptors (iGluRs). These membrane-bound protein assemblies consist of modular domains that can be genetically isolated and expressed, which has resulted in a plethora of crystal structures of individual domains in different conformations bound to different ligands. These structures have presented opportunities for molecular dynamics (MD) simulation studies. To examine the free energies that govern molecular behavior, simulation strategies and algorithms have been developed, collectively called enhanced sampling methods This review focuses on the use of enhanced sampling MD simulations of isolated iGluR ligand-binding domains to characterize thermodynamic properties important to receptor function. Copyright © 2018 Elsevier B.V. All rights reserved.

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

PubMed Central

Tamada, Taro; Honjo, Eijiro; Maeda, Yoshitake; Okamoto, Tomoyuki; Ishibashi, Matsujiro; Tokunaga, Masao; Kuroki, Ryota

2006-01-01

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 Å resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses. PMID:16492764

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Rajesh Kumar; Palm, Gottfried J.; Panjikar, Santosh

2007-04-01

Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein. Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. Inmore » the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.« less

Schmallenberg virus non-structural protein NSm: Intracellular distribution and role of non-hydrophobic domains.

PubMed

Kraatz, Franziska; Wernike, Kerstin; Reiche, Sven; Aebischer, Andrea; Reimann, Ilona; Beer, Martin

2018-03-01

Schmallenberg virus (SBV) induces fetal malformation, abortions and stillbirth in ruminants. While the non-structural protein NSs is a major virulence factor, the biological function of NSm, the second non-structural protein which consists of three hydrophobic transmembrane (I, III, V) and two non-hydrophobic regions (II, IV), is still unknown. Here, a series of NSm mutants displaying deletions of nearly the entire NSm or of the non-hydrophobic domains was generated and the intracellular distribution of NSm was assessed. SBV-NSm is dispensable for the generation of infectious virus and mutants lacking domains II - V showed growth properties similar to the wild-type virus. In addition, a comparable intracellular distribution of SBV-NSm was observed in mammalian cells infected with domain II mutants or wild-type virus. In both cases, NSm co-localized with the glycoprotein Gc in the Golgi compartment. However, domain IV-deletion mutants showed an altered distribution pattern and no co-localization of NSm and Gc. Copyright © 2018 Elsevier Inc. All rights reserved.

Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin.

PubMed

Papanikolopoulou, Katerina; Forge, Vincent; Goeltz, Pierrette; Mitraki, Anna

2004-03-05

The folding of beta-structured, fibrous proteins is a largely unexplored area. A class of such proteins is used by viruses as adhesins, and recent studies revealed novel beta-structured motifs for them. We have been studying the folding and assembly of adenovirus fibers that consist of a globular C-terminal domain, a central fibrous shaft, and an N-terminal part that attaches to the viral capsid. The globular C-terminal, or "head" domain, has been postulated to be necessary for the trimerization of the fiber and might act as a registration signal that directs its correct folding and assembly. In this work, we replaced the head of the fiber by the trimerization domain of the bacteriophage T4 fibritin, termed "foldon." Two chimeric proteins, comprising the foldon domain connected at the C-terminal end of four fiber shaft repeats with or without the use of a natural linker sequence, fold into highly stable, SDS-resistant trimers. The structural signatures of the chimeric proteins as seen by CD and infrared spectroscopy are reported. The results suggest that the foldon domain can successfully replace the fiber head domain in ensuring correct trimerization of the shaft sequences. Biological implications and implications for engineering highly stable, beta-structured nanorods are discussed.

Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes

PubMed Central

Zhan, Xuanzhi; Gimenez, Luis E.; Gurevich, Vsevolod V.; Spiller, Benjamin W.

2011-01-01

Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. Here we report the first crystal structure of arrestin-3, solved at 3.0Å. Arrestin-3 is an elongated two-domain molecule with the overall fold and key inter-domain interactions that hold free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and -3 we show that the presence of this loose structure correlates with reduced arrestin selectivity for activated receptor, consistent with a conformational change in this β-sheet upon receptor binding. PMID:21215759

Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

PubMed

Chang, Chung-ke; Chen, Chia-Min Michael; Chiang, Ming-hui; Hsu, Yen-lan; Huang, Tai-huang

2013-01-01

The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain

PubMed Central

Djordjevic, Snezana; Goudreau, Paul N.; Xu, Qingping; Stock, Ann M.; West, Ann H.

1998-01-01

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein–protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces. PMID:9465023

Is there a domain-general cognitive structuring system? Evidence from structural priming across music, math, action descriptions, and language.

PubMed

Van de Cavey, Joris; Hartsuiker, Robert J

2016-01-01

Cognitive processing in many domains (e.g., sentence comprehension, music listening, and math solving) requires sequential information to be organized into an integrational structure. There appears to be some overlap in integrational processing across domains, as shown by cross-domain interference effects when for example linguistic and musical stimuli are jointly presented (Koelsch, Gunter, Wittfoth, & Sammler, 2005; Slevc, Rosenberg, & Patel, 2009). These findings support theories of overlapping resources for integrational processing across domains (cfr. SSIRH Patel, 2003; SWM, Kljajevic, 2010). However, there are some limitations to the studies mentioned above, such as the frequent use of unnaturalistic integrational difficulties. In recent years, the idea has risen that evidence for domain-generality in structural processing might also be yielded though priming paradigms (cfr. Scheepers, 2003). The rationale behind this is that integrational processing across domains regularly requires the processing of dependencies across short or long distances in the sequence, involving respectively less or more syntactic working memory resources (cfr. SWM, Kljajevic, 2010), and such processing decisions might persist over time. However, whereas recent studies have shown suggestive priming of integrational structure between language and arithmetics (though often dependent on arithmetic performance, cfr. Scheepers et al., 2011; Scheepers & Sturt, 2014), it remains to be investigated to what extent we can also find evidence for priming in other domains, such as music and action (cfr. SWM, Kljajevic, 2010). Experiment 1a showed structural priming from the processing of musical sequences onto the position in the sentence structure (early or late) to which a relative clause was attached in subsequent sentence completion. Importantly, Experiment 1b showed that a similar structural manipulation based on non-hierarchically ordered color sequences did not yield any priming effect, suggesting that the priming effect is not based on linear order, but integrational dependency. Finally, Experiment 2 presented primes in four domains (relative clause sentences, music, mathematics, and structured descriptions of actions), and consistently showed priming within and across domains. These findings provide clear evidence for domain-general structural processing mechanisms. Copyright © 2015 Elsevier B.V. All rights reserved.

ECOD: An Evolutionary Classification of Protein Domains

PubMed Central

Kinch, Lisa N.; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V.

2014-01-01

Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies. PMID:25474468

ECOD: an evolutionary classification of protein domains.

PubMed

Cheng, Hua; Schaeffer, R Dustin; Liao, Yuxing; Kinch, Lisa N; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V

2014-12-01

Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or "fold"). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.

Natural triple beta-stranded fibrous folds.

PubMed

Mitraki, Anna; Papanikolopoulou, Katerina; Van Raaij, Mark J

2006-01-01

A distinctive family of beta-structured folds has recently been described for fibrous proteins from viruses. Virus fibers are usually involved in specific host-cell recognition. They are asymmetric homotrimeric proteins consisting of an N-terminal virus-binding tail, a central shaft or stalk domain, and a C-terminal globular receptor-binding domain. Often they are entirely or nearly entirely composed of beta-structure. Apart from their biological relevance and possible gene therapy applications, their shape, stability, and rigidity suggest they may be useful as blueprints for biomechanical design. Folding and unfolding studies suggest their globular C-terminal domain may fold first, followed by a "zipping-up" of the shaft domains. The C-terminal domains appear to be important for registration because peptides corresponding to shaft domains alone aggregate into nonnative fibers and/or amyloid structures. C-terminal domains can be exchanged between different fibers and the resulting chimeric proteins are useful as a way to solve structures of unknown parts of the shaft domains. The following natural triple beta-stranded fibrous folds have been discovered by X-ray crystallography: the triple beta-spiral, triple beta-helix, and T4 short tail fiber fold. All have a central longitudinal hydrophobic core and extensive intermonomer polar and nonpolar interactions. Now that a reasonable body of structural and folding knowledge has been assembled about these fibrous proteins, the next challenge and opportunity is to start using this information in medical and industrial applications such as gene therapy and nanotechnology.

Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity

PubMed Central

Lorenz, Sonja; Deng, Patricia; Hantschel, Oliver; Superti-Furga, Giulio; Kuriyan, John

2018-01-01

Constitutive activation of the non-receptor tyrosine kinase c-Abl (Abl1) in the Bcr-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukemia. Recent studies have indicated that an interaction between the SH2 domain and the N-lobe of the c-Abl kinase domain has a critical role in leukemogenesis. To dissect the structural basis of this phenomenon we studied c-Abl constructs comprising the SH2 and kinase domains in vitro. We present a crystal structure of an SH2-kinase domain construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the kinase domain. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2-N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the kinase domain. That the effects are small compared to the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the autoinhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity. PMID:25779001

Mesostructure of Ordered Corneal Nano-nipple Arrays: The Role of 5-7 Coordination Defects

NASA Astrophysics Data System (ADS)

Lee, Ken C.; Yu, Qi; Erb, Uwe

2016-06-01

Corneal nano-nipple structures consisting of hexagonally arranged protrusions with diameters around 200 nm have long been known for their antireflection capability and have served as biological blueprint for solar cell, optical lens and other surface designs. However, little is known about the global arrangement of these nipples on the ommatidial surface and their growth during the eye development. This study provides new insights based on the analysis of nano-nipple arrangements on the mesoscale across entire ommatidia, which has never been done before. The most important feature in the nipple structures are topological 5- and 7-fold coordination defects, which align to form dislocations and interconnected networks of grain boundaries that divide the ommatidia into crystalline domains in different orientations. Furthermore, the domain size distribution might be log-normal, and the domains demonstrate no preference in crystal orientation. Both observations suggest that the nipple growth process may be similar to the nucleation and growth mechanisms during the formation of other crystal structures. Our results are also consistent with the most recently proposed Turing-type reaction-diffusion process. In fact, we were able to produce the key structural characteristics of the nipple arrangements using Turing analysis from the nucleation to the final structure development.

Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.

PubMed

Kunys, Andrew R; Lian, Wenlong; Pei, Dehua

2012-12-01

One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.

Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis.

PubMed

Levdikov, Vladimir M; Blagova, Elena; Young, Vicki L; Belitsky, Boris R; Lebedev, Andrey; Sonenshein, Abraham L; Wilkinson, Anthony J

2017-02-17

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (c G MP-stimulated phosphodiesterases, a denylate cyclases, F hlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity.

PubMed

Ho, C S James; Rydstrom, Anna; Manimekalai, Malathy Sony Subramanian; Svanborg, Catharina; Grüber, Gerhard

2012-01-01

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.

Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

PubMed Central

Ho CS, James; Rydstrom, Anna; Manimekalai, Malathy Sony Subramanian; Svanborg, Catharina; Grüber, Gerhard

2012-01-01

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells. PMID:23300861

Domain Motion Enhanced (DoME) Model for Efficient Conformational Sampling of Multidomain Proteins.

PubMed

Kobayashi, Chigusa; Matsunaga, Yasuhiro; Koike, Ryotaro; Ota, Motonori; Sugita, Yuji

2015-11-19

Large conformational changes of multidomain proteins are difficult to simulate using all-atom molecular dynamics (MD) due to the slow time scale. We show that a simple modification of the structure-based coarse-grained (CG) model enables a stable and efficient MD simulation of those proteins. "Motion Tree", a tree diagram that describes conformational changes between two structures in a protein, provides information on rigid structural units (domains) and the magnitudes of domain motions. In our new CG model, which we call the DoME (domain motion enhanced) model, interdomain interactions are defined as being inversely proportional to the magnitude of the domain motions in the diagram, whereas intradomain interactions are kept constant. We applied the DoME model in combination with the Go model to simulations of adenylate kinase (AdK). The results of the DoME-Go simulation are consistent with an all-atom MD simulation for 10 μs as well as known experimental data. Unlike the conventional Go model, the DoME-Go model yields stable simulation trajectories against temperature changes and conformational transitions are easily sampled despite domain rigidity. Evidently, identification of domains and their interfaces is useful approach for CG modeling of multidomain proteins.

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Gustchina, Alla; Rasulova, Fatima S.

2010-10-22

The structure of a recombinant construct consisting of residues 1-245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 {angstrom} resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal {alpha}-helix. The structure of the first subdomain (residues 1-117), which consists mostly of {beta}-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas themore » second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

Domain analyses of Usher syndrome causing Clarin-1 and GPR98 protein models.

PubMed

Khan, Sehrish Haider; Javed, Muhammad Rizwan; Qasim, Muhammad; Shahzadi, Samar; Jalil, Asma; Rehman, Shahid Ur

2014-01-01

Usher syndrome is an autosomal recessive disorder that causes hearing loss, Retinitis Pigmentosa (RP) and vestibular dysfunction. It is clinically and genetically heterogeneous disorder which is clinically divided into three types i.e. type I, type II and type III. To date, there are about twelve loci and ten identified genes which are associated with Usher syndrome. A mutation in any of these genes e.g. CDH23, CLRN1, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A and DFNB31 can result in Usher syndrome or non-syndromic deafness. These genes provide instructions for making proteins that play important roles in normal hearing, balance and vision. Studies have shown that protein structures of only seven genes have been determined experimentally and there are still three genes whose structures are unavailable. These genes are Clarin-1, GPR98 and Usherin. In the absence of an experimentally determined structure, homology modeling and threading often provide a useful 3D model of a protein. Therefore in the current study Clarin-1 and GPR98 proteins have been analyzed for signal peptide, domains and motifs. Clarin-1 protein was found to be without any signal peptide and consists of prokar lipoprotein domain. Clarin-1 is classified within claudin 2 super family and consists of twelve motifs. Whereas, GPR98 has a 29 amino acids long signal peptide and classified within GPCR family 2 having Concanavalin A-like lectin/glucanase superfamily. It was found to be consists of GPS and G protein receptor F2 domains and twenty nine motifs. Their 3D structures have been predicted using I-TASSER server. The model of Clarin-1 showed only α-helix but no beta sheets while model of GPR98 showed both α-helix and β sheets. The predicted structures were then evaluated and validated by MolProbity and Ramachandran plot. The evaluation of the predicted structures showed 78.9% residues of Clarin-1 and 78.9% residues of GPR98 within favored regions. The findings of present study has resulted in the three dimensional structure prediction and conserved domain analysis which will be quite beneficial in better understanding of molecular components, protein-protein interaction, clinical heterogeneity and pathophysiology of Usher syndrome.

Domain analyses of Usher syndrome causing Clarin-1 and GPR98 protein models

PubMed Central

Khan, Sehrish Haider; Javed, Muhammad Rizwan; Qasim, Muhammad; Shahzadi, Samar; Jalil, Asma; Rehman, Shahid ur

2014-01-01

Usher syndrome is an autosomal recessive disorder that causes hearing loss, Retinitis Pigmentosa (RP) and vestibular dysfunction. It is clinically and genetically heterogeneous disorder which is clinically divided into three types i.e. type I, type II and type III. To date, there are about twelve loci and ten identified genes which are associated with Usher syndrome. A mutation in any of these genes e.g. CDH23, CLRN1, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A and DFNB31 can result in Usher syndrome or non-syndromic deafness. These genes provide instructions for making proteins that play important roles in normal hearing, balance and vision. Studies have shown that protein structures of only seven genes have been determined experimentally and there are still three genes whose structures are unavailable. These genes are Clarin-1, GPR98 and Usherin. In the absence of an experimentally determined structure, homology modeling and threading often provide a useful 3D model of a protein. Therefore in the current study Clarin-1 and GPR98 proteins have been analyzed for signal peptide, domains and motifs. Clarin-1 protein was found to be without any signal peptide and consists of prokar lipoprotein domain. Clarin-1 is classified within claudin 2 super family and consists of twelve motifs. Whereas, GPR98 has a 29 amino acids long signal peptide and classified within GPCR family 2 having Concanavalin A-like lectin/glucanase superfamily. It was found to be consists of GPS and G protein receptor F2 domains and twenty nine motifs. Their 3D structures have been predicted using I-TASSER server. The model of Clarin-1 showed only α-helix but no beta sheets while model of GPR98 showed both α-helix and β sheets. The predicted structures were then evaluated and validated by MolProbity and Ramachandran plot. The evaluation of the predicted structures showed 78.9% residues of Clarin-1 and 78.9% residues of GPR98 within favored regions. The findings of present study has resulted in the three dimensional structure prediction and conserved domain analysis which will be quite beneficial in better understanding of molecular components, protein-protein interaction, clinical heterogeneity and pathophysiology of Usher syndrome. PMID:25258483

X-ray structure determination at low resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunger, Axel T., E-mail: brunger@stanford.edu; Department of Molecular and Cellular Physiology, Stanford University; Department of Neurology and Neurological Sciences, Stanford University

2009-02-01

Refinement is meaningful even at 4 Å or lower, but with present methodologies it should start from high-resolution crystal structures whenever possible. As an example of structure determination in the 3.5–4.5 Å resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alonemore » was subsequently solved at 3 Å resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at ∼4 Å resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible.« less

Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard

Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLAmore » complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.« less

The CWB2 Cell Wall-Anchoring Module Is Revealed by the Crystal Structures of the Clostridium difficile Cell Wall Proteins Cwp8 and Cwp6.

PubMed

Usenik, Aleksandra; Renko, Miha; Mihelič, Marko; Lindič, Nataša; Borišek, Jure; Perdih, Andrej; Pretnar, Gregor; Müller, Uwe; Turk, Dušan

2017-03-07

Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helixmore » of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.« less

The reliability and validity of the English version of the Evaluation of Daily Activity Questionnaire for people with rheumatoid arthritis

PubMed Central

Tennant, Alan; Tyson, Sarah F.; Nordenskiöld, Ulla; Hawkins, Ruth; Prior, Yeliz

2015-01-01

Objectives. The Evaluation of Daily Activity Questionnaire (EDAQ) includes 138 items in 14 domains identified as important by people with RA. The aim of this study was to test the validity and reliability of the English EDAQ. Methods. A total of 502 participants completed two questionnaires 3 weeks apart. The first consisted of the EDAQ, HAQ, RA Quality of Life (RAQoL) and the Medical Outcomes Scale (MOS) 36-item Short-Form Health Survey (SF-36v2), and the second consisted of the EDAQ only. The 14 EDAQ domains were tested for: unidimensionality—using confirmatory factor analysis; fit, response dependency, invariance across groups (differential item functioning)—using Rasch analysis; internal consistency [Person Separation Index (PSI)]; concurrent validity—by correlations with the HAQ, SF-36v2 and RAQoL; and test–retest reliability (Spearman’s correlations). Results. Confirmatory factor analysis of the 14 EDAQ domains indicated unidimensionality, after adjustment for local dependency in each domain. All domains achieved a root mean square error of approximation <0.10 and satisfied Rasch model expectations for local dependency. DIF by age, gender and employment status was largely absent. The PSI was consistent with individual use (PSI = 0.94 for all 14 domains). For all domains, except Caring, concurrent validity was good: HAQ (rs = 0.72–0.91), RAQoL (rs = 0.67–0.82) and SF36v2 Physical Function scale (rs = −0.60 to −0.84) and test–retest reliability was good (rs = 0.70–0.89). Conclusion. Analysis supported a 14-domain, two-component structure (Self care and Mobility) of the EDAQ, where each domain, and both components, satisfied Rasch model requirements, and have robust reliability and validity. PMID:25863045

The prokaryotic zinc-finger: structure, function and comparison with the eukaryotic counterpart.

PubMed

Malgieri, Gaetano; Palmieri, Maddalena; Russo, Luigi; Fattorusso, Roberto; Pedone, Paolo V; Isernia, Carla

2015-12-01

Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2 His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships. © 2015 FEBS.

Neurology objective structured clinical examination reliability using generalizability theory

PubMed Central

Park, Yoon Soo; Lukas, Rimas V.; Brorson, James R.

2015-01-01

Objectives: This study examines factors affecting reliability, or consistency of assessment scores, from an objective structured clinical examination (OSCE) in neurology through generalizability theory (G theory). Methods: Data include assessments from a multistation OSCE taken by 194 medical students at the completion of a neurology clerkship. Facets evaluated in this study include cases, domains, and items. Domains refer to areas of skill (or constructs) that the OSCE measures. G theory is used to estimate variance components associated with each facet, derive reliability, and project the number of cases required to obtain a reliable (consistent, precise) score. Results: Reliability using G theory is moderate (Φ coefficient = 0.61, G coefficient = 0.64). Performance is similar across cases but differs by the particular domain, such that the majority of variance is attributed to the domain. Projections in reliability estimates reveal that students need to participate in 3 OSCE cases in order to increase reliability beyond the 0.70 threshold. Conclusions: This novel use of G theory in evaluating an OSCE in neurology provides meaningful measurement characteristics of the assessment. Differing from prior work in other medical specialties, the cases students were randomly assigned did not influence their OSCE score; rather, scores varied in expected fashion by domain assessed. PMID:26432851

Neurology objective structured clinical examination reliability using generalizability theory.

PubMed

Blood, Angela D; Park, Yoon Soo; Lukas, Rimas V; Brorson, James R

2015-11-03

This study examines factors affecting reliability, or consistency of assessment scores, from an objective structured clinical examination (OSCE) in neurology through generalizability theory (G theory). Data include assessments from a multistation OSCE taken by 194 medical students at the completion of a neurology clerkship. Facets evaluated in this study include cases, domains, and items. Domains refer to areas of skill (or constructs) that the OSCE measures. G theory is used to estimate variance components associated with each facet, derive reliability, and project the number of cases required to obtain a reliable (consistent, precise) score. Reliability using G theory is moderate (Φ coefficient = 0.61, G coefficient = 0.64). Performance is similar across cases but differs by the particular domain, such that the majority of variance is attributed to the domain. Projections in reliability estimates reveal that students need to participate in 3 OSCE cases in order to increase reliability beyond the 0.70 threshold. This novel use of G theory in evaluating an OSCE in neurology provides meaningful measurement characteristics of the assessment. Differing from prior work in other medical specialties, the cases students were randomly assigned did not influence their OSCE score; rather, scores varied in expected fashion by domain assessed. © 2015 American Academy of Neurology.

A New Model for Allosteric Regulation of Phenylalanine Hydroxylase: Implications for Disease and Therapeutics

PubMed Central

Stith, Linda; Lawrence, Sarah H.; Andrake, Mark; Dunbrack, Roland L.

2013-01-01

The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ~90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the inter-subunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic. PMID:23296088

Fas Apoptosis Inhibitory Molecule (FAIM) Contains a Novel Beta Sandwich in Contact with a Partially Ordered Domain

PubMed Central

Hemond, Michael; Rothstein, Thomas L.; Wagner, Gerhard

2009-01-01

Summary Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform (FAIM-S) is expressed in all tissue types, while an alternatively spliced long isoform (FAIM-L) is specifically expressed in the brain. Here FAIM-S is shown to consist of two independently folding domains in contact with one another. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded beta sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N- and C-terminal domains. PMID:19168072

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

PubMed

Freemont, P S; Ollis, D L; Steitz, T A; Joyce, C M

1986-09-01

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Mechanism of allosteric propagation across a β-sheet structure investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca; Thomas, Wendy E

2016-07-01

The bacterial adhesin FimH consists of an allosterically regulated mannose-binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter-domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter-domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring-like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called "population shift" model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990-1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

The structures of native celluloses, and the origin of their variability

Treesearch

R. H. Atalla

1999-01-01

The structures of native celluloses have traditionally been presented in terms of two-domain models consisting of crystalline and non-crystalline fractions. Such models have been of little help in advancing understanding of enzyme-substrate interactions. In this report we first address issues that complicate characterization of the structure of native celluloses...

The NIFSTD and BIRNLex Vocabularies: Building Comprehensive Ontologies for Neuroscience

PubMed Central

Bug, William J.; Ascoli, Giorgio A.; Grethe, Jeffrey S.; Gupta, Amarnath; Fennema-Notestine, Christine; Laird, Angela R.; Larson, Stephen D.; Rubin, Daniel; Shepherd, Gordon M.; Turner, Jessica A.; Martone, Maryann E.

2009-01-01

A critical component of the Neuroscience Information Framework (NIF) project is a consistent, flexible terminology for describing and retrieving neuroscience-relevant resources. Although the original NIF specification called for a loosely structured controlled vocabulary for describing neuroscience resources, as the NIF system evolved, the requirement for a formally structured ontology for neuroscience with sufficient granularity to describe and access a diverse collection of information became obvious. This requirement led to the NIF standardized (NIFSTD) ontology, a comprehensive collection of common neuroscience domain terminologies woven into an ontologically consistent, unified representation of the biomedical domains typically used to describe neuroscience data (e.g., anatomy, cell types, techniques), as well as digital resources (tools, databases) being created throughout the neuroscience community. NIFSTD builds upon a structure established by the BIRNLex, a lexicon of concepts covering clinical neuroimaging research developed by the Biomedical Informatics Research Network (BIRN) project. Each distinct domain module is represented using the Web Ontology Language (OWL). As much as has been practical, NIFSTD reuses existing community ontologies that cover the required biomedical domains, building the more specific concepts required to annotate NIF resources. By following this principle, an extensive vocabulary was assembled in a relatively short period of time for NIF information annotation, organization, and retrieval, in a form that promotes easy extension and modification. We report here on the structure of the NIFSTD, and its predecessor BIRNLex, the principles followed in its construction and provide examples of its use within NIF. PMID:18975148

The NIFSTD and BIRNLex vocabularies: building comprehensive ontologies for neuroscience.

PubMed

Bug, William J; Ascoli, Giorgio A; Grethe, Jeffrey S; Gupta, Amarnath; Fennema-Notestine, Christine; Laird, Angela R; Larson, Stephen D; Rubin, Daniel; Shepherd, Gordon M; Turner, Jessica A; Martone, Maryann E

2008-09-01

A critical component of the Neuroscience Information Framework (NIF) project is a consistent, flexible terminology for describing and retrieving neuroscience-relevant resources. Although the original NIF specification called for a loosely structured controlled vocabulary for describing neuroscience resources, as the NIF system evolved, the requirement for a formally structured ontology for neuroscience with sufficient granularity to describe and access a diverse collection of information became obvious. This requirement led to the NIF standardized (NIFSTD) ontology, a comprehensive collection of common neuroscience domain terminologies woven into an ontologically consistent, unified representation of the biomedical domains typically used to describe neuroscience data (e.g., anatomy, cell types, techniques), as well as digital resources (tools, databases) being created throughout the neuroscience community. NIFSTD builds upon a structure established by the BIRNLex, a lexicon of concepts covering clinical neuroimaging research developed by the Biomedical Informatics Research Network (BIRN) project. Each distinct domain module is represented using the Web Ontology Language (OWL). As much as has been practical, NIFSTD reuses existing community ontologies that cover the required biomedical domains, building the more specific concepts required to annotate NIF resources. By following this principle, an extensive vocabulary was assembled in a relatively short period of time for NIF information annotation, organization, and retrieval, in a form that promotes easy extension and modification. We report here on the structure of the NIFSTD, and its predecessor BIRNLex, the principles followed in its construction and provide examples of its use within NIF.

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

PubMed

Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek

2016-11-20

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2. Published by Elsevier Ltd.

Topological domain walls in helimagnets

NASA Astrophysics Data System (ADS)

Schoenherr, P.; Müller, J.; Köhler, L.; Rosch, A.; Kanazawa, N.; Tokura, Y.; Garst, M.; Meier, D.

2018-05-01

Domain walls naturally arise whenever a symmetry is spontaneously broken. They interconnect regions with different realizations of the broken symmetry, promoting structure formation from cosmological length scales to the atomic level1,2. In ferroelectric and ferromagnetic materials, domain walls with unique functionalities emerge, holding great promise for nanoelectronics and spintronics applications3-5. These walls are usually of Ising, Bloch or Néel type and separate homogeneously ordered domains. Here we demonstrate that a wide variety of new domain walls occurs in the presence of spatially modulated domain states. Using magnetic force microscopy and micromagnetic simulations, we show three fundamental classes of domain walls to arise in the near-room-temperature helimagnet iron germanium. In contrast to conventional ferroics, the domain walls exhibit a well-defined inner structure, which—analogous to cholesteric liquid crystals—consists of topological disclination and dislocation defects. Similar to the magnetic skyrmions that form in the same material6,7, the domain walls can carry a finite topological charge, permitting an efficient coupling to spin currents and contributions to a topological Hall effect. Our study establishes a new family of magnetic nano-objects with non-trivial topology, opening the door to innovative device concepts based on helimagnetic domain walls.

Structure of the Head of the Bartonella Adhesin BadA

PubMed Central

Szczesny, Pawel; Linke, Dirk; Ursinus, Astrid; Bär, Kerstin; Schwarz, Heinz; Riess, Tanja M.; Kempf, Volkhard A. J.; Lupas, Andrei N.; Martin, Jörg; Zeth, Kornelius

2008-01-01

Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 Å. Both domains are β-prisms, the N-terminal one formed by interleaved, five-stranded β-meanders parallel to the trimer axis and the C-terminal one by five-stranded β-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens. PMID:18688279

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase: DAWIDZIAK et al.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dawidziak, Daria M.; Sanchez, Jacint G.; Wagner, Jonathan M.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

Solution Structure of Homology Region (HR) Domain of Type II Secretion System*

PubMed Central

Gu, Shuang; Kelly, Geoff; Wang, Xiaohui; Frenkiel, Tom; Shevchik, Vladimir E.; Pickersgill, Richard W.

2012-01-01

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a β-sandwich-like fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the type II secretion system have a structural or sequence ortholog within the type IV pili system. The HR domain is shown to interact with the N0 domain of the secretin. The importance of this interaction is explored in the context of the functional secretion system. PMID:22253442

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

PubMed

Liu, Xu; Shepherd, Tyson R; Murray, Ann M; Xu, Zhen; Fuentes, Ernesto J

2013-03-05

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites. Copyright © 2013 Elsevier Ltd. All rights reserved.

Low-energy Structural Dynamics of Multiferroic Domain Walls in Hexagonal Rare-earth Manganites

NASA Astrophysics Data System (ADS)

Wu, Xiaoyu; Petralanda, Urko; Zheng, Lu; Ren, Yuan; Hu, Rongwei; Cheong, Sang-Wook; Artyukhin, Sergey; Lai, Keji

Multiferroic domain walls (DWs), the natural interfaces between domains with different order parameters, usually exhibit unconventional functionalities. For instance, recent discovery of the ferroelectric DW conduction highlights its extraordinary electronic structure that is absent in bulk domains. The structural dynamics of individual DWs in the microwave regime, however, have not been fully explored due to the lack of spatially resolved studies. Here, we report the broadband (106-1010 Hz) scanning impedance microscopy results on the interlocked anti-phase boundaries and ferroelectric DWs in hexagonal rare-earth manganites. Surprisingly, the effective conductivity of the (001) DWs displays a 106-fold increase from dc to GHz frequencies, while the effect is absent on surfaces with in-plane polarized domains. First-principles and model calculations indicate that the frequency range and selection rules are consistent with the periodic sliding of the DW around its equilibrium position. This DW acoustic-wave-like mode, which is associated with the synchronized oscillation of local polarization and apical oxygen atoms, is localized perpendicular to the DW but free to propagate along the DW plane. Our results break the ground to understand structural DW dynamics and exploit new interfacial phenomena for novel devices.

New mechanism of structuring associated with the quasi-merohedral twinning by an example of Ca{sub 1–x}La{sub x}F{sub 2+x} ordered solid solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maksimov, S. K., E-mail: maksimov-sk@comtv.ru; Maksimov, K. S., E-mail: kuros@rambler.ru; Sukhov, N. D.

Merohedry is considered an inseparable property of atomic structures, and uses for the refinement of structural data in a process of correct determination of structure of compounds. Transformation of faulty structures stimulated by decreasing of systemic cumulative energy leads to generation of merohedral twinning type. Ordering is accompanied by origin of antiphase domains. If ordering belongs to the CuAu type, it is accompanied by tetragonal distortions along different (100) directions. If a crystal consists of mosaic of nanodimensional antiphase domains, the conjugation of antiphase domains with different tetragonality leads to monoclinic distortions, at that, conjugated domains are distorted mirrorly. Similarmore » system undergoes further transformation by means of quasi-merohedral twinning. As a result of quasi-merohedry, straight-lines of lattices with different monoclinic distortions are transformed into coherent lattice broken-lines providing minimization of the cumulative energy. Structuring is controlled by regularities of the self-organization. However stochasticity of ordering predetermines the origin areas where few domains with different tetragonality contact which leads to the origin of faulty fields braking regular passage of structuring. Resulting crystal has been found structurally non-uniform, furthermore structural non-uniformity permits identifying elements and stages of a process. However there is no precondition preventing arising the origin of homogenous states. Effect has been revealed in Ca{sub 1–x}La{sub x}F{sub 2+x} solid solution, but it can be expected that distortions of regular alternation of ions similar to antiphase domains can be obtained in non-equilibrium conditions in compounds and similar effect of the quasi-merohedry can falsify results of structural analysis.« less

Contribution of trimeric autotransporter C-terminal domains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA passenger domain and virulence of Yersinia enterocolitica.

PubMed

Ackermann, Nikolaus; Tiller, Maximilian; Anding, Gisela; Roggenkamp, Andreas; Heesemann, Jürgen

2008-07-01

The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.

Molecular Dynamics Simulation of Rap1 Myb-type domain in Saccharomyces cerevisiae

PubMed Central

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2012-01-01

Telomere is a nucleoprotein complex that plays important role in stability and their maintenance and consists of random repeats of species specific motifs. In budding Saccharomyces cerevisiae, Repressor Activator Protein 1 (Rap1) is a sequence specific protein that involved in transcriptional regulation. Rap1 consist of three active domains like N-terminal BRCT-domain, DNA-binding domain and C-terminal RCT-domain. In this study the unknown 3D structure of Myb-type domain (having 61 residues) within DNAbinding domain was modeled by Modeller7, and verified using different online bioinformatics tools (ProCheck, WhatIf, Verify3D). Dynamics of Myb-type domain of Rap1was carried out through simulation studies using GROMACS software. Time dependent interactions among the molecules were analyzed by Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Root Mean Square Fluctuation (RMSF) plots. Motional properties in reduced dimension were also performed by Principal Component Analysis (PCA). Result indicated that Rap1 interacts with DNA major groove through its Helix Turn Helix motifs. Helix 3 was rigid, less amount of fluctuation was found as it interacts with DNA major groove. Helix2 and N-terminal having considerable fluctuation in the time scale. PMID:23144544

Molecular Dynamics Simulation of Rap1 Myb-type domain in Saccharomyces cerevisiae.

PubMed

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2012-01-01

Telomere is a nucleoprotein complex that plays important role in stability and their maintenance and consists of random repeats of species specific motifs. In budding Saccharomyces cerevisiae, Repressor Activator Protein 1 (Rap1) is a sequence specific protein that involved in transcriptional regulation. Rap1 consist of three active domains like N-terminal BRCT-domain, DNA-binding domain and C-terminal RCT-domain. In this study the unknown 3D structure of Myb-type domain (having 61 residues) within DNAbinding domain was modeled by Modeller7, and verified using different online bioinformatics tools (ProCheck, WhatIf, Verify3D). Dynamics of Myb-type domain of Rap1was carried out through simulation studies using GROMACS software. Time dependent interactions among the molecules were analyzed by Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Root Mean Square Fluctuation (RMSF) plots. Motional properties in reduced dimension were also performed by Principal Component Analysis (PCA). Result indicated that Rap1 interacts with DNA major groove through its Helix Turn Helix motifs. Helix 3 was rigid, less amount of fluctuation was found as it interacts with DNA major groove. Helix2 and N-terminal having considerable fluctuation in the time scale.

Factors across home, work, and school domains influence nutrition and physical activity behaviors of nontraditional college students.

PubMed

Quintiliani, Lisa M; Bishop, Hillary L; Greaney, Mary L; Whiteley, Jessica A

2012-10-01

Nontraditional college students (older, part-time, and/or working) have less healthful nutrition and physical activity behaviors compared to traditional students, yet few health promotion efforts focus on nontraditional students. The purpose of this study was to use qualitative methods to explore factors affecting nutrition and physical activity behaviors of nontraditional students. Fourteen semi-structured individual interviews were conducted with nontraditional undergraduate students attending a large university. The sample had a median age of 25 (range, 21-64), 57% were men, 43% were racial/ethnic minorities, and 57% were employed (mean 22 hours/week). Data were coded using a systematic team-based approach. Consistent themes (mentioned by 4+ students) were identified and categorized into three domains: home, work, and school. Home (themes: neighborhood characteristics, family, partners), work (theme: work environment), and school (themes: cafeteria, vending machines) factors consistently influenced positive nutrition behaviors. Similarly, home (themes: neighborhood including safety, friends from home, partner,), work (theme: work environment), and school (themes: not having a car, campus structure, campus gym, friends at school) factors consistently influenced positive physical activity. Financial resources and perceptions of autonomy had influence across domains. Results indicate consistent influences on nutrition and physical activity behaviors across home, work, and school domains for nontraditional college students. Study findings suggest possible, and sometimes unconventional, intervention strategies to promote healthful eating and physical activity. For example, when cafeteria meal plans are not offered and financial constraints limit eating at the cafeteria, encouraging healthful choices from vending machines could be preferable to not eating at all. Copyright © 2012 Elsevier Inc. All rights reserved.

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, R.; Wilton, R.; Cuff, M. E.

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes butmore » have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.« less

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain.

PubMed

Wu, R; Wilton, R; Cuff, M E; Endres, M; Babnigg, G; Edirisinghe, J N; Henry, C S; Joachimiak, A; Schiffer, M; Pokkuluri, P R

2017-04-01

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport. © 2017 The Protein Society.

Structure of the orthorhombic Al13Co4(100) surface using LEED, STM, and ab initio studies

NASA Astrophysics Data System (ADS)

Shin, Heekeun; Pussi, K.; Gaudry, É.; Ledieu, J.; Fournée, V.; Alarcón Villaseca, S.; Dubois, J.-M.; Grin, Yu.; Gille, P.; Moritz, W.; Diehl, R. D.

2011-08-01

In a combined scanning tunneling microscopy (STM), low-energy electron diffraction (LEED), and density functional theory (DFT) study of the surface of Al13Co4(100), all techniques have found that after annealing to 1165 K, the surface structure is consistent with a dense Al-rich plane with surface Co atom depletion. Various structure models were considered, and in the LEED study, the best agreement was found with a model that consists of Al-rich terminating planes with no Co atoms, and otherwise a structure similar to the bulk puckered layers. This structure was also found to be stable in the DFT study. The best-fit structural parameters are presented for the two domains of this structure, which contain bipentagons that can be related to the pentagonal bipyramidal structures in the bulk, plus additional glue atoms between them. These domains are not strictly related to each other by symmetry, as they have different surface relaxations. The STM study found significant differences in the surfaces of samples grown by different methods and is able to explain a different interpretation made in an earlier study.

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors.

PubMed

Zafra Ruano, Ana; Cilia, Elisa; Couceiro, José R; Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic; Luque, Irene; Lenaerts, Tom

2016-05-01

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis.

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors

PubMed Central

Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic

2016-01-01

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of humanmore » Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.« less

Crystal structure of a chimaeric bacterial glutamate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.

2016-05-23

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P) +as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD +versusNADP +, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies,more » as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP +cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.« less

Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant Staphylococcus aureus in the apo and cefotaxime-bound forms.

PubMed

Yoshida, Hisashi; Kawai, Fumihiro; Obayashi, Eiji; Akashi, Satoko; Roper, David I; Tame, Jeremy R H; Park, Sam-Yong

2012-10-26

Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

DOE PAGES

Long, Feng; Fong, Rachel H.; Austin, Stephen K.; ...

2015-10-26

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. In this paper, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain’s β-ribbon connector of the viral glycoprotein E2. The footprints ofmore » these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. Finally, this finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes.« less

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event.

PubMed

Kyndt, Tina; Haegeman, Annelies; Gheysen, Godelieve

2008-11-03

Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level.

The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes

PubMed Central

Himbert, Sebastian; Alsop, Richard J.; Rose, Markus; Hertz, Laura; Dhaliwal, Alexander; Moran-Mirabal, Jose M.; Verschoor, Chris P.; Bowdish, Dawn M. E.; Kaestner, Lars; Wagner, Christian; Rheinstädter, Maikel C.

2017-01-01

We prepared highly oriented, multi-lamellar stacks of human red blood cell (RBC) membranes applied on silicon wafers. RBC ghosts were prepared by hemolysis and applied onto functionalized silicon chips and annealed into multi-lamellar RBC membranes. High resolution X-ray diffraction was used to determine the molecular structure of the stacked membranes. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. Our results further support current models of RBC membranes as patchy structures and provide unprecedented structural details of the molecular organization in the different domains. PMID:28045119

The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes

NASA Astrophysics Data System (ADS)

Himbert, Sebastian; Alsop, Richard J.; Rose, Markus; Hertz, Laura; Dhaliwal, Alexander; Moran-Mirabal, Jose M.; Verschoor, Chris P.; Bowdish, Dawn M. E.; Kaestner, Lars; Wagner, Christian; Rheinstädter, Maikel C.

2017-01-01

We prepared highly oriented, multi-lamellar stacks of human red blood cell (RBC) membranes applied on silicon wafers. RBC ghosts were prepared by hemolysis and applied onto functionalized silicon chips and annealed into multi-lamellar RBC membranes. High resolution X-ray diffraction was used to determine the molecular structure of the stacked membranes. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. Our results further support current models of RBC membranes as patchy structures and provide unprecedented structural details of the molecular organization in the different domains.

Domain-to-domain coupling in voltage-sensing phosphatase.

PubMed

Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

2017-01-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

Domain-to-domain coupling in voltage-sensing phosphatase

PubMed Central

Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

2017-01-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain. PMID:28744425

A new geological framework for south-central Madagascar, and its relevance to the "out-of-Africa" hypothesis

USGS Publications Warehouse

Tucker, R.D.; Roig, J.Y.; Macey, P.H.; Delor, C.; Amelin, Y.; Armstrong, R.A.; Rabarimanana, M.H.; Ralison, A.V.

2011-01-01

The Precambrian shield of south-central Madagascar, excluding the Vohibory region, consists of three geologic domains, from north to south: Antananarivo, Ikalamavony-Itremo, and Anosyen-Androyen. The northern Antananarivo domain represents the Neoarchean sector of the Greater Dharwar Craton amalgamated at 2.52-2.48. Ga. The Greater Dharwar Craton is overlain by several groups of Meso- to Neoproterozoic supracrustal rocks (Ambatolampy, Manampotsy, Ampasary, Sahantaha, and Maha Groups) each with a common and diagnostic signature of Paleoproterozoic detrital zircons (2.2-1.8. Ga). The central domain (Ikalamavony-Itremo) consists of two distinct parts. The Itremo Sub-domain, in the east, is a structurally intercalated sequence of Neoarchean gneiss and shallow marine metasedimentary rocks of Paleo-Mesoproterozoic age (Itremo Group), the latter with Paleoproterozoic detrital zircons ranging in age between 2.2 and 1.8. Ga. The Ikalamavony Sub-domain, to the west, contains abundant volcano-clastic metasediments and lesser quartzite (Ikalamavony Group), formed between 1.03. Ga and 0.98. Ga, and intruded by igneous rocks (Dabolava Suite) of Stenian-Tonian age. Structurally intercalated with these are sheets of Neoarchean gneiss (~2.5. Ga) and Neoproterozoic metaclastic rocks (Molo Group). Like the Itremo Group, quartzite of the Ikalamavony Group has detrital zircons of Paleoproterozoic age (2.1-1.8. Ga). The southern domain of Anosyen-Androyen consists of a newly recognized suite of Paleoproterozoic igneous rocks (2.0-1.8. Ga), and stratified supracrustal rocks also having Paleoproterozoic detrital zircons (2.3-1.8. Ga). The contact between the Anosyen-Androyen and Ikalamavony-Itremo domains, formerly known as the Ranotsara-Bongolava shear zone, is a tightly folded and highly flattened boundary that was ductilely deformed in Ediacaran time. It is roughly equivalent to the Palghat-Cauvery shear zone in south India, and it defines approximately the boundary between the Archean Greater Dharwar Craton (to the north) and the Paleoproterozoic terrane of Anosyen-Androyen (to the south).

The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin.

PubMed

Sun, Y J; Chou, C C; Chen, W S; Wu, R T; Meng, M; Hsiao, C D

1999-05-11

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this studymore » we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.« less

Structural Analysis of Peptide-Analogues of Human Zona Pellucida ZP1 Protein with Amyloidogenic Properties: Insights into Mammalian Zona Pellucida Formation

PubMed Central

Louros, Nikolaos N.; Iconomidou, Vassiliki A.; Giannelou, Polina; Hamodrakas, Stavros J.

2013-01-01

Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as ‘aggregation-prone’ by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted ‘aggregation-prone’ parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization. PMID:24069181

Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains.

PubMed

Halawani, Dalia; Gogonea, Valentin; DiDonato, Joseph A; Pipich, Vitaliy; Yao, Peng; China, Arnab; Topbas, Celalettin; Vasu, Kommireddy; Arif, Abul; Hazen, Stanley L; Fox, Paul L

2018-06-08

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH- S -transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Ping; Swanson, Kurt A.; Leser, George P.

2014-10-02

The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HNmore » interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, R.; Wilton, R.; Cuff, M. E.

The tandem Per-Arnt-Sim (PAS) like sensors are commonly found in signal transduction proteins. The periplasmic solute binding protein (SBP) domains are found ubiquitously and are generally involved in solute transport. These domains are widely observed as parts of separate proteins but not within the same polypeptide chain. We report the structural and biochemical characterization of the extracellular ligand-binding receptor, Dret_0059 from Desulfohalobium retbaense DSM 5692, an organism isolated from the Retba salt lake in Senegal. The structure of Dret_0059 consists of a novel combination of SBP and TPAS sensor domains. The N-terminal region forms an SBP domain and the C-terminalmore » region folds into a tandem PAS-like domain structure. A ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS domain of the TPAS. The differential scanning flourimetry studies in solution support the ligands observed in the crystal structure. There are only two other proteins with this structural architecture in the non-redundant sequence data base and we predict that they too bind the same substrates. There is significant interaction between the SBP and TPAS domains, and it is quite conceivable that the binding of one ligand will have an effect on the binding of the other. Our attempts to remove the ligands bound to the protein during expression were not successful, therefore, it is not clear what the relative affects are. The genomic context of this receptor does not contain any protein components expected for transport function, hence, we suggest that Dret_0059 is likely involved in signal transduction and not in solute transport.« less

Thermal fluctuations of ferroelectric nanodomains in a ferroelectric-dielectric PbTiO 3 / SrTiO 3 superlattice

DOE PAGES

Zhang, Qingteng; Dufresne, Eric M.; Chen, Pice; ...

2017-02-27

Ferroelectric-dielectric superlattices consisting of alternating layers of ferroelectric PbTiO 3 and dielectric SrTiO 3 exhibit a disordered striped nanodomain pattern, with characteristic length scales of 6 nm for the domain periodicity and 30 nm for the in-plane coherence of the domain pattern. Spatial disorder in the domain pattern gives rise to coherent hard x-ray scattering patterns exhibiting intensity speckles. We show here using variable-temperature Bragg-geometry x-ray photon correlation spectroscopy that x-ray scattering patterns from the disordered domains exhibit a continuous temporal decorrelation due to spontaneous domain fluctuations. The temporal decorrelation can be described using a compressed exponential function, consistent withmore » what has been observed in other systems with arrested dynamics. The fluctuation speeds up at higher temperatures and the thermal activation energy estimated from the Arrhenius model is 0.35±0.21 eV. As a result, the magnitude of the energy barrier implies that the complicated energy landscape of the domain structures is induced by pinning mechanisms and domain patterns fluctuate via the generation and annihilation of topological defects similar to soft materials such as block copolymers.« less

Bidirectional Transfer between Metaphorical Related Domains in Implicit Learning of Form-Meaning Connections

PubMed Central

Yang, Zhiliang; Dienes, Zoltan

2013-01-01

People can implicitly learn a connection between linguistic forms and meanings, for example between specific determiners (e.g. this, that…) and the type of nouns to which they apply. Li et al (2013) recently found that transfer of form-meaning connections from a concrete domain (height) to an abstract domain (power) was achieved in a metaphor-consistent way without awareness, showing that unconscious knowledge can be abstract and flexibly deployed. The current study aims to determine whether people transfer knowledge of form-meaning connections not only from a concrete domain to an abstract one, but also vice versa, consistent with metaphor representation being bi-directional. With a similar paradigm as used by Li et al, participants learnt form- meaning connections of different domains (concrete vs. abstract) and then were tested on two kinds of generalizations (same and different domain generalization). As predicted, transfer of form-meaning connections occurred bidirectionally when structural knowledge was unconscious. Moreover, the present study also revealed that more transfer occurred between metaphorically related domains when judgment knowledge was conscious (intuition) rather than unconscious (guess). Conscious and unconscious judgment knowledge may have different functional properties. PMID:23844159

A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain

DOE PAGES

Wu, R.; Wilton, R.; Cuff, M. E.; ...

2017-02-07

The tandem Per-Arnt-Sim (PAS) like sensors are commonly found in signal transduction proteins. The periplasmic solute binding protein (SBP) domains are found ubiquitously and are generally involved in solute transport. These domains are widely observed as parts of separate proteins but not within the same polypeptide chain. We report the structural and biochemical characterization of the extracellular ligand-binding receptor, Dret_0059 from Desulfohalobium retbaense DSM 5692, an organism isolated from the Retba salt lake in Senegal. The structure of Dret_0059 consists of a novel combination of SBP and TPAS sensor domains. The N-terminal region forms an SBP domain and the C-terminalmore » region folds into a tandem PAS-like domain structure. A ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS domain of the TPAS. The differential scanning flourimetry studies in solution support the ligands observed in the crystal structure. There are only two other proteins with this structural architecture in the non-redundant sequence data base and we predict that they too bind the same substrates. There is significant interaction between the SBP and TPAS domains, and it is quite conceivable that the binding of one ligand will have an effect on the binding of the other. Our attempts to remove the ligands bound to the protein during expression were not successful, therefore, it is not clear what the relative affects are. The genomic context of this receptor does not contain any protein components expected for transport function, hence, we suggest that Dret_0059 is likely involved in signal transduction and not in solute transport.« less

Role of ANC-1 in tethering nuclei to the actin cytoskeleton.

PubMed

Starr, Daniel A; Han, Min

2002-10-11

Mutations in anc-1 (nuclear anchorage defective) disrupt the positioning of nuclei and mitochondria in Caenorhabditis elegans. ANC-1 is shown to consist of mostly coiled regions with a nuclear envelope localization domain (called the KASH domain) and an actin-binding domain; this structure was conserved with the Drosophila protein Msp-300 and the mammalian Syne proteins. Antibodies against ANC-1 localized cytoplasmically and were enriched at the nuclear periphery in an UNC-84-dependent manner. Overexpression of the KASH domain or the actin-binding domain caused a dominant negative anchorage defect. Thus, ANC-1 may connect nuclei to the cytoskeleton by interacting with UNC-84 at the nuclear envelope and with actin in the cytoplasm.

Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51.

PubMed

Kumar, Rajnish; Moche, Martin; Winblad, Bengt; Pavlov, Pavel F

2017-10-27

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

Cognitive flexibility and undergraduate physiology students: increasing advanced knowledge acquisition within an ill-structured domain.

PubMed

Rhodes, Ashley E; Rozell, Timothy G

2017-09-01

Cognitive flexibility is defined as the ability to assimilate previously learned information and concepts to generate novel solutions to new problems. This skill is crucial for success within ill-structured domains such as biology, physiology, and medicine, where many concepts are simultaneously required for understanding a complex problem, yet the problem consists of patterns or combinations of concepts that are not consistently used or needed across all examples. To succeed within ill-structured domains, a student must possess a certain level of cognitive flexibility: rigid thought processes and prepackaged informational retrieval schemes relying on rote memorization will not suffice. In this study, we assessed the cognitive flexibility of undergraduate physiology students using a validated instrument entitled Student's Approaches to Learning (SAL). The SAL evaluates how deeply and in what way information is processed, as well as the investment of time and mental energy that a student is willing to expend by measuring constructs such as elaboration and memorization. Our results indicate that students who rely primarily on memorization when learning new information have a smaller knowledge base about physiological concepts, as measured by a prior knowledge assessment and unit exams. However, students who rely primarily on elaboration when learning new information have a more well-developed knowledge base about physiological concepts, which is displayed by higher scores on a prior knowledge assessment and increased performance on unit exams. Thus students with increased elaboration skills possibly possess a higher level of cognitive flexibility and are more likely to succeed within ill-structured domains. Copyright © 2017 the American Physiological Society.

Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ito, Tasuku; Saikawa, Kyo; Kim, Seonah

2014-04-25

Graphical abstract: - Highlights: • HypBA1 β-L-arabinofuranosidase belongs to glycoside hydrolase family 127. • Crystal structure of HypBA1 was determined. • HypBA1 consists of a catalytic barrel and two additional β-sandwich domains. • The active site contains a Zn{sup 2+} coordinated by glutamate and three cysteines. • A possible reaction mechanism involving cysteine as the nucleophile is proposed. - Abstract: Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-L-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-L-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system ofmore » hydroxyproline-linked β-L-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-L-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn{sup 2+} coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-L-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.« less

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

PubMed

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the American Physiological Society.

Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

PubMed

Alvarado, John J; Betts, Laurie; Moroco, Jamie A; Smithgall, Thomas E; Yeh, Joanne I

2010-11-12

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a "conformational switch" that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that "fine-tune" their sensitivities to activation by SH3-based ligands.

Crystal structure of the Rous sarcoma virus intasome

DOE PAGES

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit; ...

2016-02-17

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

2010-11-05

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« less

Brazilian Portuguese version of the Revised Fibromyalgia Impact Questionnaire (FIQR-Br): cross-cultural validation, reliability, and construct and structural validation.

PubMed

Lupi, Jaqueline Basilio; Carvalho de Abreu, Daniela Cristina; Ferreira, Mariana Candido; Oliveira, Renê Donizeti Ribeiro de; Chaves, Thais Cristina

2017-08-01

This study aimed to culturally adapt and validate the Revised Fibromyalgia Impact Questionnaire (FIQR) to Brazilian Portuguese, by the use of analysis of internal consistency, reliability, and construct and structural validity. A total of 100 female patients with fibromyalgia participated in the validation process of the Brazilian Portuguese version of the FIQR (FIQR-Br).The intraclass correlation coefficient (ICC) was used for statistical analysis of reliability (test-retest), Cronbach's alpha for internal consistency, Pearson's rank correlation for construct validity, and confirmatory factor analysis (CFA) for structural validity. It was verified excellent levels of reliability, with ICC greater than 0.75 for all questions and domains of the FIQR-Br. For internal consistency, alpha values greater than 0.70 for the items and domains of the questionnaire were observed. Moderate (0.40 < r < 0.70) and strong (r > 0.70) correlations were observed for the scores of domains and total score between the FIQR-Br and FIQ-Br. The structure of the three domains of the FIQR-Br was confirmed by CFA. The results of this study suggest that that the FIQR-Br is a reliable and valid instrument for assessing fibromyalgia-related impact, and supports its use in clinical settings and research. The structure of the three domains of the FIQR-Br was also confirmed. Implications for Rehabilitation Fibromyalgia is a chronic musculoskeletal disorder characterized by widespread and diffuse pain, fatigue, sleep disturbances, and depression. The disease significantly impairs patients' quality of life and can be highly disabling. To be used in multicenter research efforts, the Revised Fibromyalgia Impact Questionnaire (FIQR) must be cross-culturally validated and psychometrically tested. This paper will make available a new version of the FIQR-Br since another version already exists, but there are concerns about its measurement properties. The availability of an instrument adapted to and validated for Brazilian Portuguese may make it possible to reliably verify the effects of rehabilitation programs on disability from fibromyalgia. The FIQR-Br showed results comparable with other versions of the FIQR in other languages, thereby enabling comparison of effects of rehabilitation interventions on disability from fibromyalgia conducted in Brazil with results of studies carried out in other parts of the world.

Structure of the membrane domain of respiratory complex I.

PubMed

Efremov, Rouslan G; Sazanov, Leonid A

2011-08-07

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.

Kinetic and structural characterization of amyloid-β peptide hydrolysis by human angiotensin-1-converting enzyme.

PubMed

Larmuth, Kate M; Masuyer, Geoffrey; Douglas, Ross G; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D

2016-03-01

Angiotensin-1-converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N-domain in complex with Aβ fragments. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14-Gln15. Furthermore, Aβ(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7-Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4-10)Y. Surprisingly, in contrast to Aβ(1-16) and Aβ(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards Aβ(4-10)Y. The structures of the Aβ peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal P2' position to the S2' pocket and recognizes the main chain of the P1' peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific S2' residue Thr358. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms. The atomic coordinates and structure factors for N-domain ACE with Aβ peptides 4-10 (5AM8), 10-16 (5AM9), 1-16 (5AMA), 35-42 (5AMB) and (4-10)Y (5AMC) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/). © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

The International Index of Erectile Function: development of an adapted tool for use in HIV-positive men who have sex with men.

PubMed

Coyne, Katherine; Mandalia, Sundhiya; McCullough, Sonya; Catalan, Jose; Noestlinger, Christiana; Colebunders, Robert; Asboe, David

2010-02-01

Erectile dysfunction is common in HIV-positive men who have sex with men (MSM). A standardized scale is needed to assess erectile function in clinical practice and research studies. The International Index of Erectile Function (IIEF) is a widely accepted tool for assessing erectile function designed for heterosexual men. We modified the tool for MSM. We present an analysis of internal consistency of the questionnaire in an HIV-positive cohort. The adapted questionnaire included modified questions within each of the five domains of the IIEF: (i) erectile function, (ii) intercourse satisfaction, (iii) orgasmic function, (iv) sexual desire, and (v) overall satisfaction with sex. MSM at seven European HIV treatment centers completed the questionnaire. Responses were analyzed for internal consistency using standardized Cronbach's alpha values within each of the five domains. A factor analysis was performed to confirm the domain structure of the questionnaire. Data from 486 MSM were analyzed. The factor analysis supported the domain structure described. Questions about erectile function, orgasmic function, and sexual desire performed well, with Cronbach's alpha values of 0.82, 0.83, and 0.89, respectively. Questions concerning intercourse satisfaction were less consistent (Cronbach's alpha 0.55) because frequency of attempts at sexual intercourse did not correlate with other responses. Responses about satisfaction with sex with a regular partner diverged from satisfaction with overall sex life. Frequency of morning erections diverged from other aspects of erectile function, whereas erections with masturbation correlated better. Internal consistency was high overall. This tool is suitable for HIV-positive MSM and can be used in screening, research, and monitoring treatment response.

An immersed-shell method for modelling fluid–structure interactions

PubMed Central

Viré, A.; Xiang, J.; Pain, C. C.

2015-01-01

The paper presents a novel method for numerically modelling fluid–structure interactions. The method consists of solving the fluid-dynamics equations on an extended domain, where the computational mesh covers both fluid and solid structures. The fluid and solid velocities are relaxed to one another through a penalty force. The latter acts on a thin shell surrounding the solid structures. Additionally, the shell is represented on the extended domain by a non-zero shell-concentration field, which is obtained by conservatively mapping the shell mesh onto the extended mesh. The paper outlines the theory underpinning this novel method, referred to as the immersed-shell approach. It also shows how the coupling between a fluid- and a structural-dynamics solver is achieved. At this stage, results are shown for cases of fundamental interest. PMID:25583857

Review the role of terminal domains during storage and assembly of spider silk proteins.

PubMed

Eisoldt, Lukas; Thamm, Christopher; Scheibel, Thomas

2012-06-01

Fibrous proteins in nature fulfill a wide variety of functions in different structures ranging from cellular scaffolds to very resilient structures like tendons and even extra-corporal fibers such as silks in spider webs or silkworm cocoons. Despite their different origins and sequence varieties many of these fibrous proteins share a common building principle: they consist of a large repetitive core domain flanked by relatively small non-repetitive terminal domains. Amongst protein fibers, spider dragline silk shows prominent mechanical properties that exceed those of man-made fibers like Kevlar. Spider silk fibers assemble in a spinning process allowing the transformation from an aqueous solution into a solid fiber within milliseconds. Here, we highlight the role of the non-repetitive terminal domains of spider dragline silk proteins during storage in the gland and initiation of the fiber assembly process. Copyright © 2011 Wiley Periodicals, Inc.

alpha-helical structural elements within the voltage-sensing domains of a K(+) channel.

PubMed

Li-Smerin, Y; Hackos, D H; Swartz, K J

2000-01-01

Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

PubMed

Unciuleac, Mihaela-Carmen; Smith, Paul C; Shuman, Stewart

2016-05-15

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A conserved π-cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability.

PubMed

Eek, Priit; Piht, Mari-Ann; Rätsep, Margus; Freiberg, Arvi; Järving, Ivar; Samel, Nigulas

2015-10-01

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation. Copyright © 2015 Elsevier B.V. All rights reserved.

The domain interface method: a general-purpose non-intrusive technique for non-conforming domain decomposition problems

NASA Astrophysics Data System (ADS)

Cafiero, M.; Lloberas-Valls, O.; Cante, J.; Oliver, J.

2016-04-01

A domain decomposition technique is proposed which is capable of properly connecting arbitrary non-conforming interfaces. The strategy essentially consists in considering a fictitious zero-width interface between the non-matching meshes which is discretized using a Delaunay triangulation. Continuity is satisfied across domains through normal and tangential stresses provided by the discretized interface and inserted in the formulation in the form of Lagrange multipliers. The final structure of the global system of equations resembles the dual assembly of substructures where the Lagrange multipliers are employed to nullify the gap between domains. A new approach to handle floating subdomains is outlined which can be implemented without significantly altering the structure of standard industrial finite element codes. The effectiveness of the developed algorithm is demonstrated through a patch test example and a number of tests that highlight the accuracy of the methodology and independence of the results with respect to the framework parameters. Considering its high degree of flexibility and non-intrusive character, the proposed domain decomposition framework is regarded as an attractive alternative to other established techniques such as the mortar approach.

The study of co-citation analysis and knowledge structure on healthcare domain

NASA Astrophysics Data System (ADS)

Chu, Kuo-Chung; Liu, Wen-I.; Tsai, Ming-Yu

2012-11-01

With the prevalence of Internet and digital archives, the online e-journal database facilitates scholars to search literature in a research domain, or to cross-search an inter-disciplined field; the key literature can be efficiently traced out. This study intends to build a Web-based citation analysis system, which consists of four modules, they are: 1) literature search module; (2) statistics module; (3) articles analysis module; and (4) co-citation analysis module. The system focuses on PubMed Central dataset that has 170,000 records. In a research domain, a specific keyword searches in terms of authors, journals, and core issues. In addition, we use data mining techniques for co-citation analysis. The results assist researchers with in-depth understanding of the domain knowledge. Having an automated system for co-citation analysis, it helps to understand changes, trends, and knowledge structure of research domain. For the best of our knowledge, the proposed system differentiates from existing online electronic retrieval database analysis function. Perhaps, the proposed system is going to be a value-added database of healthcare domain, and hope to contribute the researchers.

Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B.

PubMed

Masuyer, Geoffrey; Beard, Matthew; Cadd, Verity A; Chaddock, John A; Acharya, K Ravi

2011-04-01

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. BoNTs consist of three essential domains for toxicity: the cell binding domain (Hc), the translocation domain (Hn) and the catalytic domain (LC). A functional derivative (LHn) of the parent neurotoxin B composed of Hn and LC domains was recombinantly produced and characterised. LHn/B crystallographic structure at 2.8Å resolution is reported. The catalytic activity of LHn/B towards recombinant human VAMP was analysed by substrate cleavage assay and showed a higher specificity for VAMP-1, -2 compared to VAMP-3. LHn/B also showed measurable activity in living spinal cord neurons. Despite lacking the Hc (cell-targeting) domain, LHn/B retained the capacity to internalize and cleave intracellular VAMP-1 and -2 when added to the cells at high concentration. These activities of the LHn/B fragment demonstrate the utility of engineered botulinum neurotoxin fragments as analytical tools to study the mechanisms of action of BoNT neurotoxins and of SNARE proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Domain modeling and grid generation for multi-block structured grids with application to aerodynamic and hydrodynamic configurations

NASA Technical Reports Server (NTRS)

Spekreijse, S. P.; Boerstoel, J. W.; Vitagliano, P. L.; Kuyvenhoven, J. L.

1992-01-01

About five years ago, a joint development was started of a flow simulation system for engine-airframe integration studies on propeller as well as jet aircraft. The initial system was based on the Euler equations and made operational for industrial aerodynamic design work. The system consists of three major components: a domain modeller, for the graphical interactive subdivision of flow domains into an unstructured collection of blocks; a grid generator, for the graphical interactive computation of structured grids in blocks; and a flow solver, for the computation of flows on multi-block grids. The industrial partners of the collaboration and NLR have demonstrated that the domain modeller, grid generator and flow solver can be applied to simulate Euler flows around complete aircraft, including propulsion system simulation. Extension to Navier-Stokes flows is in progress. Delft Hydraulics has shown that both the domain modeller and grid generator can also be applied successfully for hydrodynamic configurations. An overview is given about the main aspects of both domain modelling and grid generation.

Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

PubMed

Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

2016-10-18

The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

Cross-cultural adaptation, reliability and construct validity of the Tampa scale for kinesiophobia for temporomandibular disorders (TSK/TMD-Br) into Brazilian Portuguese.

PubMed

Aguiar, A S; Bataglion, C; Visscher, C M; Bevilaqua Grossi, D; Chaves, T C

2017-07-01

Fear of movement (kinesiophobia) seems to play an important role in the development of chronic pain. However, for temporomandibular disorders (TMD), there is a scarcity of studies about this topic. The Tampa Scale for Kinesiophobia for TMD (TSK/TMD) is the most widely used instrument to measure fear of movement and it is not available in Brazilian Portuguese. The purpose of this study was to culturally adapt the TSK/TMD to Brazilian Portuguese and to assess its psychometric properties regarding internal consistency, reliability, and construct and structural validity. A total of 100 female patients with chronic TMD participated in the validation process of the TSK/TMD-Br. The intraclass correlation coefficient (ICC) was used for statistical analysis of reliability (test-retest), Cronbach's alpha for internal consistency, Spearman's rank correlation for construct validity and confirmatory factor analysis (CFA) for structural validity. CFA endorsed the pre-specified model with two domains and 12-items (Activity Avoidance - AA/Somatic Focus - SF) and all items obtained a loading factor greater than 0·4. Acceptable levels of reliability were found (ICC > 0·75) for all questions and domains of the TSK/TMD-Br. For internal consistency, Cronbach's α of 0·78 for both domains were found. Moderate correlations (0·40 < r < 0.60) were observed for 84% of the analyses conducted between TSK/TMD-Br scores versus catastrophising, depression and jaw functional limitation. TSK/TMD-Br 12 items and two-factor demonstrated sound psychometric properties (transcultural validity, reliability, internal consistency and structural validity). In such a way, the instrument can be used in clinical settings and for research purposes. © 2017 John Wiley & Sons Ltd.

Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

PubMed Central

Singh, S.; Folkers, G.E.; Bonvin, A.M.J.J.; Boelens, R.; Wechselberger, R.; Niztayev, A.; Kaptein, R.

2002-01-01

The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5′ incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix–hairpin–helix (HhH) motifs connected by a small connector helix. The UvrC CTD is shown to mediate structure-specific DNA binding. The domain binds to a single-stranded–double-stranded junction DNA, with a strong specificity towards looped duplex DNA that contains at least six unpaired bases per loop (‘bubble DNA’). Using chemical shift perturbation experiments, the DNA-binding surface is mapped to the first hairpin region encompassing the conserved glycine–valine–glycine residues followed by lysine–arginine–arginine, a positively charged surface patch and the second hairpin region consisting of glycine–isoleucine–serine. A model for the protein– DNA complex is proposed that accounts for this specificity. PMID:12426397

Structural Studies of the Intestinal α-Glucosidases, Maltase-glucoamylase and Sucrase-isomaltase.

PubMed

Rose, David R; Chaudet, Marcia M; Jones, Kyra

2018-06-01

Maltase-glucoamylase and sucrase-isomaltase are enzymes in the brush-border membrane of the small intestinal lumen responsible for the breakdown of postamylase starch polysaccharides to release monomeric glucose. As such, they are critical players in healthy nutrition and their malfunction can lead to severe disorders. This review covers investigations of the structures and functions of these enzymes. Each consists of 2 enzyme domains of the glycoside hydrolase family GH31 classification, yet with somewhat differing enzymatic properties. Crystallographic structures of 3 of the domains have been published. Insights into substrate binding and specificity will be discussed, along with future lines of inquiry related to the enzymes' roles in disease and potential avenues for therapeutics.

Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbB Receptor Family of Proteins

DTIC Science & Technology

2005-06-01

acid residue motif, Small-x-x-Large-G/A, consist- complexes is critical to understanding the signal ing of a small residue (Gly, Ala , Ser, Thr, or Pro...in transduction process. Recent structures of the the zero position, a large aliphatic residue ( Ala , Val, ligand-binding domains of the erbB...receptors Leu, or Ile) in position 3, followed by Gly or Ala in have begun to provide insight into the mechanisms position four.’ This motif was identified

Mapping the Structure of Knowledge for Teaching Nominal Categorical Data Analysis

ERIC Educational Resources Information Center

Groth, Randall E.; Bergner, Jennifer A.

2013-01-01

This report describes a model for mapping cognitive structures related to content knowledge for teaching. The model consists of knowledge elements pertinent to teaching a content domain, the nature of the connections among them, and a means for representing the elements and connections visually. The model is illustrated through empirical data…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, Feng; Fong, Rachel H.; Austin, Stephen K.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. In this paper, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain’s β-ribbon connector of the viral glycoprotein E2. The footprints ofmore » these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. Finally, this finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes.« less

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains♦

PubMed Central

Tanaka, Hiroaki; Akagi, Ken-ichi; Oneyama, Chitose; Tanaka, Masakazu; Sasaki, Yuichi; Kanou, Takashi; Lee, Young-Ho; Yokogawa, Daisuke; Dobenecker, Marc-Werner; Nakagawa, Atsushi; Okada, Masato; Ikegami, Takahisa

2013-01-01

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. PMID:23548896

Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pampa, K.J., E-mail: sagarikakj@gmail.com; Lokanath, N.K.; Girish, T.U.

Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme bymore » X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Q. X. H.; Dementieva, I. S. D.; Walsh, M. A. W.

2001-02-23

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom}more » based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.« less

Mutation of domain III and domain VI in L gene conserved domain of Nipah virus

NASA Astrophysics Data System (ADS)

Jalani, Siti Aishah; Ibrahim, Nazlina

2016-11-01

Nipah virus (NiV) is the etiologic agent responsible for the respiratory illness and causes fatal encephalitis in human. NiV L protein subunit is thought to be responsible for the majority of enzymatic activities involved in viral transcription and replication. The L protein which is the viral RNA dependent RNA polymerase has high sequence homology among negative sense RNA viruses. In negative stranded RNA viruses, based on sequence alignment six conserved domain (domain I-IV) have been determined. Each domain is separated on variable regions that suggest the structure to consist concatenated functional domain. To directly address the roles of domains III and VI, site-directed mutations were constructed by the substitution of bases at sequences 2497, 2500, 5528 and 5532. Each mutated L gene can be used in future studies to test the ability for expression on in vitro translation.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

PubMed

Lee, Jinwoo; Nyenhuis, David A; Nelson, Elizabeth A; Cafiso, David S; White, Judith M; Tamm, Lukas K

2017-09-19

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

Structural studies of MFE-1: the 1.9 A crystal structure of the dehydrogenase part of rat peroxisomal MFE-1.

PubMed

Taskinen, Jukka P; Kiema, Tiila R; Hiltunen, J Kalervo; Wierenga, Rik K

2006-01-27

The 1.9 A structure of the C-terminal dehydrogenase part of the rat peroxisomal monomeric multifunctional enzyme type 1 (MFE-1) has been determined. In this construct (residues 260-722 and referred to as MFE1-DH) the N-terminal hydratase part of MFE-1 has been deleted. The structure of MFE1-DH shows that it consists of an N-terminal helix, followed by a Rossmann-fold domain (domain C), followed by two tightly associated helical domains (domains D and E), which have similar topology. The structure of MFE1-DH is compared with the two known homologous structures: human mitochondrial 3-hydroxyacyl-CoA dehydrogenase (HAD; sequence identity is 33%) (which is dimeric and monofunctional) and with the dimeric multifunctional alpha-chain (alphaFOM; sequence identity is 28%) of the bacterial fatty acid beta-oxidation alpha2beta2-multienzyme complex. Like MFE-1, alphaFOM has an N-terminal hydratase part and a C-terminal dehydrogenase part, and the structure comparisons show that the N-terminal helix of MFE1-DH corresponds to the alphaFOM linker helix, located between its hydratase and dehydrogenase part. It is also shown that this helix corresponds to the C-terminal helix-10 of the hydratase/isomerase superfamily, suggesting that functionally it belongs to the N-terminal hydratase part of MFE-1.

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

PubMed Central

Noguchi, Taro Q. P.; Morimatsu, Masatoshi; Iwane, Atsuko H.; Yanagida, Toshio; Uyeda, Taro Q. P.

2015-01-01

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin. PMID:25945499

The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii.

PubMed

Yoshimitsu, Kyohei; Takatani, Nobuyuki; Miura, Yukio; Watanabe, Yoshihito; Nakajima, Hiroshi

2011-09-01

VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase-2 in Azotobacter vinelandii. VnfA consists of three domains: an N-terminal regulatory domain termed GAF, including a Cys-rich motif; a central domain from the AAA+ family; and a C-terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe-S cluster (presumably of the 3Fe-4S type) as a prosthetic group and the Cys-rich motif were possibly associated with coordination of the Fe-S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: ΔN15(VnfA) and ΔGAF(VnfA) that lack the N-terminal 15 residues and whole GAF domain, respectively, and GAF(VnfA) consisting of only the GAF domain. ΔN15(VnfA) and ΔGAF(VnfA) lost the ability to bind the Fe-S cluster, whereas GAF(VnfA) was still able to bind to the cluster, consistent with the hypothesis that the Cys-rich motif is essential for Fe-S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe-S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain. © 2011 The Authors Journal compilation © 2011 FEBS.

Structural and functional characterization of the NHR1 domain of the Drosophila neuralized E3 ligase in the notch signaling pathway.

PubMed

He, Fahu; Saito, Kohei; Kobayashi, Naohiro; Harada, Takushi; Watanabe, Satoru; Kigawa, Takanori; Güntert, Peter; Ohara, Osamu; Tanaka, Akiko; Unzai, Satoru; Muto, Yutaka; Yokoyama, Shigeyuki

2009-10-23

The Notch signaling pathway is critical for many developmental processes and requires complex trafficking of both Notch receptor and its ligands, Delta and Serrate. In Drosophila melanogaster, the endocytosis of Delta in the signal-sending cell is essential for Notch receptor activation. The Neuralized protein from D. melanogaster (Neur) is a ubiquitin E3 ligase, which binds to Delta through its first neuralized homology repeat 1 (NHR1) domain and mediates the ubiquitination of Delta for endocytosis. Tom, a Bearded protein family member, inhibits the Neur-mediated endocytosis through interactions with the NHR1 domain. We have identified the domain boundaries of the novel NHR1 domain, using a screening system based on our cell-free protein synthesis method, and demonstrated that the identified Neur NHR1 domain had binding activity to the 20-residue peptide corresponding to motif 2 of Tom by isothermal titration calorimetry experiments. We also determined the solution structure of the Neur NHR1 domain by heteronuclear NMR methods, using a (15)N/(13)C-labeled sample. The Neur NHR1 domain adopts a characteristic beta-sandwich fold, consisting of a concave five-stranded antiparallel beta-sheet and a convex seven-stranded antiparallel beta-sheet. The long loop (L6) between the beta6 and beta7 strands covers the hydrophobic patch on the concave beta-sheet surface, and the Neur NHR1 domain forms a compact globular fold. Intriguingly, in spite of the slight, but distinct, differences in the topology of the secondary structure elements, the structure of the Neur NHR1 domain is quite similar to those of the B30.2/SPRY domains, which are known to mediate specific protein-protein interactions. Further NMR titration experiments of the Neur NHR1 domain with the 20-residue Tom peptide revealed that the resonances originating from the bottom area of the beta-sandwich (the L3, L5, and L11 loops, as well as the tip of the L6 loop) were affected. In addition, a structural comparison of the Neur NHR1 domain with the first NHR domain of the human KIAA1787 protein, which is from another NHR subfamily and does not bind to the 20-residue Tom peptide, suggested the critical amino acid residues for the interactions between the Neur NHR1 domain and the Tom peptide. The present structural study will shed light on the role of the Neur NHR1 domain in the Notch signaling pathway.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guan, Rongjin; Aiyer, Sriram; Cote, Marie L.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1–44) and an HHCC zinc-finger NTD (residues 45–105), in two crystal forms are reported. The structuresmore » of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647–656.« less

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

PubMed

Orth, Peter; Xiao, Li; Hernandez, Lorraine D; Reichert, Paul; Sheth, Payal R; Beaumont, Maribel; Yang, Xiaoyu; Murgolo, Nicholas; Ermakov, Grigori; DiNunzio, Edward; Racine, Fred; Karczewski, Jerzy; Secore, Susan; Ingram, Richard N; Mayhood, Todd; Strickland, Corey; Therien, Alex G

2014-06-27

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

PubMed

Wirtz, M; Berkowitz, O; Droux, M; Hell, R

2001-02-01

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

Personality Inventory for DSM-5-Short Form (PID-5-SF): Reliability, Factorial Structure, and Relationship With Functional Impairment in Dual Diagnosis Patients.

PubMed

Díaz-Batanero, Carmen; Ramírez-López, Juan; Domínguez-Salas, Sara; Fernández-Calderón, Fermín; Lozano, Óscar M

2017-11-01

Section III of the Diagnostic and Statistical Manual of Mental Disorders-Fifth edition ( DSM-5) has generated a personality paradigm consisting of 25 personality facets identified in five domains. The developed assessment instrument Personality Inventory for DSM-5 (PID-5) has showed good psychometric properties, but the potential for certain improvements still remain. In this article, a sample of 282 dual diagnosis patients is used to provide evidence of the psychometric properties of the PID-5-Short Form. The mean value of Cronbach's alpha coefficients reached .73 on the facets and .84 for domains and test-retest values ranged between .57 to .83 for facets and .70 to .87 for the domains. Confirmatory factor analyses conducted showed good fit on both models tested: the five correlated factor structure and hierarchical structure of personality traits. The WHODAS 2.0 domains of understanding and communicating, and participating in society, appear to show the strongest relationship with personality facets. In general, the PID-5-Short Form shows adequate psychometric properties for use in dual diagnosis patients.

Structural basis for midbody targeting of spastin by the ESCRT-III protein CHMP1B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Dong; Rimanchi, Neggy; Renvoise, Benoit

2009-01-15

The endosomal sorting complex required for transport (ESCRT) machinery, including ESCRT-III, localizes to the midbody and participates in the membrane-abscission step of cytokinesis. The ESCRT-III protein charged multivesicular body protein 1B (CHMP1B) is required for recruitment of the MIT domain-containing protein spastin, a microtubule-severing enzyme, to the midbody. The 2.5-{angstrom} structure of the C-terminal tail of CHMP1B with the MIT domain of spastin reveals a specific, high-affinity complex involving a noncanonical binding site between the first and third helices of the MIT domain. The structural interface is twice as large as that of the MIT domain of the VPS4-CHMP complex,more » consistent with the high affinity of the interaction. A series of unique hydrogen-bonding interactions and close packing of small side chains discriminate against the other ten human ESCRT-III subunits. Point mutants in the CHMP1B binding site of spastin block recruitment of spastin to the midbody and impair cytokinesis.« less

Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

PubMed

Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

2016-03-01

The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

[NMR structure and dynamics of the chimeric protein SH3-F2].

PubMed

Kutyshenko, V P; Gushchina, L V; Khristoforov, V S; Prokhorov, D A; Timchenko, M A; Kudrevatykh, Iu A; Fediukina, D V; Filimonov, V V

2010-01-01

For the further elucidation of structural and dynamic principles of protein self-organization and protein-ligand interactions the design of new chimeric protein SH3-F2 was made and genetically engineered construct was created. The SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamics properties of the protein were studied by high-resolution NMR. According to NMR data the tertiary structure of the chimeric protein SH3-F2 has the topology which is typical of SH3 domains in the complex with the ligand, forming polyproline type II helix, located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However the interaction of ligand and the part of globule relative to SH3 domain is not too large because the analysis of protein dynamic characteristics points to the low amplitude, high-frequency ligand tumbling in relation to the slow intramolecular motions of the main globule. The constructed chimera permits to carry out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

A structural model of PpoA derived from SAXS-analysis-implications for substrate conversion.

PubMed

Koch, Christian; Tria, Giancarlo; Fielding, Alistair J; Brodhun, Florian; Valerius, Oliver; Feussner, Kirstin; Braus, Gerhard H; Svergun, Dmitri I; Bennati, Marina; Feussner, Ivo

2013-09-01

In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement. Copyright © 2013 Elsevier B.V. All rights reserved.

An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

PubMed Central

Chiappori, Federica; Merelli, Ivan; Milanesi, Luciano; Colombo, Giorgio; Morra, Giulia

2016-01-01

The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery. PMID:27025773

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase.

PubMed

Samai, Poulami; Shuman, Stewart

2011-06-24

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.

The unique structural and biochemical development of single cell C4 photosynthesis along longitudinal leaf gradients in Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae)

PubMed Central

Koteyeva, Nuria K.; Voznesenskaya, Elena V.; Berry, James O.; Cousins, Asaph B.; Edwards, Gerald E.

2016-01-01

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica. Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis. PMID:26957565

IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold*

PubMed Central

Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M. F.; Downes, C. Peter; Batty, Ian H.

2012-01-01

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105–107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

Oil migration in a major growth fault: Structural analysis of the Pathfinder core, South Eugene Island Block 330, offshore Louisiana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Losh, S.

1998-09-01

The Pathfinder core, collected in the South Eugene Island Block 330 field, offshore Louisiana, provides an outstanding sample of structures associated with a major growth fault that abuts a giant oil field and that is thought to have acted as a conduit for hydrocarbon migration into the producing reservoirs. The fault zone in the core consists of three structural domains, each characterized by a distinct rock type, distribution of fault dips and dip azimuths, and distribution of spacing between adjacent faults and fractures. Although all of the domains contain oil-bearing sands, only faults and fractures in the deepest domain containmore » oil, even though the oil-barren fault domains contain numerous faults and fractures that are parallel to those containing oil in the deepest domain. The deepest domain is also distinguished from the other two domains by a greater degree of structural complexity and by a well-defined power-law distribution of fault and fracture spacings. Even though oil is present in sands throughout the core, its restriction to faults and fractures in the youngest sampled portion of the fault zone implies that oil migrated only through that part of the fault that was active during the time when oil had access to it. The absence of oil in fractures or faults in the other, probably older, fault domains indicates that the oil was never sufficiently pressured to flow up the fault zone on its own, either by hydraulic fracture or by increased permeability as a result of decreased effective stress. Instead, fluid migration along faults and fractures in the Pathfinder core was enhanced by permeability created in response to relatively far-field stresses related to minibasin subsidence.« less

IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dixon, Miles J.; Gray, Alexander; Schenning, Martijn

2012-10-16

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those ofmore » the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.« less

Computational Insight Into the Structural Organization of Full-Length Toll-Like Receptor 4 Dimer in a Model Phospholipid Bilayer

PubMed Central

Patra, Mahesh Chandra; Kwon, Hyuk-Kwon; Batool, Maria; Choi, Sangdun

2018-01-01

Toll-like receptors (TLRs) are a unique category of pattern recognition receptors that recognize distinct pathogenic components, often utilizing the same set of downstream adaptors. Specific molecular features of extracellular, transmembrane (TM), and cytoplasmic domains of TLRs are crucial for coordinating the complex, innate immune signaling pathway. Here, we constructed a full-length structural model of TLR4—a widely studied member of the interleukin-1 receptor/TLR superfamily—using homology modeling, protein–protein docking, and molecular dynamics simulations to understand the differential domain organization of TLR4 in a membrane-aqueous environment. Results showed that each functional domain of the membrane-bound TLR4 displayed several structural transitions that are biophysically essential for plasma membrane integration. Specifically, the extracellular and cytoplasmic domains were partially immersed in the upper and lower leaflets of the membrane bilayer. Meanwhile, TM domains tilted considerably to overcome the hydrophobic mismatch with the bilayer core. Our analysis indicates an alternate dimerization or a potential oligomerization interface of TLR4-TM. Moreover, the helical properties of an isolated TM dimer partly agree with that of the full-length receptor. Furthermore, membrane-absorbed or solvent-exposed surfaces of the toll/interleukin-1 receptor domain are consistent with previous X-ray crystallography and biochemical studies. Collectively, we provided a complete structural model of membrane-bound TLR4 that strengthens our current understanding of the complex mechanism of receptor activation and adaptor recruitment in the innate immune signaling pathway. PMID:29593733

The N253F mutant structure of trehalose synthase from Deinococcus radiodurans reveals an open active-site topology.

PubMed

Chow, Sih Yao; Wang, Yung Lin; Hsieh, Yu Chiao; Lee, Guan Chiun; Liaw, Shwu Huey

2017-11-01

Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures of Deinococcus radiodurans TS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes.

Functional Mapping of the Lectin Activity Site on the β-Prism Domain of Vibrio cholerae Cytolysin

PubMed Central

Rai, Anand Kumar; Paul, Karan; Chattopadhyay, Kausik

2013-01-01

Vibrio cholerae cytolysin (VCC) is a prominent member in the family of β-barrel pore-forming toxins. It induces lysis of target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the β-Prism domain, we demonstrate that the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the β-Prism domain's lectin activity, implicated in the process of membrane pore formation by VCC. PMID:23209283

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Ordine, Robert L.; Rydel, Timothy J.; Storek, Michael J.

2009-09-08

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O{sub 2} into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer ({alpha}{sub 3}) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While themore » Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co{sup 2+}, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 {angstrom}, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.« less

α-Helical Structural Elements within the Voltage-Sensing Domains of a K+ Channel

PubMed Central

Li-Smerin, Yingying; Hackos, David H.; Swartz, Kenton J.

2000-01-01

Voltage-gated K+ channels are tetramers with each subunit containing six (S1–S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5–S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1–S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K+ channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of α-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting α-helical secondary structure. In addition, both the S1–S2 and S3–S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain. PMID:10613917

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

PubMed

Prigozhin, Daniil M; Papavinasasundaram, Kadamba G; Baer, Christina E; Murphy, Kenan C; Moskaleva, Alisa; Chen, Tony Y; Alber, Tom; Sassetti, Christopher M

2016-10-28

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

PubMed Central

Pan, Di; Song, Yuhua

2010-01-01

Abstract N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities. PMID:20655849

Music acquisition: effects of enculturation and formal training on development.

PubMed

Hannon, Erin E; Trainor, Laurel J

2007-11-01

Musical structure is complex, consisting of a small set of elements that combine to form hierarchical levels of pitch and temporal structure according to grammatical rules. As with language, different systems use different elements and rules for combination. Drawing on recent findings, we propose that music acquisition begins with basic features, such as peripheral frequency-coding mechanisms and multisensory timing connections, and proceeds through enculturation, whereby everyday exposure to a particular music system creates, in a systematic order of acquisition, culture-specific brain structures and representations. Finally, we propose that formal musical training invokes domain-specific processes that affect salience of musical input and the amount of cortical tissue devoted to its processing, as well as domain-general processes of attention and executive functioning.

Slush-like polar structures in single-crystal relaxors

NASA Astrophysics Data System (ADS)

Takenaka, Hiroyuki; Grinberg, Ilya; Liu, Shi; Rappe, Andrew M.

2017-06-01

Despite more than 50 years of investigation, it is still unclear how the underlying structure of relaxor ferroelectrics gives rise to their defining properties, such as ultrahigh piezoelectric coefficients, high permittivity over a broad temperature range, diffuse phase transitions, strong frequency dependence in dielectric response, and phonon anomalies. The model of polar nanoregions inside a non-polar matrix has been widely used to describe the structure of relaxor ferroelectrics. However, the lack of precise knowledge about the shapes, growth and dipole patterns of polar nanoregions has led to the characterization of relaxors as “hopeless messes”, and no predictive model for relaxor behaviour is currently available. Here we use molecular dynamics simulations of the prototypical Pb(Mg1/3,Nb2/3)O3-PbTiO3 relaxor material to examine its structure and the spatial and temporal polarization correlations. Our simulations show that the unusual properties of relaxors stem from the presence of a multi-domain state with extremely small domain sizes (2-10 nanometres), and no non-polar matrix, owing to the local dynamics. We find that polar structures in the multi-domain state in relaxors are analogous to those of the slush state of water. The multi-domain structure of relaxors that is revealed by our molecular dynamics simulations is consistent with recent experimental diffuse scattering results and indicates that relaxors have a high density of low-angle domain walls. This insight explains the recently discovered classes of relaxors that cannot be described by the polar nanoregion model, and provides guidance for the design and synthesis of new relaxor materials.

Crystallized N-terminal domain of influenza virus matrix protein M1 and method of determining and using same

NASA Technical Reports Server (NTRS)

Luo, Ming (Inventor); Sha, Bingdong (Inventor)

2000-01-01

The matrix protein, M1, of influenza virus strain A/PR/8/34 has been purified from virions and crystallized. The crystals consist of a stable fragment (18 Kd) of the M1 protein. X-ray diffraction studies indicated that the crystals have a space group of P3.sub.t 21 or P3.sub.2 21. Vm calculations showed that there are two monomers in an asymmetric unit. A crystallized N-terminal domain of M1, wherein the N-terminal domain of M1 is crystallized such that the three dimensional structure of the crystallized N-terminal domain of M1 can be determined to a resolution of about 2.1 .ANG. or better, and wherein the three dimensional structure of the uncrystallized N-terminal domain of M1 cannot be determined to a resolution of about 2.1 .ANG. or better. A method of purifying M1 and a method of crystallizing M1. A method of using the three-dimensional crystal structure of M1 to screen for antiviral, influenza virus treating or preventing compounds. A method of using the three-dimensional crystal structure of M1 to screen for improved binding to or inhibition of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the manufacture of an inhibitor of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the screening of candidates for inhibition of influenza virus M1.

Crystal structure of the Mus81-Eme1 complex.

PubMed

Chang, Jeong Ho; Kim, Jeong Joo; Choi, Jung Min; Lee, Jung Hoon; Cho, Yunje

2008-04-15

The Mus81-Eme1 complex is a structure-specific endonuclease that plays an important role in rescuing stalled replication forks and resolving the meiotic recombination intermediates in eukaryotes. We have determined the crystal structure of the Mus81-Eme1 complex. Both Mus81 and Eme1 consist of a central nuclease domain, two repeats of the helix-hairpin-helix (HhH) motif at their C-terminal region, and a linker helix. While each domain structure resembles archaeal XPF homologs, the overall structure is significantly different from those due to the structure of a linker helix. We show that a flexible intradomain linker that formed with 36 residues in the nuclease domain of Eme1 is essential for the recognition of DNA. We identified several basic residues lining the outer surface of the active site cleft of Mus81 that are involved in the interaction with a flexible arm of a nicked Holliday junction (HJ). These interactions might contribute to the optimal positioning of the opposite junction across the nick into the catalytic site, which provided the basis for the "nick and counternick" mechanism of Mus81-Eme1 and for the nicked HJ to be the favored in vitro substrate of this enzyme.

Development and validation of the Chinese version of dry eye related quality of life scale.

PubMed

Zheng, Bang; Liu, Xiao-Jing; Sun, Yue-Qian Fiona; Su, Jia-Zeng; Zhao, Yang; Xie, Zheng; Yu, Guang-Yan

2017-07-17

To develop the Chinese version of quality of life scale for dry eye patients based on the Impact of Dry Eye on Everyday Life (IDEEL) questionnaire and to assess the reliability and validity of the developed scale. The original IDEEL was adapted cross-culturally to Chinese language and further developed following standard procedures. A total of 100 Chinese patients diagnosed with dry eye syndrome were included to investigate the psychometric properties of the Chinese version of scale. Psychometric tests included internal consistency (Cronbach's ɑ coefficients), construct validity (exploratory factor analysis), and known-groups validity (the analysis of variance). The Chinese version of Dry Eye Related Quality of Life (CDERQOL) Scale contains 45 items classified into 5 domains. Good to excellent internal consistency reliability was demonstrated for all 5 domains (Cronbach's ɑ coefficients range from 0.716 to 0.913). Construct validity assessment indicated a consistent factorial structure of the CDERQOL scale with hypothesized construct, with the exception of "Dry Eye Symptom-Bother" domain. All domain scores were detected with significant difference across three severity groups of dry eye patients (P < 0.05) except for "Satisfaction with Treatment" domain, indicating good known-groups validity. The results indicated that the CDERQOL scale is a reliable and valid instrument for patients with dry eye syndrome among Chinese population, and could be used as a supplementary diagnostic and treatment-effectiveness measure.

X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bornholdt, Zachary A.; Prasad, B.V. Venkataram

2009-04-08

The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains - a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker - is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60%more » of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-{angstrom}-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.« less

Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX.

PubMed

Argentaro, Anthony; Yang, Ji-Chun; Chapman, Lynda; Kowalczyk, Monika S; Gibbons, Richard J; Higgs, Douglas R; Neuhaus, David; Rhodes, Daniela

2007-07-17

The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

PubMed

Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

2013-10-01

The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

PubMed

Zhu, Hui; Shuman, Stewart

2005-04-01

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Threading structural model of the manganese-stabilizing protein PsbO reveals presence of two possible beta-sandwich domains.

PubMed

Pazos, F; Heredia, P; Valencia, A; de las Rivas, J

2001-12-01

The manganese-stabilizing protein (PsbO) is an essential component of photosystem II (PSII) and is present in all oxyphotosynthetic organisms. PsbO allows correct water splitting and oxygen evolution by stabilizing the reactions driven by the manganese cluster. Despite its important role, its structure and detailed functional mechanism are still unknown. In this article we propose a structural model based on fold recognition and molecular modeling. This model has additional support from a study of the distribution of characteristics of the PsbO sequence family, such as the distribution of conserved, apolar, tree-determinants, and correlated positions. Our threading results consistently showed PsbO as an all-beta (beta) protein, with two homologous beta domains of approximately 120 amino acids linked by a flexible Proline-Glycine-Glycine (PGG) motif. These features are compatible with a general elongated and flexible architecture, in which the two domains form a sandwich-type structure with Greek key topology. The first domain is predicted to include 8 to 9 beta-strands, the second domain 6 to 7 beta-strands. An Ig-like beta-sandwich structure was selected as a template to build the 3-D model. The second domain has, between the strands, long-loops rich in Pro and Gly that are difficult to model. One of these long loops includes a highly conserved region (between P148 and P174) and a short alpha-helix (between E181 and N188)). These regions are characteristic parts of PsbO and show that the second domain is not so similar to the template. Overall, the model was able to account for much of the experimental data reported by several authors, and it would allow the detection of key residues and regions that are proposed in this article as essential for the structure and function of PsbO. Copyright 2001 Wiley-Liss, Inc.

Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1.

PubMed Central

Gijsbers, Rik; Ceulemans, Hugo; Bollen, Mathieu

2003-01-01

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1-3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1-3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1-NPP3, suggesting that the nuclease-like domain of NPP1-3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1-3. PMID:12533192

Surface structural conformations of fibrinogen polypeptides for improved biocompatibility.

PubMed

Yaseen, Mohammed; Zhao, Xiubo; Freund, Amy; Seifalian, Alexander M; Lu, Jian R

2010-05-01

This work reports on how incorporation of silica nanocages into poly(urethane) copolymers (PU) affects conformational orientations of adsorbed fibrinogen and how different surfaces subsequently influenced HeLa cell attachment and proliferation. Incorporation of 2 wt% silica nanocages into poly(urethane) (PU4) substantially altered the surface topography of the films and some 50% of the surface was covered with the nanocages due to their preferential exposure. AFM studies revealed the deposition of a dense protein network on the soft polymeric domains of PU4 and much reduced fibrinogen adsorption on the hard nanocage domains. As on the bare SiO(2) control surface, fibrinogen molecules adsorbed on top of the hard nanocages mainly took the dominant trinodular structures in monomeric and dimeric forms. In addition, net positively charged long alpha chains were prone to being hidden beneath the D domains whilst gamma chains predominantly remained exposed. Dynamic interfacial adsorption as probed by spectroscopic ellipsometry revealed fast changes in interfacial conformation induced by electrostatic interactions between different segments of fibrinogen and the surface, consistent with the AFM imaging. On the PU surfaces without nanocage incorporation (PUA), however, adsorbed fibrinogen molecules formed beads-like chain networks, consistent with the structure featured on the soft PU4 domains, showing very different effects of surface chemical nature. Monoclonal antibodies specific to the alpha and gamma chains showed reduced alpha but increased gamma chain binding at the silicon oxide control and PU4 surfaces, whilst on the PUA, C18 and amine surfaces (organic surface controls) the opposite binding trend was detected with alpha chain binding dominant, showing different fibrinogen conformations. Cell attachment studies revealed differences in cell attachment and proliferation, consistent with the different polypeptide conformations on the two types of surfaces, showing a strong preference to the extent of exposure of gamma chains. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

Sequence analyses reveal that a TPR-DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR-DP domains and prokaryotic GerD proteins.

PubMed

Hernández Torres, Jorge; Papandreou, Nikolaos; Chomilier, Jacques

2009-05-01

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR-DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR-DP domains.

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

PubMed

Wissmann, Ralph; Bildl, Wolfgang; Oliver, Dominik; Beyermann, Michael; Kalbitzer, Hans-Robert; Bentrop, Detlef; Fakler, Bernd

2003-05-02

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Study of Raft Domains in Model Membrane of DPPC/PE/Cholesterol

NASA Astrophysics Data System (ADS)

Lor, Chai; Hirst, Linda

2010-10-01

Raft domains in bilayer membrane are thought to play an important role in many cell functions such as cell signaling or trans-membrane protein activation. Here we use a model membrane consisting of DPPC/PE/cholesterol to examine the structure of membrane rafts and phase interactions. In particular we are interested in lipids containing the highly polyunsaturated fatty acid DHA. We use both atomic force microscopy (AFM) and fluorescence microscopy to obtain information on the structural properties of raft regions and track cholesterol. As expected, we find phase separation of raft regions between saturated and unsaturated lipids. Moreover, we find that the roughness of the domains change with varying cholesterol concentration possibly due to overpacking. This model study provides further understanding of the role of cholesterol in bilayer membrane leading towards a better knowledge of cell membranes.

Fullerene data mining using bibliometrics and database tomography

PubMed

Kostoff; Braun; Schubert; Toothman; Humenik

2000-01-01

Database tomography (DT) is a textual database analysis system consisting of two major components: (1) algorithms for extracting multiword phrase frequencies and phrase proximities (physical closeness of the multiword technical phrases) from any type of large textual database, to augment (2) interpretative capabilities of the expert human analyst. DT was used to derive technical intelligence from a fullerenes database derived from the Science Citation Index and the Engineering Compendex. Phrase frequency analysis by the technical domain experts provided the pervasive technical themes of the fullerenes database, and phrase proximity analysis provided the relationships among the pervasive technical themes. Bibliometric analysis of the fullerenes literature supplemented the DT results with author/journal/institution publication and citation data. Comparisons of fullerenes results with past analyses of similarly structured near-earth space, chemistry, hypersonic/supersonic flow, aircraft, and ship hydrodynamics databases are made. One important finding is that many of the normalized bibliometric distribution functions are extremely consistent across these diverse technical domains and could reasonably be expected to apply to broader chemical topics than fullerenes that span multiple structural classes. Finally, lessons learned about integrating the technical domain experts with the data mining tools are presented.

Free energy landscapes for initiation and branching of protein aggregation.

PubMed

Zheng, Weihua; Schafer, Nicholas P; Wolynes, Peter G

2013-12-17

Experiments on artificial multidomain protein constructs have probed the early stages of aggregation processes, but structural details of the species that initiate aggregation remain elusive. Using the associative-memory, water-mediated, structure and energy model known as AWSEM, a transferable coarse-grained protein model, we performed simulations of fused constructs composed of up to four copies of the Titin I27 domain or its mutant I27* (I59E). Free energy calculations enable us to quantify the conditions under which such multidomain constructs will spontaneously misfold. Consistent with experimental results, the dimer of I27 is found to be the smallest spontaneously misfolding construct. Our results show how structurally distinct misfolded states can be stabilized under different thermodynamic conditions, and this result provides a plausible link between the single-molecule misfolding experiments under native conditions and aggregation experiments under denaturing conditions. The conditions for spontaneous misfolding are determined by the interplay among temperature, effective local protein concentration, and the strength of the interdomain interactions. Above the folding temperature, fusing additional domains to the monomer destabilizes the native state, and the entropically stabilized amyloid-like state is favored. Because it is primarily energetically stabilized, the domain-swapped state is more likely to be important under native conditions. Both protofibril-like and branching structures are found in annealing simulations starting from extended structures, and these structures suggest a possible connection between the existence of multiple amyloidogenic segments in each domain and the formation of branched, amorphous aggregates as opposed to linear fibrillar structures.

Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding.

PubMed

Boëda, Batiste; Briggs, David C; Higgins, Theresa; Garvalov, Boyan K; Fadden, Andrew J; McDonald, Neil Q; Way, Michael

2007-12-28

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.

Comparative Reliability of Structured Versus Unstructured Interviews in the Admission Process of a Residency Program

PubMed Central

Blouin, Danielle; Day, Andrew G.; Pavlov, Andrey

2011-01-01

Background Although never directly compared, structured interviews are reported as being more reliable than unstructured interviews. This study compared the reliability of both types of interview when applied to a common pool of applicants for positions in an emergency medicine residency program. Methods In 2008, one structured interview was added to the two unstructured interviews traditionally used in our resident selection process. A formal job analysis using the critical incident technique guided the development of the structured interview tool. This tool consisted of 7 scenarios assessing 4 of the domains deemed essential for success as a resident in this program. The traditional interview tool assessed 5 general criteria. In addition to these criteria, the unstructured panel members were asked to rate each candidate on the same 4 essential domains rated by the structured panel members. All 3 panels interviewed all candidates. Main outcomes were the overall, interitem, and interrater reliabilities, the correlations between interview panels, and the dimensionality of each interview tool. Results Thirty candidates were interviewed. The overall reliability reached 0.43 for the structured interview, and 0.81 and 0.71 for the unstructured interviews. Analyses of the variance components showed a high interrater, low interitem reliability for the structured interview, and a high interrater, high interitem reliability for the unstructured interviews. The summary measures from the 2 unstructured interviews were significantly correlated, but neither was correlated with the structured interview. Only the structured interview was multidimensional. Conclusions A structured interview did not yield a higher overall reliability than both unstructured interviews. The lower reliability is explained by a lower interitem reliability, which in turn is due to the multidimensionality of the interview tool. Both unstructured panels consistently rated a single dimension, even when prompted to assess the 4 specific domains established as essential to succeed in this residency program. PMID:23205201

Comparative reliability of structured versus unstructured interviews in the admission process of a residency program.

PubMed

Blouin, Danielle; Day, Andrew G; Pavlov, Andrey

2011-12-01

Although never directly compared, structured interviews are reported as being more reliable than unstructured interviews. This study compared the reliability of both types of interview when applied to a common pool of applicants for positions in an emergency medicine residency program. In 2008, one structured interview was added to the two unstructured interviews traditionally used in our resident selection process. A formal job analysis using the critical incident technique guided the development of the structured interview tool. This tool consisted of 7 scenarios assessing 4 of the domains deemed essential for success as a resident in this program. The traditional interview tool assessed 5 general criteria. In addition to these criteria, the unstructured panel members were asked to rate each candidate on the same 4 essential domains rated by the structured panel members. All 3 panels interviewed all candidates. Main outcomes were the overall, interitem, and interrater reliabilities, the correlations between interview panels, and the dimensionality of each interview tool. Thirty candidates were interviewed. The overall reliability reached 0.43 for the structured interview, and 0.81 and 0.71 for the unstructured interviews. Analyses of the variance components showed a high interrater, low interitem reliability for the structured interview, and a high interrater, high interitem reliability for the unstructured interviews. The summary measures from the 2 unstructured interviews were significantly correlated, but neither was correlated with the structured interview. Only the structured interview was multidimensional. A structured interview did not yield a higher overall reliability than both unstructured interviews. The lower reliability is explained by a lower interitem reliability, which in turn is due to the multidimensionality of the interview tool. Both unstructured panels consistently rated a single dimension, even when prompted to assess the 4 specific domains established as essential to succeed in this residency program.

The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I).

PubMed

Salinas, Gustavo; Gao, Wei; Wang, Yang; Bonilla, Mariana; Yu, Long; Novikov, Andrey; Virginio, Veridiana G; Ferreira, Henrique B; Vieites, Marisol; Gladyshev, Vadim N; Gambino, Dinorah; Dai, Shaodong

2017-12-20

New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with Au I -MPO, a novel gold inhibitor, together with inhibition assays were performed. Au I -MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys 519 and Cys 573 in the Au I -TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys 519 and Cys 573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.

The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salinas, Gustavo; Gao, Wei; Wang, Yang

Aims: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. Results: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer–monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxinmore » (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. Innovation: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. Conclusions: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491–1504.« less

Anthrax Toxin Receptor 1 / Tumor Endothelial Marker 8: Mutation of Conserved Inserted Domain Residues Overrides Cytosolic Control of Protective Antigen Binding†

PubMed Central

Ramey, Jordan D.; Villareal, Valerie A.; Ng, Charles; Ward, Sabrina; Xiong, Jian-Ping; Clubb, Robert T.; Bradley, Kenneth A.

2010-01-01

Anthrax toxin receptor 1 (ANTXR1) / tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS-mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the KD and total amount of PA bound by the isolated ANTXR1 I domain was not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W and WT I domains were minor despite a greater than 103-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for anti-tumor therapies. PMID:20690680

Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains.

PubMed

Konno, Kotaro; Shimura, Sachiko; Ueno, Chihiro; Arakawa, Toru; Nakamura, Masatoshi

2018-03-01

MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cross-over between discrete and continuous protein structure space: insights into automatic classification and networks of protein structures.

PubMed

Pascual-García, Alberto; Abia, David; Ortiz, Angel R; Bastolla, Ugo

2009-03-01

Structural classifications of proteins assume the existence of the fold, which is an intrinsic equivalence class of protein domains. Here, we test in which conditions such an equivalence class is compatible with objective similarity measures. We base our analysis on the transitive property of the equivalence relationship, requiring that similarity of A with B and B with C implies that A and C are also similar. Divergent gene evolution leads us to expect that the transitive property should approximately hold. However, if protein domains are a combination of recurrent short polypeptide fragments, as proposed by several authors, then similarity of partial fragments may violate the transitive property, favouring the continuous view of the protein structure space. We propose a measure to quantify the violations of the transitive property when a clustering algorithm joins elements into clusters, and we find out that such violations present a well defined and detectable cross-over point, from an approximately transitive regime at high structure similarity to a regime with large transitivity violations and large differences in length at low similarity. We argue that protein structure space is discrete and hierarchic classification is justified up to this cross-over point, whereas at lower similarities the structure space is continuous and it should be represented as a network. We have tested the qualitative behaviour of this measure, varying all the choices involved in the automatic classification procedure, i.e., domain decomposition, alignment algorithm, similarity score, and clustering algorithm, and we have found out that this behaviour is quite robust. The final classification depends on the chosen algorithms. We used the values of the clustering coefficient and the transitivity violations to select the optimal choices among those that we tested. Interestingly, this criterion also favours the agreement between automatic and expert classifications. As a domain set, we have selected a consensus set of 2,890 domains decomposed very similarly in SCOP and CATH. As an alignment algorithm, we used a global version of MAMMOTH developed in our group, which is both rapid and accurate. As a similarity measure, we used the size-normalized contact overlap, and as a clustering algorithm, we used average linkage. The resulting automatic classification at the cross-over point was more consistent than expert ones with respect to the structure similarity measure, with 86% of the clusters corresponding to subsets of either SCOP or CATH superfamilies and fewer than 5% containing domains in distinct folds according to both SCOP and CATH. Almost 15% of SCOP superfamilies and 10% of CATH superfamilies were split, consistent with the notion of fold change in protein evolution. These results were qualitatively robust for all choices that we tested, although we did not try to use alignment algorithms developed by other groups. Folds defined in SCOP and CATH would be completely joined in the regime of large transitivity violations where clustering is more arbitrary. Consistently, the agreement between SCOP and CATH at fold level was lower than their agreement with the automatic classification obtained using as a clustering algorithm, respectively, average linkage (for SCOP) or single linkage (for CATH). The networks representing significant evolutionary and structural relationships between clusters beyond the cross-over point may allow us to perform evolutionary, structural, or functional analyses beyond the limits of classification schemes. These networks and the underlying clusters are available at http://ub.cbm.uam.es/research/ProtNet.php.

Current induced incoherent magnetization dynamics in ferromagnetic/non-magnetic metallic multilayer nanowires

NASA Astrophysics Data System (ADS)

Al-Rashid, Md Mamun; Maqableh, Mazin; Stadler, Bethanie; Atulasimha, Jayasimha

High density arrays of electrodeposited nanowires consisting of ferromagnetic/non-magnetic (Co/Cu) multilayers are promising as magnetic memory devices. For individual nanowires containing multiple (Co/Cu) bilayers, the stable magnetization orientations of the Co layers (with respect to each other and the nanowire axis) are dependent on the Cu layer thickness, even when the Co layer dimensions are fixed. This dependence is a result of the competition between shape anisotropy, magneto-crystalline anisotropy and intra-wire dipole coupling. However, when the nanowires are closely packed in arrays, inter-wire dipole coupling can result in complex and tunable domain structures comprising segments of multiple nanowires. This work explores the dependence of these domain structures and their switching on the non-magnetic layer thickness and intra-wire spacing both experimentally and via rigorous micromagnetic simulation. These domain structures play a crucial role in determining the current and time required for STT switching. NSF CAREER Grant CCF-1253370.

Respiratory complex I: 'steam engine' of the cell?

PubMed

Efremov, Rouslan G; Sazanov, Leonid A

2011-08-01

Complex I is the first enzyme of the respiratory chain and plays a central role in cellular energy production. It has been implicated in many human neurodegenerative diseases, as well as in ageing. One of the biggest membrane protein complexes, it is an L-shaped assembly consisting of hydrophilic and membrane domains. Previously, we have determined structures of the hydrophilic domain in several redox states. Last year was marked by fascinating breakthroughs in the understanding of the complete structure. We described the architecture of the membrane domain and of the entire bacterial complex I. X-ray analysis of the larger mitochondrial enzyme has also been published. The core subunits of the bacterial and mitochondrial enzymes have remarkably similar structures. The proposed mechanism of coupling between electron transfer and proton translocation involves long-range conformational changes, coordinated in part by a long α-helix, akin to the coupling rod of a steam engine. Copyright © 2011 Elsevier Ltd. All rights reserved.

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain.

PubMed

Shi, Hang; Rojas, Raul; Bonifacino, Juan S; Hurley, James H

2006-06-01

The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29 and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1-A resolution reveals two curved beta-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unpredicted structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a glycine in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.

Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.

PubMed

Xu, Qingping; Chiu, Hsiu-Ju; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Lesley, Scott A; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

2014-01-09

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state). © 2013.

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

PubMed

Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

2017-08-08

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

Structure of the EMMPRIN N-terminal domain 1: Dimerization via [beta]-strand swapping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Jinquan; Teplyakov, Alexey; Obmolova, Galina

2010-09-27

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residuesmore » 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which has a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last {beta}-strand (strand G). {beta}-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell-cell adhesion by cadherins. 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains and amyloidogenesis. In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state. It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations. Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis-trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell-cell adhesion.« less

Structure of the TRPV1 ion channel determined by electron cryo-microscopy.

PubMed

Liao, Maofu; Cao, Erhu; Julius, David; Cheng, Yifan

2013-12-05

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop, which is flanked by S1-S4 voltage-sensor-like domains. TRPV1 has a wide extracellular 'mouth' with a short selectivity filter. The conserved 'TRP domain' interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

Quantitative Three-Dimensional Characterization of Block Copolymer Directed Self-Assembly on Combined Chemical and Topographical Prepatterned Templates.

PubMed

Segal-Peretz, Tamar; Ren, Jiaxing; Xiong, Shisheng; Khaira, Gurdaman; Bowen, Alec; Ocola, Leonidas E; Divan, Ralu; Doxastakis, Manolis; Ferrier, Nicola J; de Pablo, Juan; Nealey, Paul F

2017-02-28

Characterization of the three-dimensional (3D) structure in directed self-assembly (DSA) of block copolymers is crucial for understanding the complex relationships between the guiding template and the resulting polymer structure so DSA could be successfully implemented for advanced lithography applications. Here, we combined scanning transmission electron microscopy (STEM) tomography and coarse-grain simulations to probe the 3D structure of P2VP-b-PS-b-P2VP assembled on prepatterned templates using solvent vapor annealing. The templates consisted of nonpreferential background and raised guiding stripes that had PS-preferential top surfaces and P2VP-preferential sidewalls. The full 3D characterization allowed us to quantify the shape of the polymer domains and the interface between domains as a function of depth in the film and template geometry and offered important insights that were not accessible with 2D metrology. Sidewall guiding was advantageous in promoting the alignment and lowering the roughness of the P2VP domains over the sidewalls, but incommensurate confinement from the increased topography could cause roughness and intermittent dislocations in domains over the background region at the bottom of the film. The 3D characterization of bridge structures between domains over the background and breaks within domains on guiding lines sheds light on possible origins of common DSA defects. The positional fluctuations of the PS/P2VP interface between domains showed a depth-dependent behavior, with high levels of fluctuations near both the free surface of the film and the substrate and lower fluctuation levels in the middle of the film. This research demonstrates how 3D characterization offers a better understanding of DSA processes, leading to better design and fabrication of directing templates.

Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

PubMed Central

Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

2016-01-01

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase*

PubMed Central

Samai, Poulami; Shuman, Stewart

2011-01-01

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5′-phosphate nucleotide and the 3′-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps. PMID:21527793

Ribosomal small subunit domains radiate from a central core

NASA Astrophysics Data System (ADS)

Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

2016-02-01

The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

PubMed Central

Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

2016-01-01

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

Electron microscope studies of nano-domain structures in Ru-based magneto-superconductors: RuSr(2)Gd(1.5)Ce(0.5)Cu(2)O(10-delta) (Ru-1222) and RuSr(2)GdCu(2)O(8) (Ru-1212).

PubMed

Yokosawa, Tadahiro; Awana, V P S Veer Pal Singh; Kimoto, Koji; Takayama-Muromachi, Eiji; Karppinen, Maarit; Yamauchi, Hisao; Matsui, Yoshio

2004-01-01

Microstructures of the RuSr(2)Gd(1.5)Ce(0.5)Cu(2)O(10-delta) (Ru-1222) and RuSr(2)GdCu(2)O(8) (Ru-1212) magneto-superconductors have been investigated by using selected-area electron diffraction, convergent-beam electron diffraction, dark-field electron microscopy and high-resolution electron microscopy at room temperature. Both Ru-1212 and Ru-1222 consist of nm-size domains stacked along the [Formula: see text] direction, where the domains are formed by two types of superstructures due to ordering of rotated RuO(6) octahedra about the c-axis. In Ru-1212, both primitive-and body-centered tetragonal superstructures (the possible space groups: P4/mbm and I4/mcm) are derived to form the corresponding nm-domains. It is of great interest that Ru-1212 consists of domains of two crystallographically different superstructures, while the similar domains observed in Ru-1222 have crystallographically identical superstructure with an orthorhombic symmetry (possible space group: Aeam), related by 90 degrees rotation around the c-axis (Yokosawa et al., 2003, submitted for publication).

Changes at the KinA PAS-A Dimerization Interface Influence Histidine Kinase Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, James; Tomchick, Diana R.; Brautigam, Chad A.

2008-11-12

The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatorymore » role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using {beta}-sheet/{beta}-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.« less

Psychometric properties of the WHOQOL-BREF in an Iranian adult sample.

PubMed

Yousefy, A R; Usefy, A R; Ghassemi, Gh R; Sarrafzadegan, N; Mallik, S; Baghaei, A M; Rabiei, K

2010-04-01

To evaluate discriminant validity, reliability, internal consistency, and dimensional structure of the World Health Organization Quality of Life-BREF (WHOQOL-BREF) in a heterogeneous Iranian population. A clustered randomized sample of 2,956 healthy with 2,936 unhealthy rural and urban inhabitants aged 30 and above from two dissimilar Iranian provinces during 2006 completed the Persian version of the WHOQOL-BREF. We performed descriptive and analytical analysis including t-student, correlation matrix, Cronbach's Alpha, and factor analysis with principal components method and Varimax rotation with SPSS.15. The mean age of the participants was 42.2 +/- 12.1 years and the mean years of education was 9.3 +/- 3.8. The Iranian version of the WHOQOL-BREF domain scores demonstrated good internal consistency, criterion validity, and discriminant validity. The physical health domain contributed most in overall quality of life, while the environment domain made the least contribution. Factor analysis provided evidence for construct validity for four-factor model of the instrument. The scores of all domains discriminated between healthy persons and the patients. The WHOQOL-BREF has adequate psychometric properties and is, therefore, an adequate measure for assessing quality of life at the domain level in an adult Iranian population.

Crystal structures of sampatrilat and sampatrilat-Asp in complex with human ACE - a molecular basis for domain selectivity.

PubMed

Cozier, Gyles E; Schwager, Sylva L; Sharma, Rajni K; Chibale, Kelly; Sturrock, Edward D; Acharya, K Ravi

2018-04-01

Angiotensin-1-converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (known as nACE and cACE) with different substrate specificities. Based on kinetic studies it was previously reported that sampatrilat, a tight-binding inhibitor of ACE, K i = 13.8 nm and 171.9 nm for cACE and nACE respectively [Sharma et al., Journal of Chemical Information and Modeling (2016), 56, 2486-2494], was 12.4-fold more selective for cACE. In addition, samAsp, in which an aspartate group replaces the sampatrilat lysine, was found to be a nonspecific and lower micromolar affinity inhibitor. Here, we report a detailed three-dimensional structural analysis of sampatrilat and samAsp binding to ACE using high-resolution crystal structures elucidated by X-ray crystallography, which provides a molecular basis for differences in inhibitor affinity and selectivity for nACE and cACE. The structures show that the specificity of sampatrilat can be explained by increased hydrophobic interactions and a H-bond from Glu403 of cACE with the lysine side chain of sampatrilat that are not observed in nACE. In addition, the structures clearly show a significantly greater number of hydrophilic and hydrophobic interactions with sampatrilat compared to samAsp in both cACE and nACE consistent with the difference in affinities. Our findings provide new experimental insights into ligand binding at the active site pockets that are important for the design of highly specific domain selective inhibitors of ACE. The atomic coordinates and structure factors for N- and C-domains of ACE bound to sampatrilat and sampatrilat-Asp complexes (6F9V, 6F9R, 6F9T and 6F9U respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). © 2018 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Modulation of the magnetic domain size induced by an electric field

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ando, F.; Kakizakai, H.; Yamada, K.

2016-07-11

The electric field (EF) effect on the magnetic domain structure of a Pt/Co system was studied, where an EF was applied to the top surface of the Co layer. The width of the maze domain was significantly modified by the application of the EF at a temperature slightly below the Curie temperature. After a detailed analysis, a change in the microscopic exchange stiffness induced by the EF application was suggested to dominate the modulation of the domain width observed in the experiment. The accumulation of electrons at the surface of the Co layer resulted in an increase in the microscopicmore » exchange stiffness and the Curie temperature. The result was consistent with the recent theoretical prediction.« less

SAbDab: the structural antibody database

PubMed Central

Dunbar, James; Krawczyk, Konrad; Leem, Jinwoo; Baker, Terry; Fuchs, Angelika; Georges, Guy; Shi, Jiye; Deane, Charlotte M.

2014-01-01

Structural antibody database (SAbDab; http://opig.stats.ox.ac.uk/webapps/sabdab) is an online resource containing all the publicly available antibody structures annotated and presented in a consistent fashion. The data are annotated with several properties including experimental information, gene details, correct heavy and light chain pairings, antigen details and, where available, antibody–antigen binding affinity. The user can select structures, according to these attributes as well as structural properties such as complementarity determining region loop conformation and variable domain orientation. Individual structures, datasets and the complete database can be downloaded. PMID:24214988

Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems

PubMed Central

Espina, Marianela; Ausar, S. Fernando; Middaugh, C. Russell; Baxter, M. Aaron; Picking, William D.; Picking, Wendy L.

2007-01-01

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are α-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins. PMID:17327391

The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

PubMed Central

1996-01-01

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line. PMID:8603916

Structural analysis of the DAP5 MIF4G domain and its interaction with eIF4A

PubMed Central

Virgili, Geneviève; Frank, Filipp; Feoktistova, Kateryna; Sawicki, Maxime; Sonenberg, Nahum; Fraser, Christopher S.; Nagar, Bhushan

2013-01-01

Summary Death-associated protein 5 (DAP5/p97) is a homolog of the eukaryotic initiation factor 4G (eIF4G) that promotes the IRES-driven translation of multiple cellular mRNAs. Central to its function is the middle domain (MIF4G), which recruits the RNA helicase eIF4A. The middle domain of eIF4G consists of tandem HEAT repeats that coalesce to form a solenoid-type structure. Here, we report the crystal structure of the DAP5 MIF4G domain. Its overall fold is very similar to that of eIF4G, however, significant conformational variations impart distinct surface properties that could explain the observed differences in IRES binding between the two proteins. Interestingly, quantitative analysis of the DAP5-eIF4A interaction using isothermal titration calorimetry reveals a 10-fold lower affinity than with the eIF4G-eIF4A interaction that appears to affect their ability to stimulate eIF4A RNA unwinding activity in vitro. This difference in stability of the complex may have functional implications in selecting the mode of translation initiation. PMID:23478064

Methodology for testing and validating knowledge bases

NASA Technical Reports Server (NTRS)

Krishnamurthy, C.; Padalkar, S.; Sztipanovits, J.; Purves, B. R.

1987-01-01

A test and validation toolset developed for artificial intelligence programs is described. The basic premises of this method are: (1) knowledge bases have a strongly declarative character and represent mostly structural information about different domains, (2) the conditions for integrity, consistency, and correctness can be transformed into structural properties of knowledge bases, and (3) structural information and structural properties can be uniformly represented by graphs and checked by graph algorithms. The interactive test and validation environment have been implemented on a SUN workstation.

Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain.

PubMed

Hung, Huynh Minh; Hang, Tran Dieu; Nguyen, Minh Tho

2016-09-09

Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

The Structure of Neurexin 1[alpha] Reveals Features Promoting a Role as Synaptic Organizer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Fang; Venugopal, Vandavasi; Murray, Beverly

{alpha}-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The {alpha}-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how {alpha}-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1{alpha} extracellular domain (n1{alpha}) to 2.65 {angstrom}. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 {angstrom} structure of n1{alpha}more » carrying splice insert SS3 in LNS4 reveals that SS3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables {alpha}-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.« less

Medical Group Structural Integration May Not Ensure That Care Is Integrated, From The Patient's Perspective.

PubMed

Kerrissey, Michaela J; Clark, Jonathan R; Friedberg, Mark W; Jiang, Wei; Fryer, Ashley K; Frean, Molly; Shortell, Stephen M; Ramsay, Patricia P; Casalino, Lawrence P; Singer, Sara J

2017-05-01

Structural integration is increasing among medical groups, but whether these changes yield care that is more integrated remains unclear. We explored the relationships between structural integration characteristics of 144 medical groups and perceptions of integrated care among their patients. Patients' perceptions were measured by a validated national survey of 3,067 Medicare beneficiaries with multiple chronic conditions across six domains that reflect knowledge and support of, and communication with, the patient. Medical groups' structural characteristics were taken from the National Study of Physician Organizations and included practice size, specialty mix, technological capabilities, and care management processes. Patients' survey responses were most favorable for the domain of test result communication and least favorable for the domain of provider support for medication and home health management. Medical groups' characteristics were not consistently associated with patients' perceptions of integrated care. However, compared to patients of primary care groups, patients of multispecialty groups had strong favorable perceptions of medical group staff knowledge of patients' medical histories. Opportunities exist to improve patient care, but structural integration of medical groups might not be sufficient for delivering care that patients perceive as integrated. Project HOPE—The People-to-People Health Foundation, Inc.

Terahertz pulsed imaging for the monitoring of dental caries: a comparison with x-ray imaging

NASA Astrophysics Data System (ADS)

Karagoz, Burcu; Kamburoglu, Kıvanc; Altan, Hakan

2017-07-01

Dental caries in sliced samples are investigated using terahertz pulsed imaging. Frequency domain terahertz response of these structures consistent with X-ray imaging results show the potential of this technique in the detection of early caries.

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

PubMed Central

1992-01-01

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1469047

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

PubMed Central

Chouquet, Anne; Païdassi, Helena; Ling, Wai Li; Frachet, Philippe; Houen, Gunnar; Arlaud, Gérard J.; Gaboriaud, Christine

2011-01-01

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism. PMID:21423620

Structure and dimerization of the catalytic domain of the protein phosphatase Cdc14p, a key regulator of mitotic exit in Saccharomyces cerevisiae.

PubMed

Kobayashi, Junya; Matsuura, Yoshiyuki

2017-10-01

In the budding yeast Saccharomyces cerevisiae, the protein phosphatase Cdc14p orchestrates various events essential for mitotic exit. We have determined the X-ray crystal structures at 1.85 Å resolution of the catalytic domain of Cdc14p in both the apo state, and as a complex with S160-phosphorylated Swi6p peptide. Each asymmetric unit contains two Cdc14p chains arranged in an intimately associated homodimer, consistent with its oligomeric state in solution. The dimerization interface is located on the backside of the substrate-binding cleft. Structure-based mutational analyses indicate that the dimerization of Cdc14p is required for normal growth of yeast cells. © 2017 The Protein Society.

Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.

PubMed

Lin, Lianyun; Liu, Chen; Qin, Juan; Wang, Jie; Dong, Shengjie; Chen, Wei; He, Weiyi; Gao, Qingzhi; You, Minsheng; Yuchi, Zhiguang

2018-01-01

Ryanodine receptors (RyRs) are large calcium-release channels located in sarcoplasmic reticulum membrane. They play a central role in excitation-contraction coupling of muscle cells. Three commercialized insecticides targeting pest RyRs generate worldwide sales over 2 billion U.S. dollars annually, but the structure of insect RyRs remains elusive, hindering our understanding of the mode of action of RyR-targeting insecticides and the development of insecticide resistance in pests. Here we present the crystal structure of RyR N-terminal domain (NTD) (residue 1-205) at 2.84 Å resolution from the diamondback moth (DBM), Plutella xylostella, a destructive pest devouring cruciferous crops all over the world. Similar to its mammalian homolog, DBM RyR NTD consists of a beta-trefoil folding motif and a flanking alpha helix. Interestingly, two regions in NTD interacting with neighboring domains showed distinguished conformations in DBM relative to mammalian RyRs. Using homology modeling and molecular dynamics simulation, we created a structural model of the N-terminal three domains, showing two unique binding pockets that could be targeted by potential species-specific insecticides. Thermal melt experiment showed that the stability of DBM RyR NTD was higher than mammalian RyRs, probably due to a stable intra-domain disulfide bond observed in the crystal structure. Previously DBM NTD was shown to be one of the two critical regions to interact with insecticide flubendiamide, but isothermal titration calorimetry experiments negated DBM NTD alone as a major binding site for flubendiamide. Copyright © 2017 Elsevier Ltd. All rights reserved.

Common fold in helix–hairpin–helix proteins

PubMed Central

Shao, Xuguang; Grishin, Nick V.

2000-01-01

Helix–hairpin–helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein–protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)2 domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)2 domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each α-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the α-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glyco­s­y­lases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)2 domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)2 functional unit. PMID:10908318

Anatomy of triply-periodic network assemblies: characterizing skeletal and inter-domain surface geometry of block copolymer gyroids.

PubMed

Prasad, Ishan; Jinnai, Hiroshi; Ho, Rong-Ming; Thomas, Edwin L; Grason, Gregory M

2018-05-09

Triply-periodic networks (TPNs), like the well-known gyroid and diamond network phases, abound in soft matter assemblies, from block copolymers (BCPs), lyotropic liquid crystals and surfactants to functional architectures in biology. While TPNs are, in reality, volume-filling patterns of spatially-varying molecular composition, physical and structural models most often reduce their structure to lower-dimensional geometric objects: the 2D interfaces between chemical domains; and the 1D skeletons that thread through inter-connected, tubular domains. These lower-dimensional structures provide a useful basis of comparison to idealized geometries based on triply-periodic minimal, or constant-mean curvature surfaces, and shed important light on the spatially heterogeneous packing of molecular constituents that form the networks. Here, we propose a simple, efficient and flexible method to extract a 1D skeleton from 3D volume composition data of self-assembled networks. We apply this method to both self-consistent field theory predictions as well as experimental electron microtomography reconstructions of the double-gyroid phase of an ABA triblock copolymer. We further demonstrate how the analysis of 1D skeleton, 2D inter-domain surfaces, and combinations therefore, provide physical and structural insight into TPNs, across multiple length scales. Specifically, we propose and compare simple measures of network chirality as well as domain thickness, and analyze their spatial and statistical distributions in both ideal (theoretical) and non-ideal (experimental) double gyroid assemblies.

Structure of the C-terminal domain of nsp4 from feline coronavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh

2009-08-01

The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes.more » In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.« less

Membrane Microdomain Structures of Liposomes and Their Contribution to the Cellular Uptake Efficiency into HeLa Cells.

PubMed

Onuki, Yoshinori; Obata, Yasuko; Kawano, Kumi; Sano, Hiromu; Matsumoto, Reina; Hayashi, Yoshihiro; Takayama, Kozo

2016-02-01

The purpose of this study is to obtain a comprehensive relationship between membrane microdomain structures of liposomes and their cellular uptake efficiency. Model liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (Ch) were prepared with various lipid compositions. To detect distinct membrane microdomains in the liposomes, fluorescence-quenching assays were performed at temperatures ranging from 25 to 60 °C using 1,6-diphenyl-1,3,5-hexatriene-labeled liposomes and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl. From the data analysis using the response surface method, we gained a better understanding of the conditions for forming distinct domains (Lo, Ld, and gel phase membranes) as a function of lipid composition. We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its essence. As a result, DPPC/DOPC/Ch liposomes in any lipid composition were integrated into five distinct clusters in terms of similarity of the domain structure. In addition, the findings from synchrotron small-angle X-ray scattering analysis offered further insight into the domain structures. As a last phase of this study, an in vitro cellular uptake study using HeLa cells was conducted using SOM clusters' liposomes with/without PEGylation. As a consequence of this study, higher cellular uptake was observed from liposomes having Ch-rich ordered domains.

Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Misono, K. S.; Philo, J. S.; Arakawa, T.

2011-06-01

Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures ofmore » the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.« less

Consolidation of glycosyl hydrolase family 30 : a dual domain 4/7 hydrolase family consisting of two structurally distinct groups

Treesearch

Franz J. St John; Javier M. Gonzalez; Edwin Pozharski

2010-01-01

In this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement...

Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.

PubMed

Solomon, Hodaya V; Tabachnikov, Orly; Lansky, Shifra; Salama, Rachel; Feinberg, Hadar; Shoham, Yuval; Shoham, Gil

2015-12-01

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moore, J.; Hendrickson, W

Histidine kinase receptors respond to diverse signals and mediate signal transduction across the plasma membrane in all prokaryotes and certain eukaryotes. Each receptor is part of a two-component system that regulates a particular cellular process. Organisms that use trimethylamine-N-oxide (TMAO) as a terminal electron acceptor typically control their anaerobic respiration through the TMAO reductase (Tor) pathway, which the TorS histidine kinase activates when sensing TMAO in the environment. We have determined crystal structures for the periplasmic sensor domains of TorS receptors from Escherichia coli and Vibrio parahaemolyticus. TorS sensor domains have a novel fold consisting of a membrane-proximal right-handed four-helicalmore » bundle and a membrane-distal left-handed four-helical bundle, but conformational dispositions differ significantly in the two structures. Isolated TorS sensor domains dimerize in solution; and from comparisons with dimeric NarX and Tar sensors, we postulate that signaling through TorS dimers involves a piston-type displacement between helices.« less

Aeroelastic Modeling of X-56A Stiff-Wing Configuration Flight Test Data

NASA Technical Reports Server (NTRS)

Grauer, Jared A.; Boucher, Matthew J.

2017-01-01

Aeroelastic stability and control derivatives for the X-56A Multi-Utility Technology Testbed (MUTT), in the stiff-wing configuration, were estimated from flight test data using the output-error method. Practical aspects of the analysis are discussed. The orthogonal phase-optimized multisine inputs provided excellent data information for aeroelastic modeling. Consistent parameter estimates were determined using output error in both the frequency and time domains. The frequency domain analysis converged faster and was less sensitive to starting values for the model parameters, which was useful for determining the aeroelastic model structure and obtaining starting values for the time domain analysis. Including a modal description of the structure from a finite element model reduced the complexity of the estimation problem and improved the modeling results. Effects of reducing the model order on the short period stability and control derivatives were investigated.

Electroformation of Janus and patchy capsules

NASA Astrophysics Data System (ADS)

Rozynek, Zbigniew; Mikkelsen, Alexander; Dommersnes, Paul; Fossum, Jon Otto

2014-05-01

Janus and patchy particles have designed heterogeneous surfaces that consist of two or several patches with different materials properties. These particles are emerging as building blocks for a new class of soft matter and functional materials. Here we introduce a route for forming heterogeneous capsules by producing highly ordered jammed colloidal shells of various shapes with domains of controlled size and composition. These structures combine the functionalities offered by Janus or patchy particles, and those given by permeable shells such as colloidosomes. The simple assembly route involves the synergetic action of electro-hydrodynamic flow and electro-coalescence. We demonstrate that the method is robust and straightforwardly extendable to production of multi-patchy capsules. This forms a starting point for producing patchy colloidosomes with domains of anisotropic chemical surface properties, permeability or mixed liquid-solid phase domains, which could be exploited to produce functional emulsions, light and hollow supra-colloidosome structures, or scaffolds.

Chimera States in Continuous Media: Existence and Distinctness

NASA Astrophysics Data System (ADS)

Nicolaou, Zachary G.; Riecke, Hermann; Motter, Adilson E.

2017-12-01

The defining property of chimera states is the coexistence of coherent and incoherent domains in systems that are structurally and spatially homogeneous. The recent realization that such states might be common in oscillator networks raises the question of whether an analogous phenomenon can occur in continuous media. Here, we show that chimera states can exist in continuous systems even when the coupling is strictly local, as in many fluid and pattern forming media. Using the complex Ginzburg-Landau equation as a model system, we characterize chimera states consisting of a coherent domain of a frozen spiral structure and an incoherent domain of amplitude turbulence. We show that in this case, in contrast with discrete network systems, fluctuations in the local coupling field play a crucial role in limiting the coherent regions. We suggest these findings shed light on new possible forms of coexisting order and disorder in fluid systems.

Estrogen receptor of primary breast cancers: evidence for intracellular proteolysis.

PubMed

Maaroufi, Y; Lacroix, M; Lespagnard, L; Journé, F; Larsimont, D; Leclercq, G

2000-01-01

Iodinated oestradiol-labeled oestrogen receptor (ER) isoforms devoid of amino-terminal ABC domains represent about two-thirds of the whole receptor population detected in cytosol samples from human breast cancers. This high frequency could not be ascribed to the expression of truncated mRNAs, or to the proteolysis of the native ER peptide at the time of homogenization or assay, suggesting an intracellular proteolysis. Free amino-terminal and ligand-binding domains maintained together within oligomeric structure(s); increase of ionic strength separated them. The amino-terminal region was consistently detected in the cell nucleus by specific immunohistochemistry leading to the concept of a potential intranuclear association between ER cleavage products and/or other regulatory proteins.

Structure and Function of the Haemophilus influenzae Autotransporters

PubMed Central

Spahich, Nicole A.; St. Geme, Joseph W.

2011-01-01

Autotransporters are a large class of proteins that are found in the outer membrane of Gram-negative bacteria and are almost universally implicated in virulence. These proteins consist of a C-terminal β-domain that is embedded in the outer membrane and an N-terminal domain that is exposed on the bacterial surface and is endowed with effector function. In this article, we review and compare the structural and functional characteristics of the Haemophilus influenzae IgA1 protease and Hap monomeric autotransporters and the H. influenzae Hia and Hsf trimeric autotransporters. All of these proteins play a role in colonization of the upper respiratory tract and in the pathogenesis of H. influenzae disease. PMID:22919571

Parametric effects of syntactic-semantic conflict in Broca's area during sentence processing.

PubMed

Thothathiri, Malathi; Kim, Albert; Trueswell, John C; Thompson-Schill, Sharon L

2012-03-01

The hypothesized role of Broca's area in sentence processing ranges from domain-general executive function to domain-specific computation that is specific to certain syntactic structures. We examined this issue by manipulating syntactic structure and conflict between syntactic and semantic cues in a sentence processing task. Functional neuroimaging revealed that activation within several Broca's area regions of interest reflected the parametric variation in syntactic-semantic conflict. These results suggest that Broca's area supports sentence processing by mediating between multiple incompatible constraints on sentence interpretation, consistent with this area's well-known role in conflict resolution in other linguistic and non-linguistic tasks. Copyright © 2011 Elsevier Inc. All rights reserved.

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry.

PubMed

Martin, Douglas S; Fathi, Reza; Mitchison, Timothy J; Gelles, Jeff

2010-03-23

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."

3D Structure and Interaction of p24β and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport.

PubMed

Nagae, Masamichi; Hirata, Tetsuya; Morita-Matsumoto, Kana; Theiler, Romina; Fujita, Morihisa; Kinoshita, Taroh; Yamaguchi, Yoshiki

2016-10-09

The p24 family consists of four subfamilies (p24α, p24β, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (K d = ~10 -4 M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deformation Partitioning: The Missing Link Between Outcrop-Scale Observations And Orogen-Scale Processes

NASA Astrophysics Data System (ADS)

Attia, S.; Paterson, S. R.; Jiang, D.; Miller, R. B.

2017-12-01

Structural studies of orogenic deformation fields are mostly based on small-scale structures ubiquitous in field exposures, hand samples, and under microscopes. Relating deformation histories derived from such structures to changing lithospheric-scale deformation and boundary conditions is not trivial due to vast scale separation (10-6 107 m) between characteristic lengths of small-scale structures and lithospheric plates. Rheological heterogeneity over the range of orogenic scales will lead to deformation partitioning throughout intervening scales of structural development. Spectacular examples of structures documenting deformation partitioning are widespread within hot (i.e., magma-rich) orogens such as the well-studied central Sierra Nevada and Cascades core of western North America: (1) deformation partitioned into localized, narrow, triclinic shear zones separated by broad domains of distributed pure shear at micro- to 10 km scales; (2) deformation partitioned between plutons and surrounding metamorphic host rocks as shown by pluton-wide magmatic fabrics consistently oriented differently than coeval host rock fabrics; (3) partitioning recorded by different fabric intensities, styles, and orientations established from meter-scale grid mapping to 100 km scale domainal analyses; and (4) variations in the causes of strain and kinematics within fold-dominated domains. These complex, partitioned histories require synthesized mapping, geochronology, and structural data at all scales to evaluate partitioning and in the absence of correct scaling can lead to incorrect interpretations of histories. Forward modeling capable of addressing deformation partitioning in materials containing multiple scales of rheologically heterogeneous elements of varying characteristic lengths provides the ability to upscale the large synthesized datasets described above to plate-scale tectonic processes and boundary conditions. By comparing modeling predictions from the recently developed self-consistent Multi-Order Power-Law Approach (MOPLA) to multi-scale field observations, we constrain likely paleo-tectonic controls of orogenic structural evolution rather than predicting a unique, but likely incorrect deformation history.

Role of the Dc domain of the bacterial hook protein FlgE in hook assembly and function

PubMed Central

Moriya, Nao; Minamino, Tohru; Ferris, Hedda U.; Morimoto, Yusuke V.; Ashihara, Masamichi; Kato, Takayuki; Namba, Keiichi

2013-01-01

The bacterial flagellar hook acts as a universal joint to smoothly transmit torque produced by the motor to the filament. The hook protein FlgE assembles into a 55 nm tubular structure with the help of the hook cap (FlgD). FlgE consists of four domains, D0, Dc, D1 and D2, arranged from the inner to the outer part of the tubular structure of the hook. The Dc domain contributes to the structural stability of the hook, but it is unclear how this Dc domain is responsible for the universal joint mechanism. Here, we carried out a deletion analysis of the FlgE Dc domain. FlgEΔ4/5 with deletion of residues 30 to 49 was not secreted into the culture media. FlgEΔ5 and FlgEΔ6 with deletions of residues 40 to 49 and 50 to 59, respectively, still formed hooks, allowing the export apparatus to export the hook-filament junction proteins FlgK and FlgL and flagellin FliC. However, these deletions inhibited the replacement of the FlgD hook cap by FlgK at the hook tip, thereby abolishing filament formation. Deletion of residues 50 to 59 significantly affected hook morphology. These results suggest that the Dc domain is responsible not only for hook assembly but also for FlgE export, the interaction with FlgK, and the polymorphic supercoiling mechanism of the hook. PMID:27493542

Separate but correlated: The latent structure of space and mathematics across development.

PubMed

Mix, Kelly S; Levine, Susan C; Cheng, Yi-Ling; Young, Chris; Hambrick, D Zachary; Ping, Raedy; Konstantopoulos, Spyros

2016-09-01

The relations among various spatial and mathematics skills were assessed in a cross-sectional study of 854 children from kindergarten, third, and sixth grades (i.e., 5 to 13 years of age). Children completed a battery of spatial mathematics tests and their scores were submitted to exploratory factor analyses both within and across domains. In the within domain analyses, all of the measures formed single factors at each age, suggesting consistent, unitary structures across this age range. Yet, as in previous work, the 2 domains were highly correlated, both in terms of overall composite score and pairwise comparisons of individual tasks. When both spatial and mathematics scores were submitted to the same factor analysis, the 2 domain specific factors again emerged, but there also were significant cross-domain factor loadings that varied with age. Multivariate regressions replicated the factor analysis and further revealed that mental rotation was the best predictor of mathematical performance in kindergarten, and visual-spatial working memory was the best predictor of mathematical performance in sixth grade. The mathematical tasks that predicted the most variance in spatial skill were place value (K, 3rd, 6th), word problems (3rd, 6th), calculation (K), fraction concepts (3rd), and algebra (6th). Thus, although spatial skill and mathematics each have strong internal structures, they also share significant overlap, and have particularly strong cross-domain relations for certain tasks. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

PubMed

Draeger, Christian; Ndinyanka Fabrice, Tohnyui; Gineau, Emilie; Mouille, Grégory; Kuhn, Benjamin M; Moller, Isabel; Abdou, Marie-Therese; Frey, Beat; Pauly, Markus; Bacic, Antony; Ringli, Christoph

2015-06-24

Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

Use of a Molecular Decoy to Segregate Transport from Antigenicity in the FrpB Iron Transporter from Neisseria meningitidis

PubMed Central

Saleem, Muhammad; Prince, Stephen M.; Rigby, Stephen E. J.; Imran, Muhammad; Patel, Hema; Chan, Hannah; Sanders, Holly; Maiden, Martin C. J.; Feavers, Ian M.; Derrick, Jeremy P.

2013-01-01

FrpB is an outer membrane transporter from Neisseria meningitidis, the causative agent of meningococcal meningitis. It is a member of the TonB-dependent transporter (TBDT) family and is responsible for iron uptake into the periplasm. FrpB is subject to a high degree of antigenic variation, principally through a region of hypervariable sequence exposed at the cell surface. From the crystal structures of two FrpB antigenic variants, we identify a bound ferric ion within the structure which induces structural changes on binding which are consistent with it being the transported substrate. Binding experiments, followed by elemental analysis, verified that FrpB binds Fe3+ with high affinity. EPR spectra of the bound Fe3+ ion confirmed that its chemical environment was consistent with that observed in the crystal structure. Fe3+ binding was reduced or abolished on mutation of the Fe3+-chelating residues. FrpB orthologs were identified in other Gram-negative bacteria which showed absolute conservation of the coordinating residues, suggesting the existence of a specific TBDT sub-family dedicated to the transport of Fe3+. The region of antigenic hypervariability lies in a separate, external sub-domain, whose structure is conserved in both the F3-3 and F5-1 variants, despite their sequence divergence. We conclude that the antigenic sub-domain has arisen separately as a result of immune selection pressure to distract the immune response from the primary transport function. This would enable FrpB to function as a transporter independently of antibody binding, by using the antigenic sub-domain as a ‘molecular decoy’ to distract immune surveillance. PMID:23457610

Use of a molecular decoy to segregate transport from antigenicity in the FrpB iron transporter from Neisseria meningitidis.

PubMed

Saleem, Muhammad; Prince, Stephen M; Rigby, Stephen E J; Imran, Muhammad; Patel, Hema; Chan, Hannah; Sanders, Holly; Maiden, Martin C J; Feavers, Ian M; Derrick, Jeremy P

2013-01-01

FrpB is an outer membrane transporter from Neisseria meningitidis, the causative agent of meningococcal meningitis. It is a member of the TonB-dependent transporter (TBDT) family and is responsible for iron uptake into the periplasm. FrpB is subject to a high degree of antigenic variation, principally through a region of hypervariable sequence exposed at the cell surface. From the crystal structures of two FrpB antigenic variants, we identify a bound ferric ion within the structure which induces structural changes on binding which are consistent with it being the transported substrate. Binding experiments, followed by elemental analysis, verified that FrpB binds Fe(3+) with high affinity. EPR spectra of the bound Fe(3+) ion confirmed that its chemical environment was consistent with that observed in the crystal structure. Fe(3+) binding was reduced or abolished on mutation of the Fe(3+)-chelating residues. FrpB orthologs were identified in other Gram-negative bacteria which showed absolute conservation of the coordinating residues, suggesting the existence of a specific TBDT sub-family dedicated to the transport of Fe(3+). The region of antigenic hypervariability lies in a separate, external sub-domain, whose structure is conserved in both the F3-3 and F5-1 variants, despite their sequence divergence. We conclude that the antigenic sub-domain has arisen separately as a result of immune selection pressure to distract the immune response from the primary transport function. This would enable FrpB to function as a transporter independently of antibody binding, by using the antigenic sub-domain as a 'molecular decoy' to distract immune surveillance.

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding

PubMed Central

Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.

2015-01-01

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C)more » domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.« less

Producing genome structure populations with the dynamic and automated PGS software.

PubMed

Hua, Nan; Tjong, Harianto; Shin, Hanjun; Gong, Ke; Zhou, Xianghong Jasmine; Alber, Frank

2018-05-01

Chromosome conformation capture technologies such as Hi-C are widely used to investigate the spatial organization of genomes. Because genome structures can vary considerably between individual cells of a population, interpreting ensemble-averaged Hi-C data can be challenging, in particular for long-range and interchromosomal interactions. We pioneered a probabilistic approach for the generation of a population of distinct diploid 3D genome structures consistent with all the chromatin-chromatin interaction probabilities from Hi-C experiments. Each structure in the population is a physical model of the genome in 3D. Analysis of these models yields new insights into the causes and the functional properties of the genome's organization in space and time. We provide a user-friendly software package, called PGS, which runs on local machines (for practice runs) and high-performance computing platforms. PGS takes a genome-wide Hi-C contact frequency matrix, along with information about genome segmentation, and produces an ensemble of 3D genome structures entirely consistent with the input. The software automatically generates an analysis report, and provides tools to extract and analyze the 3D coordinates of specific domains. Basic Linux command-line knowledge is sufficient for using this software. A typical running time of the pipeline is ∼3 d with 300 cores on a computer cluster to generate a population of 1,000 diploid genome structures at topological-associated domain (TAD)-level resolution.

The Design and Development of a Potent and Selective Novel Diprolyl Derivative That Binds to the N-Domain of Angiotensin-I Converting Enzyme.

PubMed

Fienberg, Stephen; Cozier, Gyles E; Acharya, K Ravi; Chibale, Kelly; Sturrock, Edward D

2018-01-11

Angiotensin-I converting enzyme (ACE) is a zinc metalloprotease consisting of two catalytic domains (N- and C-). Most clinical ACE inhibitor(s) (ACEi) have been shown to inhibit both domains nonselectively, resulting in adverse effects such as cough and angioedema. Selectively inhibiting the individual domains is likely to reduce these effects and potentially treat fibrosis in addition to hypertension. ACEi from the GVK Biosciences database were inspected for possible N-domain selective binding patterns. From this set, a diprolyl chemical series was modeled using docking simulations. The series was expanded based on key target interactions involving residues known to impart N-domain selectivity. In total, seven diprolyl compounds were synthesized and tested for N-domain selective ACE inhibition. One compound with an aspartic acid in the P 2 position (compound 16) displayed potent inhibition (K i = 11.45 nM) and was 84-fold more selective toward the N-domain. A high-resolution crystal structure of compound 16 in complex with the N-domain revealed the molecular basis for the observed selectivity.

Structural insights into the multifunctional protein VP3 of birnaviruses.

PubMed

Casañas, Arnau; Navarro, Aitor; Ferrer-Orta, Cristina; González, Dolores; Rodríguez, José F; Verdaguer, Núria

2008-01-01

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92-220, consisting of two alpha-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors.

Polarized light microscopy study on the reentrant phase transition in a (Ba 1–xK x)Fe 2As 2 single crystal with x = 0.24

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yong; Tanatar, Makariy A.; Timmons, Erik

In this study, a sequence of structural/magnetic transitions on cooling is reported in the literature for hole-doped iron-based superconductor (Ba 1–xK x)Fe 2As 2 with x = 0.24. By using polarized light microscopy, we directly observe the formation of orthorhombic domains in (Ba 1–xK x)Fe 2As 2 (x = 0.24) single crystal below a temperature of simultaneous structural/magnetic transition T N ~ 80 K. The structural domains vanish below ~30 K, but reappear below T = 15 K. Our results are consistent with reentrance transformation sequence from high-temperature tetragonal (HTT) to low temperature orthorhombic (LTO1) structure at T N ~more » 80 K, LTO1 to low temperature tetragonal (LTT) structure at T c ~ 25 K, and LTT to low temperature orthorhombic (LTO2) structure at T ~ 15 K.« less

Polarized light microscopy study on the reentrant phase transition in a (Ba 1–xK x)Fe 2As 2 single crystal with x = 0.24

DOE PAGES

Liu, Yong; Tanatar, Makariy A.; Timmons, Erik; ...

2016-11-09

In this study, a sequence of structural/magnetic transitions on cooling is reported in the literature for hole-doped iron-based superconductor (Ba 1–xK x)Fe 2As 2 with x = 0.24. By using polarized light microscopy, we directly observe the formation of orthorhombic domains in (Ba 1–xK x)Fe 2As 2 (x = 0.24) single crystal below a temperature of simultaneous structural/magnetic transition T N ~ 80 K. The structural domains vanish below ~30 K, but reappear below T = 15 K. Our results are consistent with reentrance transformation sequence from high-temperature tetragonal (HTT) to low temperature orthorhombic (LTO1) structure at T N ~more » 80 K, LTO1 to low temperature tetragonal (LTT) structure at T c ~ 25 K, and LTT to low temperature orthorhombic (LTO2) structure at T ~ 15 K.« less

1.55 Å resolution X-ray crystal structure of Rv3902c from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, Bharat G.; Moates, Derek B.; Kim, Heung-Bok

The 1.55 Å resolution X-ray crystal structure of Rv3902c from M. tuberculosis reveals a novel fold. The crystallographic structure of the Mycobacterium tuberculosis (TB) protein Rv3902c (176 residues; molecular mass of 19.8 kDa) was determined at 1.55 Å resolution. The function of Rv3902c is unknown, although several TB genes involved in bacterial pathogenesis are expressed from the operon containing the Rv3902c gene. The unique structural fold of Rv3902c contains two domains, each consisting of antiparallel β-sheets and α-helices, creating a hand-like binding motif with a small binding pocket in the palm. Structural homology searches reveal that Rv3902c has an overallmore » structure similar to that of the Salmonella virulence-factor chaperone InvB, with an r.m.s.d. for main-chain atoms of 2.3 Å along an aligned domain.« less

Structural Insight into the Assembly of TRPV Channels

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Chakrapani, Sudha; Holdaway, Heather A.; Stewart, Phoebe L.; Moiseenkova-Bell, Vera Y.

2017-01-01

SUMMARY Transient receptor potential (TRP) proteins are a large family of polymodal nonselective cation channels. The TRP vanilloid (TRPV) subfamily consists of six homologous members with diverse functions. TRPV1–TRPV4 are nonselective cation channels proposed to play a role in nociception, while TRPV5 and TRPV6 are involved in epithelial Ca2+ homeostasis. Here we present the cryo-electron microscopy (cryo-EM) structure of functional, full-length TRPV2 at 13.6 Å resolution. The map reveals that the TRPV2 cytoplasmic domain displays a 4-fold petal-like shape in which high-resolution N-terminal ankyrin repeat domain (ARD) structures can be unambiguously fitted. Fitting of the available ARD structures for other TRPV subfamily members into the TRPV2 EM map suggests that TRPV subfamily members have highly homologous structural topologies. These results allowed us to postulate a structural explanation for the functional diversity among TRPV channels and their differential regulation by proteins and ligands. PMID:24373766

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system

PubMed Central

Zoltner, Martin; Ng, Wui M.A.V.; Money, Jillian J.; Fyfe, Paul K.; Kneuper, Holger; Palmer, Tracy; Hunter, William N.

2016-01-01

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein. PMID:27130157

Examination of the factor structure of the Schizotypal Personality Questionnaire among British and Trinidadian adults.

PubMed

Barron, David; Swami, Viren; Towell, Tony; Hutchinson, Gerard; Morgan, Kevin D

2015-01-01

Much debate in schizotypal research has centred on the factor structure of the Schizotypal Personality Questionnaire (SPQ), with research variously showing higher-order dimensionality consisting of two to seven dimensions. In addition, cross-cultural support for the stability of those factors remains limited. Here, we examined the factor structure of the SPQ among British and Trinidadian adults. Participants from a White British subsample (n = 351) resident in the UK and from an African Caribbean subsample (n = 284) resident in Trinidad completed the SPQ. The higher-order factor structure of the SPQ was analysed through confirmatory factor analysis, followed by multiple-group analysis for the model of best fit. Between-group differences for sex and ethnicity were investigated using multivariate analysis of variance in relation to the higher-order domains. The model of best-fit was the four-factor structure, which demonstrated measurement invariance across groups. Additionally, these data had an adequate fit for two alternative models: (a) 3-factor and (b) modified 4-factor model. The British subsample had significantly higher scores across all domains than the Trinidadian group, and men scored significantly higher on the disorganised domain than women. The four-factor structure received confirmatory support and, importantly, support for use with populations varying in ethnicity and culture.

Properties and structure of a low-potential, penta-heme cytochrome c 552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum.

PubMed

Chen, Jing-Hua; Yu, Long-Jiang; Boussac, Alain; Wang-Otomo, Zheng-Yu; Kuang, Tingyun; Shen, Jian-Ren

2018-04-24

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c 552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c 552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

The TDP-43 N-terminal domain structure at high resolution.

PubMed

Mompeán, Miguel; Romano, Valentina; Pantoja-Uceda, David; Stuani, Cristiana; Baralle, Francisco E; Buratti, Emanuele; Laurents, Douglas V

2016-04-01

Transactive response DNA-binding protein 43 kDa (TDP-43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C-terminal domain of this protein plays a key role in mediating this process. However, the N-terminal domain (residues 1-77) is needed to effectively recruit TDP-43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete (1) H, (15) N and (13) C NMR assignments and the structure of the N-terminal domain determined on the basis of 26 hydrogen-bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an α-helix and six β-strands. Two β-strands form a β-hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25 °C. At 4 kcal·mol(-1) , the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcal·mol(-1) ). The β-strands, α-helix, and three of four turns are generally rigid, although the loop formed by residues 47-53 is mobile, as determined by model-free analysis of the (15) N{(1) H}NOE, as well as the translational and transversal relaxation rates. Structural data have been deposited in the Protein Data Bank under accession code: 2n4p. The NMR assignments have been deposited in the BMRB database under access code: 25675. © 2016 Federation of European Biochemical Societies.

Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity.

PubMed

Muto, Shinsuke; Senda, Miki; Akai, Yusuke; Sato, Lui; Suzuki, Toru; Nagai, Ryozo; Senda, Toshiya; Horikoshi, Masami

2007-03-13

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

PubMed Central

Papandreou, Nikolaos; Chomilier, Jacques

2008-01-01

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0083-8) contains supplementary material, which is available to authorized users. PMID:18987995

Beltless translocation domain of botulinum neurotoxin A embodies a minimum ion-conductive channel.

PubMed

Fischer, Audrey; Sambashivan, Shilpa; Brunger, Axel T; Montal, Mauricio

2012-01-13

Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the "beltless" TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian

2011-09-06

A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the othermore » with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.« less

Measuring cancer caregiver health literacy: Validation of the Health Literacy of Caregivers Scale-Cancer (HLCS-C) in an Australian population.

PubMed

Yuen, Eva; Knight, Tess; Dodson, Sarity; Chirgwin, Jacqueline; Busija, Lucy; Ricciardelli, Lina A; Burney, Susan; Parente, Phillip; Livingston, Patricia M

2018-05-01

Caregivers have been largely neglected in health literacy measurement. We assess the construct validity, and internal consistency of the Health Literacy of Caregivers Scale-Cancer (HLCS-C), and present a revised, psychometrically robust scale. Using data from 297 cancer caregivers (12.4% response rate) recruited from Melbourne, Australia between January-July 2014, confirmatory factor analysis (CFA) was conducted to evaluate the HLCS-C's proposed factor structure. Items were evaluated for: item difficulty, unidimensionality and overall item fit within their domain. Item-threshold-ordering was examined though one-parameter Item Response Theory models. Internal consistency was assessed using Raykov's reliability coefficient. CFA results identified 42 poorly performing/redundant items which were subsequently removed. A 10-factor model was fitted to 46 acceptable items with no correlated residuals or factor cross-loadings accepted. Adequate fit was revealed (χ 2 WLSMV  = 1463.807[df = 944], p < .001, RMSEA = 0.043, CFI = 0.980, TLI = 0.978, WRMR = 1.00). Ten domains were identified: Proactivity and determination to seek information; Adequate information about cancer and cancer management; Supported by healthcare providers (HCP) to understand information; Social support; Cancer-related communication with the care recipient (CR); Understanding CR needs and preferences; Self-care; Understanding the healthcare system; Capacity to process health information; and Active engagement with HCP. Internal consistency was adequate across domains (0.78-0.92). The revised HLCS-C demonstrated good structural, convergent, and discriminant validity, and high internal consistency. The scale may be useful for the development and evaluation of caregiver interventions. © 2017 John Wiley & Sons Ltd.

Quantitative three-dimensional characterization of block copolymer directed self-assembly on combined chemical and topographical prepatterned templates

DOE PAGES

Segal-Peretz, Tamar; Ren, Jiaxing; Xiong, Shisheng; ...

2016-12-22

Characterization of the three-dimensional (3D) structure in directed self-assembly (DSA) of block copolymers is crucial for understanding the complex relationships between the guiding template and the resulting polymer structure so DSA could be successfully implemented for advanced lithography applications. Here, we combined scanning transmission electron microscopy (STEM) tomography and coarse-grain simulations to probe the 3D structure of P2VP- b-PS- b-P2VP assembled on prepatterned templates using solvent vapor annealing. The templates consisted of nonpreferential background and raised guiding stripes that had PS-preferential top surfaces and P2VP-preferential sidewalls. The full 3D characterization allowed us to quantify the shape of the polymer domainsmore » and the interface between domains as a function of depth in the film and template geometry and offered important insights that were not accessible with 2D metrology. Sidewall guiding was advantageous in promoting the alignment and lowering the roughness of the P2VP domains over the sidewalls, but incommensurate confinement from the increased topography could cause roughness and intermittent dislocations in domains over the background region at the bottom of the film. The 3D characterization of bridge structures between domains over the background and breaks within domains on guiding lines sheds light on possible origins of common DSA defects. The positional fluctuations of the PS/P2VP interface between domains showed a depth-dependent behavior, with high levels of fluctuations near both the free surface of the film and the substrate and lower fluctuation levels in the middle of the film. As a result, this research demonstrates how 3D characterization offers a better understanding of DSA processes, leading to better design and fabrication of directing templates.« less

Structure of the glucagon receptor in complex with a glucagon analogue.

PubMed

Zhang, Haonan; Qiao, Anna; Yang, Linlin; Van Eps, Ned; Frederiksen, Klaus S; Yang, Dehua; Dai, Antao; Cai, Xiaoqing; Zhang, Hui; Yi, Cuiying; Cao, Can; He, Lingli; Yang, Huaiyu; Lau, Jesper; Ernst, Oliver P; Hanson, Michael A; Stevens, Raymond C; Wang, Ming-Wei; Reedtz-Runge, Steffen; Jiang, Hualiang; Zhao, Qiang; Wu, Beili

2018-01-03

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

Atomic interaction networks in the core of protein domains and their native folds.

PubMed

Soundararajan, Venkataramanan; Raman, Rahul; Raguram, S; Sasisekharan, V; Sasisekharan, Ram

2010-02-23

Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be "signature" of a domain's native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1-2 angstroms (mean 1.61A) C(alpha) RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the 'twilight' and 'midnight' zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence-identity-independent applications demonstrated in this work, we suggest that the PCAIN is a fundamental fold feature that could be a valuable addition to the arsenal of protein modeling and analysis tools.

Atomic Interaction Networks in the Core of Protein Domains and Their Native Folds

PubMed Central

Soundararajan, Venkataramanan; Raman, Rahul; Raguram, S.; Sasisekharan, V.; Sasisekharan, Ram

2010-01-01

Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be “signature” of a domain's native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1–2 angstroms (mean 1.61A) Cα RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the ‘twilight’ and ‘midnight’ zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence-identity-independent applications demonstrated in this work, we suggest that the PCAIN is a fundamental fold feature that could be a valuable addition to the arsenal of protein modeling and analysis tools. PMID:20186337

Solution structure of the C-terminal domain of Ole e 9, a major allergen of olive pollen

PubMed Central

Treviño, Miguel Á.; Palomares, Oscar; Castrillo, Inés; Villalba, Mayte; Rodríguez, Rosalía; Rico, Manuel; Santoro, Jorge; Bruix, Marta

2008-01-01

Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-β-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-β-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel α-helices forming an angle of ∼55°, a small antiparallel β-sheet with two short strands, and a 3–10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure–allergenicity relationships. PMID:18096638

The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin

2010-07-20

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar tomore » the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.« less

Local conductance: A means to extract polarization and depolarizing fields near domain walls in ferroelectrics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Douglas, A. M.; Kumar, A.; Gregg, J. M.

Conducting atomic force microscopy images of bulk semiconducting BaTiO{sub 3} surfaces show clear stripe domain contrast. High local conductance correlates with strong out-of-plane polarization (mapped independently using piezoresponse force microscopy), and current-voltage characteristics are consistent with dipole-induced alterations in Schottky barriers at the metallic tip-ferroelectric interface. Indeed, analyzing current-voltage data in terms of established Schottky barrier models allows relative variations in the surface polarization, and hence the local domain structure, to be determined. Fitting also reveals the signature of surface-related depolarizing fields concentrated near domain walls. Domain information obtained from mapping local conductance appears to be more surface-sensitive than thatmore » from piezoresponse force microscopy. In the right materials systems, local current mapping could therefore represent a useful complementary technique for evaluating polarization and local electric fields with nanoscale resolution.« less

Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization

PubMed Central

Gao, Yong-Gui; Suzuki, Hiroaki; Itou, Hiroshi; Zhou, Yong; Tanaka, Yoshikazu; Wachi, Masaaki; Watanabe, Nobuhisa; Tanaka, Isao; Yao, Min

2008-01-01

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed. PMID:18988622

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S-adenosyl-l-methionine-dependent methyltransferase.

PubMed

Im, Ha Na; Kim, Hyoun Sook; An, Doo Ri; Jang, Jun Young; Kim, Jieun; Yoon, Hye-Jin; Yang, Jin Kuk; Suh, Se Won

2016-03-01

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site. Copyright © 2016 Elsevier Inc. All rights reserved.

Lorentz force in water: evidence that hydronium cyclotron resonance enhances polymorphism.

PubMed

D'Emilia, E; Giuliani, L; Lisi, A; Ledda, M; Grimaldi, S; Montagnier, L; Liboff, A R

2015-01-01

There is an ongoing question regarding the structure forming capabilities of water at ambient temperatures. To probe for different structures, we studied effects in pure water following magnetic field exposures corresponding to the ion cyclotron resonance of H3O(+). Included were measurements of conductivity and pH. We find that under ion cyclotron resonance (ICR) stimulation, water undergoes a transition to a form that is hydroxonium-like, with the subsequent emission of a transient 48.5 Hz magnetic signal, in the absence of any other measurable field. Our results indicate that hydronium resonance stimulation alters the structure of water, enhancing the concentration of EZ-water. These results are not only consistent with Del Giudice's model of electromagnetically coherent domains, but they can also be interpreted to show that these domains exist in quantized spin states.

Interplay between I308 and Y310 residues in the third repeat of microtubule-binding domain is essential for tau filament formation.

PubMed

Naruto, Keiko; Minoura, Katsuhiko; Okuda, Ryouhei; Taniguchi, Taizo; In, Yasuko; Ishida, Toshimasa; Tomoo, Koji

2010-10-08

Investigation of the mechanism of tau polymerization is indispensable for finding inhibitory conditions or identifying compounds preventing the formation of paired helical filament or oligomers. Tau contains a microtubule-binding domain consisting of three or four repeats in its C-terminal half. It has been considered that the key event in tau polymerization is the formation of a β-sheet structure arising from a short hexapeptide (306)VQIVYK(311) in the third repeat of tau. In this paper, we report for the first time that the C-H⋯π interaction between Ile308 and Tyr310 is the elemental structural scaffold essential for forming a dry "steric zipper" structure in tau amyloid fibrils. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Pikwitonei granulite domain: A lower crustal level along the Churchill-Superior boundary in central Manitoba

NASA Technical Reports Server (NTRS)

Weber, W.

1983-01-01

The greenschist to amphibolite facies tonalite-greenstone terrain of the Gods Lake subprovince grades - in a northwesterly direction - into the granulite facies Pikwitonei domain at the western margins of the Superior Province. The transition is the result of prograde metamorphism and takes place over 50 - 100 km without any structural or lithological breaks. Locally the orthopyroxene isograd is oblique to the structural grain and transects greenstone belts, e.g., the Cross Lake belt. The greenstone belts in the granulite facies and adjacent lower grade domain consist mainly of mafic and (minor) ultramafic metavolcanics, and clastic and chemical metasedimentary rocks. Typical for the greenstone belts crossed by the orthopyroxene isograd are anorthositic gabbros and anorthosites, and plagiophyric mafic flows. The Pikwitonei granulite domain has been interpreted as to represent a lower crustal level which was uplifted to the present level of erosion. On the basis of gravimetric data this uplift has been modelled as an obduction onto the Churchill Province during the Hudsonian orogeny, similar to the Ivrea Zone. The fault between the Churchill and Superior Province is described.

The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

PubMed Central

Dimitrov, S I; Russanova, V R; Pashev, I G

1987-01-01

The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber. Images Fig. 1. Fig. 2. PMID:2444434

Conformational entropic maps of functional coupling domains in GPCR activation: A case study with beta2 adrenergic receptor

NASA Astrophysics Data System (ADS)

Liu, Fan; Abrol, Ravinder; Goddard, William, III; Dougherty, Dennis

2014-03-01

Entropic effect in GPCR activation is poorly understood. Based on the recent solved structures, researchers in the GPCR structural biology field have proposed several ``local activating switches'' that consisted of a few number of conserved residues, but have long ignored the collective dynamical effect (conformational entropy) of a domain comprised of an ensemble of residues. A new paradigm has been proposed recently that a GPCR can be viewed as a composition of several functional coupling domains, each of which undergoes order-to-disorder or disorder-to-order transitions upon activation. Here we identified and studied these functional coupling domains by comparing the local entropy changes of each residue between the inactive and active states of the β2 adrenergic receptor from computational simulation. We found that agonist and G-protein binding increases the heterogeneity of the entropy distribution in the receptor. This new activation paradigm and computational entropy analysis scheme provides novel ways to design functionally modified mutant and identify new allosteric sites for GPCRs. The authors thank NIH and Sanofi for funding this project.

Intrinsic motions in the N-terminal domain of an ionotropic glutamate receptor detected by fluorescence correlation spectroscopy.

PubMed

Jensen, Mette H; Sukumaran, Madhav; Johnson, Christopher M; Greger, Ingo H; Neuweiler, Hannes

2011-11-18

Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Arc is a flexible modular protein capable of reversible self-oligomerization

PubMed Central

Myrum, Craig; Baumann, Anne; Bustad, Helene J.; Flydal, Marte Innselset; Mariaule, Vincent; Alvira, Sara; Cuéllar, Jorge; Haavik, Jan; Soulé, Jonathan; Valpuesta, José Maria; Márquez, José Antonio; Martinez, Aurora; Bramham, Clive R.

2015-01-01

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes. PMID:25748042

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bera, Asim K.; Aukema, Kelly G.; Elias, Mikael

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas themore » two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.« less

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacik, John-Paul; Mekasha, Sophanit; Forsberg, Zarah

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. We found that both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). Furthermore, in the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND 2 and ND –, suggesting a role for the copper ion inmore » shifting the pK a of the amino terminus.« less

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation

DOE PAGES

Bacik, John-Paul; Mekasha, Sophanit; Forsberg, Zarah; ...

2017-05-08

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. We found that both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). Furthermore, in the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND 2 and ND –, suggesting a role for the copper ion inmore » shifting the pK a of the amino terminus.« less

A pH-dependent conformational ensemble mediates proton transport through the influenza A/M2 protein†

PubMed Central

Polishchuk, Alexei L.; Lear, James D.; Ma, Chunlong; Lamb, Robert A.; Pinto, Lawrence H.; DeGrado, William F.

2010-01-01

The influenza A M2 protein exhibits inwardly rectifying, pH-activated proton transport that saturates at low pH. A comparison of high-resolution structures of the transmembrane domain at high and low pH suggests that pH-dependent conformational changes may facilitate proton conduction by alternately changing the accessibility of the N-terminal and C-terminal regions of the channel as a proton transits through the transmembrane domain. Here, we show that M2 functionally reconstituted in liposomes populates at least three different conformational states over a physiologically relevant pH range, with transition midpoints that are consistent with previously reported His37 pKas. We then develop and test two similar, quantitative mechanistic models of proton transport, where protonation shifts the equilibrium between structural states having different proton affinities and solvent accessibilities. The models account well for a collection of experimental data sets over a wide range of pHs and voltages and require only a small number of adjustable parameters to accurately describe the data. While the kinetic models do not require any specific conformation for the protein, they nevertheless are consistent with a large body of structural information based on high-resolution NMR and crystallographic structures, optical spectroscopy, and MD calculations. PMID:20968306

Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments.

PubMed

Hanson, Brendon J; Hong, Wanjin

2003-09-05

Sorting nexins (SNXs) are a growing family of proteins characterized by the presence of a PX domain. The PX domain mediates membrane association by interaction with phosphoinositides. The SNXs are generally believed to participate in membrane trafficking, but information regarding the function of individual proteins is limited. In this report, we describe the major characteristics of one member, SNX16. SNX16 is a novel 343-amino acid protein consisting of a central PX domain followed by a potential coiled-coil domain and a C-terminal region. Like other sorting nexins, SNX16 associates with the membrane via the PX domain which interacts with the phospholipid phosphatidylinositol 3-phosphate. We show via biochemical and cellular studies that SNX16 is distributed in both early and late endosome/lysosome structures. The coiled-coil domain is necessary for localization to the later endosomal structures, as mutant SNX16 lacking this domain was found only in early endosomes. Trafficking of internalized epidermal growth factor was also delayed by this SNX16 mutant, as these cells showed a delay in the segregation of epidermal growth factor in the early endosome for its delivery to later compartments. In addition, the coiled-coil domain is shown here to be important for homo-oligomerization of SNX16. Taken together, these results suggest that SNX16 is a sorting nexin that may function in the trafficking of proteins between the early and late endosomal compartments.

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

PubMed Central

Schäper, Simon; Steinchen, Wieland; Krol, Elizaveta; Altegoer, Florian; Skotnicka, Dorota; Bange, Gert; Becker, Anke

2017-01-01

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins. PMID:28559336

The X-ray Crystal Structures of Human {alpha}-Phosphomannomutase 1 Reveal the Structural Basis of Congenital Disorder of Glycosylation Type 1a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silvaggi,N.; Zhang, C.; Lu, Z.

2006-01-01

Carbohydrate-deficient glycoprotein syndrome type 1a (CDG-1a) is a congenital disease characterized by severe defects in nervous system development. It is caused by mutations in alpha -phosphomannomutase (of which there are two isozymes, {alpha}-PMM1 and {alpha}-PPM2). Here we report the X-ray crystal structures of human {alpha}-PMM1 in the open conformation, with and without the bound substrate, {alpha}-D-mannose 1-phosphate. {alpha}-PMM1, like most Haloalkanoic Acid Dehalogenase Superfamily (HADSF) members, consists of two domains, the cap and core, which open to bind substrate and then close to provide a solvent exclusive environment for catalysis. The substrate phosphate group is observed at a positively chargedmore » site of the cap domain, rather than at the core domain phosphoryl-transfer site defined by the D19 nucleophile and Mg{sup 2+} cofactor. This suggests that substrate binds first to the cap and then is swept into the active site upon cap closure. The orientation of the acid/base residue D21 suggests that {alpha}-PMM uses a different method of protecting the aspartylphosphate from hydrolysis than the HADSF member {beta}-phosphoglucomutase. It is hypothesized that the electrostatic repulsion of positive charges at the interface of the cap and core domains stabilizes {alpha}-PMM1 in the open conformation, and that the negatively charged substrate binds to the cap, thereby facilitating its closure over the core domain. The two isozymes {alpha}-PMM1 and {alpha}-PMM2 are shown to have a conserved active-site structure and to display similar kinetic properties. Analysis of the known mutation sites in the context of the structures reveals the genotype-phenotype relationship underlying CDG-1a.« less

Large-scale model quality assessment for improving protein tertiary structure prediction.

PubMed

Cao, Renzhi; Bhattacharya, Debswapna; Adhikari, Badri; Li, Jilong; Cheng, Jianlin

2015-06-15

Sampling structural models and ranking them are the two major challenges of protein structure prediction. Traditional protein structure prediction methods generally use one or a few quality assessment (QA) methods to select the best-predicted models, which cannot consistently select relatively better models and rank a large number of models well. Here, we develop a novel large-scale model QA method in conjunction with model clustering to rank and select protein structural models. It unprecedentedly applied 14 model QA methods to generate consensus model rankings, followed by model refinement based on model combination (i.e. averaging). Our experiment demonstrates that the large-scale model QA approach is more consistent and robust in selecting models of better quality than any individual QA method. Our method was blindly tested during the 11th Critical Assessment of Techniques for Protein Structure Prediction (CASP11) as MULTICOM group. It was officially ranked third out of all 143 human and server predictors according to the total scores of the first models predicted for 78 CASP11 protein domains and second according to the total scores of the best of the five models predicted for these domains. MULTICOM's outstanding performance in the extremely competitive 2014 CASP11 experiment proves that our large-scale QA approach together with model clustering is a promising solution to one of the two major problems in protein structure modeling. The web server is available at: http://sysbio.rnet.missouri.edu/multicom_cluster/human/. © The Author 2015. Published by Oxford University Press.

Epitaxial structure and transport in LaTiO3+x films on (001) SrTiO3

NASA Astrophysics Data System (ADS)

Kim, K. H.; Norton, D. P.; Budai, J. D.; Chisholm, M. F.; Sales, B. C.; Christen, D. K.; Cantoni, C.

2003-12-01

The structure and transport properties of LaTiO3+x epitaxial thin films grown on (001) SrTiO3 by pulsed-laser deposition is examined. Four-circle X-ray diffraction indicates that the films possess the defect perovskite LaTiO3 structure when deposited in vacuum, with the higher X compounds forming at moderate oxygen pressures. The crystal structure of the LaTiO3 films is tetragonal in the epitaxial films, in contrast to the orthorhombic structure observed in bulk materials. A domain structure is observed in the films, consisting of LaTiO3 oriented either with the [110] or [001] directions perpendicular to the substrate surface. Z-contrast scanning transmission electron microscopy reveals that this domain structure is not present in the first few unit cells of the film, but emerges approximately 2-3 nm from the SrTiO3/LaTiO3 interface. Upon increasing the oxygen pressure during growth, a shift in the lattice d-spacing parallel to the substrate surface is observed, and is consistent with the growth of the La2Ti2O7 phase. However, van der Pauw measurements show that the films with the larger d-spacing remain conductive, albeit with a resistivity that is significantly higher than that for the perovskite LaTiO3 films. The transport behavior suggests that the films grown at higher oxygen pressures are LaTiO3+x with 0.4 < x < 0.5. (

Reverse-mode thermoresponsive light attenuators produced by optical anisotropic composites of nematic liquid crystals and reactive mesogens

NASA Astrophysics Data System (ADS)

Kakiuchida, Hiroshi; Ogiwara, Akifumi

2018-04-01

Polymer network liquid crystals (PNLCs) whose optical transmittance state switches between transparence at low temperatures and haze at high temperatures were fabricated from mixtures of nematic liquid crystals (LCs) and reactive mesogens (RMs). This PNLC structure is simple but effective, namely, consists of micro-scale domains of orientation-ordered LCs and anisotropically polymerized RMs. The domains form through photopolymerization induced phase separation with inhomogeneous irradiation projected by laser speckling techniques. This irradiation method enables you to control the size and shape of phase-separation domains, and these PNLCs can be applied to novel thermoresponsive optical devices; optical isolators, thermometric sheets, and smart windows.

Crystal Structure of a Histidine Kinase Sensor Domain with Similarity to Periplasmic Binding Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheung, J.; Le-Khac, M; Hendrickson, W

2009-01-01

Histidine kinase receptors are elements of the two-component signal transduction systems commonly found in bacteria and lower eukaryotes, where they are crucial for environmental adaption through the coupling of extracellular changes to intracellular responses. The typical two-component system consists of a membrane-spanning histidine kinase sensor and a cytoplasmic response regulator. In the calssic system, extracellular signals such as small molecule ligands and ions are detected by the periplasmic sensor domain of the histidine kinase receptor, which modulates the catalytic activity of the cytoplasmic histidine kinase domain and promotes ATP-dependent autophosphorylation of a conserved histidine residue. G. sulfurreducens genomic DNA wasmore » used.« less

Sequence Analysis and Domain Motifs in the Porcine Skin Decorin Glycosaminoglycan Chain*

PubMed Central

Zhao, Xue; Yang, Bo; Solakylidirim, Kemal; Joo, Eun Ji; Toida, Toshihiko; Higashi, Kyohei; Linhardt, Robert J.; Li, Lingyun

2013-01-01

Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences. PMID:23423381

Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs

PubMed Central

Stoeber, Miriam; Schellenberger, Pascale; Siebert, C. Alistair; Leyrat, Cedric; Helenius, Ari

2016-01-01

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture. PMID:27834731

Structural Basis of PP2A Inhibition by Small t Antigen

PubMed Central

Cho, Uhn Soo; Morrone, Seamus; Sablina, Anna A; Arroyo, Jason D; Hahn, William C; Xu, Wenqing

2007-01-01

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST. PMID:17608567

An efficient solution procedure for the thermoelastic analysis of truss space structures

NASA Technical Reports Server (NTRS)

Givoli, D.; Rand, O.

1992-01-01

A solution procedure is proposed for the thermal and thermoelastic analysis of truss space structures in periodic motion. In this method, the spatial domain is first descretized using a consistent finite element formulation. Then the resulting semi-discrete equations in time are solved analytically by using Fourier decomposition. Geometrical symmetry is taken advantage of completely. An algorithm is presented for the calculation of heat flux distribution. The method is demonstrated via a numerical example of a cylindrically shaped space structure.

Structural organization and chromosomal assignment of the mouse embryonic TEA domain-containing factor (ETF) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Kazuo; Yasunami, Michio; Matsuda, Yoichi

1996-09-01

Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed that Etdf spans approximately 17.9 kb and consists of 12 exons. The exon-intron structure of Etdf closely resembles that of the Drosophila scalloped gene, indicating that these genes may have evolved from a common ancestor. Then multiple transcription initiation sites revealed by S1 protection and primer extension analyses are consistent with the absence of the canonical TATA and CAAT boxes in themore » 5{prime}-flanking region, which contains many potential regulatory sequences, such as the E-box, N-box, Sp1 element, GATA-1 element, TAATGARAT element, and B2 short interspersed element (SINE) as well as several direct and inverted repeat sequences. The Etdf locus was assigned to the proximal region of mouse chromosome 7 using fluorescence in situ hybridization and linkage mapping analyses. These results provide the molecular basis for studying the regulation, in vivo function, and evolution of Etdf. 29 refs., 5 figs., 1 tab.« less

Structural organization and chromosomal assignment of the mouse embryonic TEA domain-containing factor (ETF) gene.

PubMed

Suzuki, K; Yasunami, M; Matsuda, Y; Maeda, T; Kobayashi, H; Terasaki, H; Ohkubo, H

1996-09-01

Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed that Etdf spans approximately 17.9 kb and consists of 12 exons. The exon-intron structure of Etdf closely resembles that of the Drosophila scalloped gene, indicating that these genes may have evolved from a common ancestor. The multiple transcription initiation sites revealed by S1 protection and primer extension analyses are consistent with the absence of the canonical TATA and CAAT boxes in the 5'-flanking region, which contains many potential regulatory sequences, such as the E-box, N-box, Sp1 element, GATA-1 element, TAATGARAT element, and B2 short interspersed element (SINE) as well as several direct and inverted repeat sequences. The Etdf locus was assigned to the proximal region of mouse chromosome 7 using fluorescence in situ hybridization and linkage mapping analyses. These results provide the molecular basis for studying the regulation, in vivo function, and evolution of Etdf.

Structure of Bombyx mori densovirus 1, a silkworm pathogen.

PubMed

Kaufmann, Bärbel; El-Far, Mohamed; Plevka, Pavel; Bowman, Valorie D; Li, Yi; Tijssen, Peter; Rossmann, Michael G

2011-05-01

Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-Å resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel "jelly roll" fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a "domain-swapped" conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae.

Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

PubMed

Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

2017-04-01

Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Self-Consistent Field Theory for the Design of Thermoplastic Elastomers from Miktoarm Block Copolymer - Homopolymer Blends

NASA Astrophysics Data System (ADS)

Hamilton, Andrew Lawrence

We have used self-consistent field theory to study the morphological characteristics of blends of miktoarm block copolymers and homopolymers. More specifically, we have studied the effects of segregation strength, miktoarm block copolymer composition, and homopolymer size and volume fraction on the phase diagrams of these systems. A15 domains with discrete A-monomer spherical domains were found to be stable with A-monomer loading fractions of at least as high as 52%. Hexagonally-packed cylindrical domains were found to be stable at A-monomer loadings of at least as high as 72%. These findings represent a significant improvement from the loading fractions of 43% and 60% reported by Lynd et al. for spherical and cylindrical domains in neat miktoarm block copolymers, respectively. It is also quite possible that even greater loading fractions are achievable in systems too large for our simulations. These results predict exciting new materials for next-generation thermoplastic elastomers, since the ideal TPE has a large loading of A monomers in discrete, crystalline or glassy domains, surrounded by a continuous matrix of elastomeric B domains. Additionally, we have performed SCFT simulations modelled after experimental blends of polystyrene and polyisoprene-based miktoarm block copolymers and homopolymers. Certain experimental samples showed fascinating new "bricks and mortar" phases and swollen asymmetric lamellar phases. In both cases, the A domains are highly swollen with homopolymer, forcing the miktoarm block copolymer to segregate near the interface and adopt the role of a surfactant. The resulting structures maintain separate A and B domains, but lack long-range order. While it is not possible to study these mesophases using SCFT, since they lack long-range order and therefore well-defined symmetry, our SCFT results show the onset of macrophase separation at similar homopolymer loadings, for both the bricks and mortar phases and the highly swollen lamellae. This supports the theory that both phases are fluctuation-induced mesophases, similar to microemulsions in character, that lie in between the typical ordered structures and full macrophase separation.

Assessing implementation difficulties in tobacco use prevention and cessation counselling among dental providers.

PubMed

Amemori, Masamitsu; Michie, Susan; Korhonen, Tellervo; Murtomaa, Heikki; Kinnunen, Taru H

2011-05-26

Tobacco use adversely affects oral health. Clinical guidelines recommend that dental providers promote tobacco abstinence and provide patients who use tobacco with brief tobacco use cessation counselling. Research shows that these guidelines are seldom implemented, however. To improve guideline adherence and to develop effective interventions, it is essential to understand provider behaviour and challenges to implementation. This study aimed to develop a theoretically informed measure for assessing among dental providers implementation difficulties related to tobacco use prevention and cessation (TUPAC) counselling guidelines, to evaluate those difficulties among a sample of dental providers, and to investigate a possible underlying structure of applied theoretical domains. A 35-item questionnaire was developed based on key theoretical domains relevant to the implementation behaviours of healthcare providers. Specific items were drawn mostly from the literature on TUPAC counselling studies of healthcare providers. The data were collected from dentists (n = 73) and dental hygienists (n = 22) in 36 dental clinics in Finland using a web-based survey. Of 95 providers, 73 participated (76.8%). We used Cronbach's alpha to ascertain the internal consistency of the questionnaire. Mean domain scores were calculated to assess different aspects of implementation difficulties and exploratory factor analysis to assess the theoretical domain structure. The authors agreed on the labels assigned to the factors on the basis of their component domains and the broader behavioural and theoretical literature. Internal consistency values for theoretical domains varied from 0.50 ('emotion') to 0.71 ('environmental context and resources'). The domain environmental context and resources had the lowest mean score (21.3%; 95% confidence interval [CI], 17.2 to 25.4) and was identified as a potential implementation difficulty. The domain emotion provided the highest mean score (60%; 95% CI, 55.0 to 65.0). Three factors were extracted that explain 70.8% of the variance: motivation (47.6% of variance, α = 0.86), capability (13.3% of variance, α = 0.83), and opportunity (10.0% of variance, α = 0.71). This study demonstrated a theoretically informed approach to identifying possible implementation difficulties in TUPAC counselling among dental providers. This approach provides a method for moving from diagnosing implementation difficulties to designing and evaluating interventions.

Indexing Learning Objects: Vocabularies and Empirical Investigation of Consistency

ERIC Educational Resources Information Center

Kabel, Suzanne; De Hoog, Robert; Wielinga, Bob; Anjewierden, Anjo

2004-01-01

In addition to the LOM standard and instructional design specifications, as well as domain specific indexing vocabularies, a structured indexing vocabulary for the more elementary learning objects is advisable in order to support retrieval tasks of developers. Furthermore, because semantic indexing is seen as a difficult task, three issues…

Folding of a LysM Domain: Entropy-Enthalpy Compensation in the Transition State of an Ideal Two-state Folder

PubMed Central

Nickson, Adrian A.; Stoll, Kate E.; Clarke, Jane

2008-01-01

Protein-engineering methods (Φ-values) were used to investigate the folding transition state of a lysin motif (LysM) domain from Escherichia coli membrane-bound lytic murein transglycosylase D. This domain consists of just 48 structured residues in a symmetrical βααβ arrangement and is the smallest αβ protein yet investigated using these methods. An extensive mutational analysis revealed a highly robust folding pathway with no detectable transition state plasticity, indicating that LysM is an example of an ideal two-state folder. The pattern of Φ-values denotes a highly polarised transition state, with significant formation of the helices but no structure within the β-sheet. Remarkably, this transition state remains polarised after circularisation of the domain, and exhibits an identical Φ-value pattern; however, the interactions within the transition state are uniformly weaker in the circular variant. This observation is supported by results from an Eyring analysis of the folding rates of the two proteins. We propose that the folding pathway of LysM is dominated by enthalpic rather than entropic considerations, and suggest that the lower entropy cost of formation of the circular transition state is balanced, to some extent, by the lower enthalpy of contacts within this structure. PMID:18538343

C terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

South, T.L.; Blake, P.R.; Hare, D.R.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retriviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 (Zn(HIV1-F2)). Unlike results obtained for the first retroviral-type zinc finger peptide, Zn (HIV1-F1) broad signals indicative of confomational lability were observed in the {sup 1}H NMR spectrum of An(HIV1-F2) at 25 C. The NMR signals narrowed upon cooling to {minus}2 C, enabling complete {sup 1}H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhausermore » effect (NOESY) data were sued to generate 30 distance geometry (DG) structures with penalties in the range 0.02-0.03 {angstrom}{sup 2}. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. These results indicate that the r.t. zinc finger sequences observed in retroviral NCPs, simple plant virus coat proteins, and in a human single-stranded nucleic acid binding protein share a common structural motif.« less

Strain-relaxed structure in (001)/(100)-oriented epitaxial PbTiO3 films grown on (100) SrTiO3 substrates by metal organic chemical vapor deposition

NASA Astrophysics Data System (ADS)

Nakaki, Hiroshi; Kim, Yong Kwan; Yokoyama, Shintaro; Ikariyama, Rikyu; Funakubo, Hiroshi; Nishida, Ken; Saito, Keisuke

2007-09-01

The authors grew (001)- and (001)/(100)-oriented epitaxial PbTiO3 films with various thicknesses on (100)SrTiO3 substrates. They used x-ray diffraction to measure the angles between surface normal [001] of (001)-oriented domains and [100] of (100)-oriented domains. The angles were found to be approximately 3.6° when the film thickness exceeded 1100nm. This value is consistent with the value obtained by a geometric calculation for strain-free PbTiO3. This result suggests that thick epitaxial PbTiO3 films grown on (100)SrTiO3 substrates have a fully strain-relaxed structure.

Paleomagnetic Evaluation of the Resurgent Dome at Valles Caldera, Jemez Mountains, New Mexico

NASA Astrophysics Data System (ADS)

Rhode, A.; Geissman, J. W.; Goff, F. E.

2016-12-01

The Redondo Peak structural dome, located within the ca. 1.25 Ma Valles Caldera, Jemez Mountains, New Mexico, is a well documented example of post-caldera resurgence and is a fundamental part of the famous model of Smith and Bailey (1968). The NE/SW elongated structural resurgent dome, with over 1000 m of uplift, and its medial graben now occupied by Redondo Creek, parallel the NE orientation of the Jemez fault zone, a key boundary structure of the Rio Grande rift. Our paleomagnetic research quantifies the magnitude of structural tilt (i.e. rotation about a horizontal axis) as a component of any deformation of the resurgent dome to determine if uplift was accommodated by block uplift or by simple doming. Independently oriented samples from 43 sites located on two main structural domains that comprise the resurgent dome (the Redondo Border block and the Redondo Peak block) and within the Redondo Creek graben were obtained from the intracaldera facies of the Tshirege Member of the Bandelier Tuff and overlying lower members of post-Bandelier Valles Rhyolite. Magnetic mineralogy consists of low titanium magnetite and maghemite, consistent with previous paleomagnetic studies on flat-lying outflow facies tuff. In situ estimated directions of sites from the Redondo Border structural domain are generally steeper in inclination than the reference direction (D = 175.6, I = -35.7) (Doell et al., 1968; Sussman et al., 2011), with an average inclination of Ig = -42.5, and show a westward deflection in average declination (Dg = 184.2). In situ estimated directions of sites from the Redondo Peak structural domain are generally shallower in inclination than the reference direction (average inclination of Ig = -27.6) and show an eastward deflection in mean declination values, Dg = 160. Overall, paleomagnetic results show that the pattern of deformation is more pronounced parallel to the long axis of the dome and that the Redondo Border block exhibits some 12 degrees of down to the northwest tilt, while the Redondo Peak block exhibits some 9 degrees of down to the southeast tilt. Furthermore, paleomagnetic fold test results are consistent with a doming mechanism of deformation, and preclude the notion of "syntilting", or magnetization acquisition taking place concurrent with doming.

Structural polarity in the Chara rhizoid: a reevaluation

NASA Technical Reports Server (NTRS)

Kiss, J. Z.; Staehelin, L. A.

1993-01-01

The Chara rhizoid is a useful model system to study gravitropism since all phases of gravitropism occur in a single cell. Despite years of study, a complete description of the distinctive ultrastructure of Chara rhizoids is not available. Therefore, in this paper, we reevaluate the ultrastructural features of vertically grown rhizoids, which have a structural polarity consisting of seven distinct zones. We also characterize the apical vesicles and the cell wall in these rhizoids by using antibodies against pectic polysaccharides. These studies demonstrate that the cell wall consists of two pectinaceous domains and that a distinct population of apical vesicles contain methyl esterified pectin.

Changes in Quaternary Structure in the Signaling Mechanisms of PAS Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ayers, Rebecca A.; Moffat, Keith

2008-12-15

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group (P1) and at resolutions (1.5--1.8 {angstrom}) higher than the resolutions of those previously obtained. Interestingly, bjFixLHmore » can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated {approx}175 deg. relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to 2 deg. relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.« less

Conservation-Oriented Hbim. The Bimexplorer Web Tool

NASA Astrophysics Data System (ADS)

Quattrini, R.; Pierdicca, R.; Morbidoni, C.; Malinverni, E. S.

2017-05-01

The application of (H)BIM within the domain of Architectural Historical Heritage has huge potential that can be even exploited within the restoration domain. The work presents a novel approach to solve the widespread interoperability issue related to the data enrichment in BIM environment, by developing and testing a web tool based on a specific workflow experienced choosing as the case study a Romanic church in Portonovo, Ancona, Italy. Following the need to make the data, organized in a BIM environment, usable for the different actors involved in the restoration phase, we have created a pipeline that take advantage of BIM existing platforms and semantic-web technologies, enabling the end user to query a repository composed of semantically structured data. The pipeline of work consists in four major steps: i) modelling an ontology with the main information needs for the domain of interest, providing a data structure that can be leveraged to inform the data-enrichment phase and, later, to meaningfully query the data; ii) data enrichment, by creating a set of shared parameters reflecting the properties in our domain ontology; iii) structuring data in a machine-readable format (through a data conversion) to represent the domain (ontology) and analyse data of specific buildings respectively; iv) development of a demonstrative data exploration web application based on the faceted browsing paradigm and allowing to exploit both structured metadata and 3D visualization. The application can be configured by a domain expert to reflect a given domain ontology, and used by an operator to query and explore the data in a more efficient and reliable way. With the proposed solution the analysis of data can be reused together with the 3D model, providing the end-user with a non proprietary tool; in this way, the planned maintenance or the restoration project became more collaborative and interactive, optimizing the whole process of HBIM data collection.

Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

PubMed Central

2012-01-01

Background The NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches. Results Here we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day. Conclusions This approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain annotation by identifying those pattern residues that most distinguish each protein domain subgroup from other related subgroups. PMID:22726767

Photonic crystal based 1-bit full-adder optical circuit by using ring resonators in a nonlinear structure

NASA Astrophysics Data System (ADS)

Alipour-Banaei, Hamed; Seif-Dargahi, Hamed

2017-05-01

In this paper we proposed a novel design for realizing all optical 1*bit full-adder based on photonic crystals. The proposed structure was realized by cascading two optical 1-bit half-adders. The final structure is consisted of eight optical waveguides and two nonlinear resonant rings, created inside rod type two dimensional photonic crystal with square lattice. The structure has ;X;, ;Y; and ;Z; as input and ;SUM; and ;CARRY; as output ports. The performance and functionality of the proposed structure was validated by means of finite difference time domain method.

Cooption of secretory phospholipase (SPLA2) for different aspects of gravity receptor-associated mineralization in vertebrate phylogeny

NASA Astrophysics Data System (ADS)

Thalmann, R.; Lu, W.

2009-04-01

Vertebrate gravity-associated minerals consists of either a single large stone (otolith), or an assembly of minute biomineral particles, otoconia ("ear dust"). Otoliths and both, amphibian and reptilian otoconia, consist of aragonite, whereas avian and mammalian otoconia consist of calcite. Vertebrate gravity-associated minerals are the product of site-directed biologically-controlled mineralization. Insoluble frame work molecules specify sites of nucleation and direction of crystal growth. Soluble matrix proteins modulate growth kinetics and crystal morphology. It is most remarkable that the principal insoluble frame work protein, otolin, is the same for both, otolith and otoconia. Otolin is a novel type of collagen, homologous to the network-forming collagen type X prevalent in mature chondrocytes. The principal soluble matrix proteins of calcitic, aragonitic, and most likely also of vateritic otoconia are all homologs of SPLA2, which is most prevalent in pancreatic secretion and snake venoms. Otonin90 (OC90), the principal soluble matrix protein of calcitic otoconia consists of two SPLA-like (SPLAL) domains, which are connected by a sizeable linker segment and contain significant terminal extensions. The MW of the protein backbone amounts to approximately 50 kDa. The molecule contains, in addition massive post-translational modifications, 80% of which are accounted for by sulfated GAGs, resulting in a total MW of 100 KDa. The protein backbone is moderately acidic, pI 4.4, but the pI of the whole molecule is 2.9, indicating a substantial acidity of the GAG component. In adapting SPLA2 for mineral modulation the enzymatic site is modified and presumed nonfunctional. The seven SH- bonds are rigorously conserved in both, OC90 and otoconin22 (OC22). It appears that the SH-bonds of the parent SPLA2 are intended to stabilize the molecule to ensure continued enzymatic activity in the hostile environment of the gut. It therefore seems logical that SPLA2 was coopted for mineral modulation not because of its enzymatic activity but to provide a rigid interface conducive to mineral interaction. To provide sufficient matrix protein for in vitro experimentation, we generated recombinant proteins. Circular dichroidism (CD) spectra indicate that the alpha helical structure of the parent SPLA2 is conserved in the SPLAL domains. A precedent of alpha helical structure for provision of a rigid interface was demonstrated to be essential for the activity of the antifreeze protein of the winter flounder. Support for alpha helical structure as signature property of the SPLAL domains of OC90 is the fact that rOC90, when exposed to calcium or carbonate-rich ionic solutions resulted in marked conformational changes, with the largest effects seen by combined application of both ions. The capacity to induce reproducible conformational changes is a testament to the quality and authenticity of rOC90. Alpha helical structure as signature characteristic of OC90 is contrary to the traditional paradigm of beta sheet structure as the essential agent in mineral interaction of highly acidic mollusk shell proteins. Apart from the alpha helical regions of the SPLAL domains, homology-based molecular modeling indicates that most of the linker segment and the terminal extensions consist of unordered structure. The significance of unordered structure in mineral interaction has recently been pointed out by several authors. For instance, the linker segment exhibits a 20 amino residue regions, dominated by hydrogen bonding and charged residues, in other words a hydrophilic segment, suitable for mineral interaction; the same applies to the C-terminal extension. Homology-based molecular models of the SPLAL domains exhibit a spherical surface with a uniform negative electrostatic potential that should be effective in attracting calcium. OC22, the principal soluble aragonitic matrix protein, consists of a single SPLAL domain, with a minor N-linked glycoside. rOC22 in vitro does not induce formation of aragonite, but of calcite as default option, analogous to other aragonitic matrix proteins, e.g. AP8 alpha and beta of the avalone nacre. It has been shown that aragonitic proteins are able to express aragonite only in association with the appropriate insoluble matrix. The effects of the protein upon calcite modification are qualitatively identical to the effects of OC90 (nucleation density, crystal size and morphologic change), but are quantitatively much less. The most unexpected SPLA2 domain-related aspect is the recent discovery of an OC90 ortholog (Otoc1) in otoliths even though OMP, a derivative of melanotransferin is the principal soluble matrix protein of otoliths. We, heretofore, assumed that OC22, as a single SPLAL domain, constituted the earliest manifestation of this cooption in vertebrate phylogeny. Significantly, the presence of Otoc1 is not incidental. Knock-down of the gene results in agenesis or malformation of otolith with a change from aragonite to calcite. In summary, this presentation serves to illustrate the wide distribution and varied function of a coopted lipolytic enzyme in a wide range of mineralization-related contexts.

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species

PubMed Central

Liu, Liyun; Hastings, J. Woodland

2007-01-01

Noctiluca scintillans, a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size (≈100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca. PMID:17130452

Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

NASA Astrophysics Data System (ADS)

Filikov, Anton V.; James, Thomas L.

1998-05-01

A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with peptidic and peptidomimetic linkers. The linkers were refined by varying the length and side chains of the linking residues, carrying out BPMC simulations, and evaluation of the binding free energy for the best energy conformation. The dissociation constant of the best ligand designed is estimated to be in the low- to mid-nanomolar range.

Solution structure of the DNA-binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy.

PubMed Central

Damberger, F. F.; Pelton, J. G.; Harrison, C. J.; Nelson, H. C.; Wemmer, D. E.

1994-01-01

The solution structure of the 92-residue DNA-binding domain of the heat shock transcription factor from Kluyveromyces lactis has been determined using multidimensional NMR methods. Three-dimensional (3D) triple resonance, 1H-13C-13C-1H total correlation spectroscopy, and 15N-separated total correlation spectroscopy-heteronuclear multiple quantum correlation experiments were used along with various 2D spectra to make nearly complete assignments for the backbone and side-chain 1H, 15N, and 13C resonances. Five-hundred eighty-three NOE constraints identified in 3D 13C- and 15N-separated NOE spectroscopy (NOESY)-heteronuclear multiple quantum correlation spectra and a 4-dimensional 13C/13C-edited NOESY spectrum, along with 35 phi, 9 chi 1, and 30 hydrogen bond constraints, were used to calculate 30 structures by hybrid distance geometry/stimulated annealing protocol, of which 24 were used for structural comparison. The calculations revealed that a 3-helix bundle packs against a small 4-stranded antiparallel beta-sheet. The backbone RMS deviation (RMSD) for the family of structures was 1.03 +/- 0.19 A with respect to the average structure. The topology is analogous to that of the C-terminal domain of the catabolite gene activator protein and appears to be in the helix-turn-helix family of DNA-binding proteins. The overall fold determined by the NMR data is consistent with recent crystallographic work on this domain (Harrison CJ, Bohm AA, Nelson HCM, 1994, Science 263:224) as evidenced by RMSD between backbone atoms in the NMR and X-ray structures of 1.77 +/- 0.20 A. Several differences were identified some of which may be due to protein-protein interactions in the crystal. PMID:7849597

Coupling of conformational transitions in the N-terminal domain of the 51-kDa FK506-binding protein (FKBP51) near its site of interaction with the steroid receptor proteins

DOE PAGES

LeMaster, David M.; Mustafi, Sourajit M.; Brecher, Matthew; ...

2015-05-07

Interchanging Leu-119 for Pro-119 at the tip of the β 4-β 5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the β 4-β 5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying β 2 andmore » β 3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the β 2 and β 3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. As a result, the contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.« less

Basis for ligand discrimination between ON and OFF state riboswitch conformations: The case of the SAM-I riboswitch

PubMed Central

Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-ela, Fareed

2012-01-01

Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming “hybrid” structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding. PMID:22543867

Basis for ligand discrimination between ON and OFF state riboswitch conformations: the case of the SAM-I riboswitch.

PubMed

Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-Ela, Fareed

2012-06-01

Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming "hybrid" structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding.

Use of the Behavior Rating Inventory of Executive Function and Child Behavior Checklist in Ugandan Children with HIV or a History of Severe Malaria.

PubMed

Familiar, Itziar; Ruisenor-Escudero, Horacio; Giordani, Bruno; Bangirana, Paul; Nakasujja, Noeline; Opoka, Robert; Boivin, Michael

2015-05-01

To assess the structural overlap between the Behavior Rating Inventory of Executive Function (BRIEF) and Achenbach Child Behavior Checklist (CBCL) among children in Uganda. Caregiver ratings for the BRIEF and CBCL were obtained for 2 independent samples of school-aged children: 106 children (5-12 years old, 50% males) with a history of severe malaria and on 144 HIV-infected children (5-12 years old, 58% males) in Uganda. Exploratory factor analysis was used to evaluate the factor structure of the 8 subscales for the BRIEF and the 8 scales of the CBCL to determine correlation. Overall, children in the severe malaria group had higher (increased symptom) BRIEF and CBCL scores than those in the HIV-infected group. Three factors that provided a reasonable fit to the data and could be characterized as 3 specific domains were identified: (1) Metacognition, which consisted of the scales in the BRIEF Metacognition domain, (2) Behavioral Adjustment, which comprised of the scales in the BRIEF Behavioral Regulation domain and the Externalizing Symptoms scales in the CBCL, and (3) Emotional Adjustment, which mainly consisted of the Internalizing Symptoms scales in the CBCL. The BRIEF Behavior Regulation and CBCL Externalizing Symptoms scales, however, did overlap in terms of assessing similar behavior symptoms. These findings were consistent across the severe malaria and HIV-infected samples of children. The BRIEF and CBCL instruments offer distinct, yet complementary, assessments of behavior in clinical pediatric populations in the Ugandan context, supporting the use of these measures for similar research settings.

Use of the BRIEF and CBCL in Ugandan Children with HIV or a History of Severe Malaria

PubMed Central

Itziar, Familiar; Horacio, Ruisenor-Escudero; Bruno, Giordani; Paul, Bangirana; Noeline, Nakasujja; Robert, Opoka; Michael, Boivin

2015-01-01

Objective To assess the structural overlap between the Behavior Rating Inventory of Executive Function (BRIEF) and Achenbach Child Behavior Check List (CBCL) among children in Uganda. Methods Caregiver ratings for the BRIEF and CBCL were obtained for two independent samples of school-aged children: 106 children (5-12 years old, 50% males) with a history of severe malaria, and on 144 HIV-infected children (5-12 years old, 58% males) in Uganda. Exploratory factor analysis was used to evaluate the factor structure of the 8 sub-scales for the BRIEF and the 8 scales of the CBCL to determine correlation. Results Overall, children in the severe malaria group had higher (increased symptom) BRIEF and CBCL scores than children in the HIV-infected group. We identified 3 factors that provided a reasonable fit to the data and could be characterized as 3 specific domains: 1) Metacognition, which consisted of the scales in the BRIEF Metacognition domain; 2) Behavioral Adjustment, which was comprised of the scales in the BRIEF Behavioral Regulation domain and the Externalizing Symptoms scales in the CBCL; and 3) Emotional Adjustment, which mainly consisted of the Internalizing Symptoms scales in the CBCL. The BRIEF Behavior Regulation and CBCL Externalizing Symptoms scales, however, did overlap in terms of assessing similar behavior symptoms. These findings were consistent across the severe malaria and HIV-infected samples of children. Conclusion The BRIEF and CBCL instruments offer distinct, yet complementary, assessments of behavior in clinical pediatric populations in the Ugandan context, supporting the use of these measures for similar research settings. PMID:25738440

Probing domain switching dynamics in ferroelectric thick films by small field e31,f piezoelectric measurement

NASA Astrophysics Data System (ADS)

Cheng, Hongbo; Ouyang, Jun; Kanno, Isaku

2017-07-01

Epitaxial Pb(Zr0.53Ti0.47)O3 films were grown on (001) Pt/(001) MgO via rf-magnetron sputtering. Switching dynamics of 90° and 180° domains under bi-polar electric fields were probed by using small-field e31 ,f measurements in which the evolution of the transverse piezoelectric response with the bias voltage represents a set of fingerprints of the evolving domain structure. Furthermore, the asymmetric e31 ,f-V curves revealed a strong built-in electric field, which was verified by the standard polarization-electric field hysteresis measurement. Finally, X-ray 2θ-scan patterns under DC bias voltages were collected for the piezoelectric specimen. The domain switching sequence indicated by the XRD results is consistent with that revealed by the e31 ,f measurement.

Short forms of the Utrecht-Management of Identity Commitments Scale (U-MICS) with the domains of job, romantic relationship, and region.

PubMed

Schubach, Elisabeth; Zimmermann, Julia; Noack, Peter; Neyer, Franz J

2017-01-01

The U-MICS is a self-report questionnaire designed to assess the identity dimensions from a domain-specific perspective. The present study reports on the development of a short-form version for the domains of job and romantic relationship in young adults from Germany and extends this scale to include the domain of region (n Sample1  = 95, 84% female, mean age 22.45 years; n Sample2  = 1,795, 71% female, mean age 24.53 years). We found the short form to possess adequate psychometric properties and to demonstrate a factor structure congruent to the long-form version. Regarding validity, the small correlations across domains within dimensions support a domain-specific approach to identity. The associations between the different identity domains with personality traits are similar, indicating a consistent pattern of convergent validity for all domains. We conclude that "region" provides a valuable complement to the established domains that can all be reliably assessed with the U-MICS-Short Form. Copyright © 2016 The Foundation for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.

Conformational dynamics and ligand binding in the multi-domain protein PDC109.

PubMed

Kim, Hyun Jin; Choi, Moo Young; Kim, Hyung J; Llinás, Miguel

2010-02-18

PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2) repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs), a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD) simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1), estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Xiaomin; Meehan, Edward J.; Xie, Jieming

2008-10-27

A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04more » {angstrom}, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven {alpha}-helices and eight {beta}-strands, and a smaller C-terminal domain consisting of three {alpha}-helices and two {beta}-strands. The high resolution structure established a glycosylation pattern of GlcNAc{sub 2}Man3Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.« less

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

PubMed Central

Sborgi, Lorenzo; Ravotti, Francesco; Dandey, Venkata P.; Dick, Mathias S.; Mazur, Adam; Reckel, Sina; Chami, Mohamed; Scherer, Sebastian; Huber, Matthias; Böckmann, Anja; Egelman, Edward H.; Stahlberg, Henning; Broz, Petr; Meier, Beat H.; Hiller, Sebastian

2015-01-01

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms. PMID:26464513

Identification of a "glycine-loop"-like coiled structure in the 34 AA Pro,Gly,Met repeat domain of the biomineral-associated protein, PM27.

PubMed

Wustman, Brandon A; Santos, Rudolpho; Zhang, Bo; Evans, John Spencer

2002-12-05

Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain. Copyright 2002 Wiley Periodicals, Inc.

Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

NASA Astrophysics Data System (ADS)

Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron; Zhu, Allen; Jungmann, Ralf; Bellot, Gaetan; Ke, Yonggang; Yin, Peng

2017-12-01

Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair ‘voxels’ that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

Phason space analysis and structure modelling of 100 Å-scale dodecagonal quasicrystal in Mn-based alloy

NASA Astrophysics Data System (ADS)

Ishimasa, Tsutomu; Iwami, Shuhei; Sakaguchi, Norihito; Oota, Ryo; Mihalkovič, Marek

2015-11-01

The dodecagonal quasicrystal classified into the five-dimensional space group P126/mmc, recently discovered in a Mn-Cr-Ni-Si alloy, has been analysed using atomic-resolution spherical aberration-corrected electron microscopy, i.e. high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and conventional transmission electron microscopy. By observing along the 12-fold axis, non-periodic tiling consisting of an equilateral triangle and a square has been revealed, of which common edge length is a = 4.560 Å. These tiles tend to form a network of dodecagons of which size is ?a ≈ 17 Å in diameter. The tiling was interpreted as an aggregate of 100 Å-scale oriented domains of high- and low-quality quasicrystals with small crystallites appearing at their boundaries. The quasicrystal domains exhibited a densely filled circular acceptance region in the phason space. This is the first observation of the acceptance region in an actual dodecagonal quasicrystal. Atomic structure model consistent with the electron microscopy images is a standard Frank-Kasper decoration of the triangle and square tiles that can be inferred from the crystal structures of Zr4Al3 and Cr3Si. Four kinds of layers located at z = 0, ±1/4 and 1/2 are stacked periodically along the 12-fold axis, and the atoms at z = 0 and 1/2 form hexagonal anti-prisms consistently with the 126-screw axis. The validity of this structure model was examined by means of powder X-ray diffraction.

Modeling of the N-terminal Section and the Lumenal Loop of Trimeric Light Harvesting Complex II (LHCII) by Using EPR*

PubMed Central

Fehr, Niklas; Dietz, Carsten; Polyhach, Yevhen; von Hagens, Tona; Jeschke, Gunnar; Paulsen, Harald

2015-01-01

The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photosynthesis, and in preventing photodamage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment are only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is introduced to prepare LHCII trimers in which only one monomer is spin-labeled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron spin echo envelope modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the “Velcro” hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kühlbrandt, W. (2005) EMBO J. 24, 919–928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound. PMID:26316535

Imaging the Dynamics of the Ferroelectric Stripe Phase Near a Field-Driven Phase Transition in Bismuth Ferrite

NASA Astrophysics Data System (ADS)

Laanait, Nouamane; Li, Qian; Zhang, Zhan; Kalinin, Sergei

Electric field-driven phase transitions in multiferroic systems such as Bismuth Ferrite could potentially host interesting domain dynamics due to the coexistence of multiple order parameters. Structural imaging of these dynamics under a host of elastic and electric boundary conditions is therefore of interest. Here, we present X-ray diffraction microscopy (XDM) studies of the domain wall dynamics in a bismuth ferrite thin-film near the field-driven transition from rhombohedral to monoclinic (R to M). XDM is a novel full-field imaging technique that uses Bragg diffraction contrast to image structural configurations with sub-100nm lateral resolutions and fast acquisition times (milliseconds to seconds per image). We find that under electric fields 100 kV/cm, a bismuth ferrite thin-film (100 nm BiFeO3/DyScO3 (110)) undergoes a structural phase transition but that this new phase (M) is pinned by the preexisting ferroelectric/ferroelastic stripe phase (R). At higher fields ( 300 kV/cm), we observe unusually slow domain wall dynamics in the stripe phase, consisting of periodicity doubling, domain wall roughening and crowding. These observed ferroelastic domain wall spatial dynamics are weakly constrained by the crystal symmetry of the orthorhombic substrate but exhibit nonlinear dynamics more commonly associated with disordered nematic systems. This work was supported by the Eugene P. Wigner Fellowship program at Oak Ridge National Laboratory, a U.S. Department of Energy facility.

The cryo-electron microscopy structure of huntingtin

NASA Astrophysics Data System (ADS)

Guo, Qiang; Bin Huang; Cheng, Jingdong; Seefelder, Manuel; Engler, Tatjana; Pfeifer, Günter; Oeckl, Patrick; Otto, Markus; Moser, Franziska; Maurer, Melanie; Pautsch, Alexander; Baumeister, Wolfgang; Fernández-Busnadiego, Rubén; Kochanek, Stefan

2018-03-01

Huntingtin (HTT) is a large (348 kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington’s disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely α-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.

Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity.

PubMed

Lorenz, Sonja; Deng, Patricia; Hantschel, Oliver; Superti-Furga, Giulio; Kuriyan, John

2015-06-01

Constitutive activation of the non-receptor tyrosine kinase c-Abl (cellular Abelson tyrosine protein kinase 1, Abl1) in the Bcr (breakpoint cluster region)-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukaemia (CML). Recent studies have indicated that an interaction between the SH2 (Src-homology 2) domain and the N-lobe (N-terminal lobe) of the c-Abl kinase domain (KD) has a critical role in leukaemogenesis [Grebien et al. (2011) Cell 147, 306-319; Sherbenou et al. (2010) Blood 116, 3278-3285]. To dissect the structural basis of this phenomenon, we studied c-Abl constructs comprising the SH2 and KDs in vitro. We present a crystal structure of an SH2-KD construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the KD. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2/N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the KD. That the effects are small compared with the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the auto-inhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.

The European DISABKIDS project: development of seven condition-specific modules to measure health related quality of life in children and adolescents

PubMed Central

Baars, Rolanda M; Atherton, Clare I; Koopman, Hendrik M; Bullinger, Monika; Power, Mick

2005-01-01

Background The European DISABKIDS project aims to enhance the Health Related Quality of Life (HRQoL) of children and adolescents with chronic medical conditions and their families. We describe the development of the seven cross-nationally tested condition-specific modules of the European DISABKIDS HRQoL instrument in a population of children and adolescents. The condition-specific modules are intended for use in conjunction with the DISABKIDS chronic generic module. Methods Focus groups were used to construct the pilot version of the DISABKIDS condition-specific HRQoL modules for asthma, juvenile idiopathic arthritis, atopic dermatitis, cerebral palsy, cystic fibrosis, diabetes and epilepsy. Analyses were conducted on pilot test data in order to construct field test versions of the modules. A series of factor analyses were run, first, to determine potential structures for each condition-specific module, and, secondly, to select a reduced number of items from the pilot test to be included in the field test. Post-field test analyses were conducted to retest the domain structure for the final DISABKIDS condition-specific modules. Results The DISABKIDS condition-specific modules were tested in a pilot study of 360 respondents, and subsequently in a field test of 1152 respondents in 7 European countries. The final condition-specific modules consist of an 'Impact' domain and an additional domain (e.g. worry, stigma, treatment) with between 10 to 12 items in total. The Cronbach's alpha of the final domains was found to vary from 0.71 to 0.90. Conclusion The condition-specific modules of the DISABKIDS instrument were developed through a step-by-step process including cognitive interview, clinical expertise, factor analysis, correlations and internal consistency. A cross-national pilot and field test were necessary to collect these data. In general, the internal consistency of the domains was satisfactory to high. In future, the DISABKIDS instrument may serve as a useful tool with which to assess HRQoL in children and adolescents with a chronic condition. The condition-specific modules can be used in conjunction with the DISABKIDS chronic generic module. PMID:16283947

The European DISABKIDS project: development of seven condition-specific modules to measure health related quality of life in children and adolescents.

PubMed

Baars, Rolanda M; Atherton, Clare I; Koopman, Hendrik M; Bullinger, Monika; Power, Mick

2005-11-13

The European DISABKIDS project aims to enhance the Health Related Quality of Life (HRQoL) of children and adolescents with chronic medical conditions and their families. We describe the development of the seven cross-nationally tested condition-specific modules of the European DISABKIDS HRQoL instrument in a population of children and adolescents. The condition-specific modules are intended for use in conjunction with the DISABKIDS chronic generic module. Focus groups were used to construct the pilot version of the DISABKIDS condition-specific HRQoL modules for asthma, juvenile idiopathic arthritis, atopic dermatitis, cerebral palsy, cystic fibrosis, diabetes and epilepsy. Analyses were conducted on pilot test data in order to construct field test versions of the modules. A series of factor analyses were run, first, to determine potential structures for each condition-specific module, and, secondly, to select a reduced number of items from the pilot test to be included in the field test. Post-field test analyses were conducted to retest the domain structure for the final DISABKIDS condition-specific modules. The DISABKIDS condition-specific modules were tested in a pilot study of 360 respondents, and subsequently in a field test of 1152 respondents in 7 European countries. The final condition-specific modules consist of an 'Impact' domain and an additional domain (e.g. worry, stigma, treatment) with between 10 to 12 items in total. The Cronbach's alpha of the final domains was found to vary from 0.71 to 0.90. The condition-specific modules of the DISABKIDS instrument were developed through a step-by-step process including cognitive interview, clinical expertise, factor analysis, correlations and internal consistency. A cross-national pilot and field test were necessary to collect these data. In general, the internal consistency of the domains was satisfactory to high. In future, the DISABKIDS instrument may serve as a useful tool with which to assess HRQoL in children and adolescents with a chronic condition. The condition-specific modules can be used in conjunction with the DISABKIDS chronic generic module.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

PubMed

Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

2017-06-06

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

PubMed Central

Mandal, Soumit Sankar; Buchsteiner, Maximilian; Dima, Ruxandra I.; Rief, Matthias; Žoldák, Gabriel

2017-01-01

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements. PMID:28533394

Conserved and variable domains of RNase MRP RNA.

PubMed

Dávila López, Marcela; Rosenblad, Magnus Alm; Samuelsson, Tore

2009-01-01

Ribonuclease MRP is a eukaryotic ribonucleoprotein complex consisting of one RNA molecule and 7-10 protein subunits. One important function of MRP is to catalyze an endonucleolytic cleavage during processing of rRNA precursors. RNase MRP is evolutionary related to RNase P which is critical for tRNA processing. A large number of MRP RNA sequences that now are available have been used to identify conserved primary and secondary structure features of the molecule. MRP RNA has structural features in common with P RNA such as a conserved catalytic core, but it also has unique features and is characterized by a domain highly variable between species. Information regarding primary and secondary structure features is of interest not only in basic studies of the function of MRP RNA, but also because mutations in the RNA give rise to human genetic diseases such as cartilage-hair hypoplasia.

Finite element analysis and optimization of composite structures

NASA Technical Reports Server (NTRS)

Thomsen, Jan

1990-01-01

Linearly elastic fiber reinforced composite discs and laminates in plane stress with variable local orientation and concentration of one or two fiber fields embedded in the matrix material, are considered. The thicknesses and the domain of the discs or laminates are assumed to be given, together with prescribed boundary conditions and in-plane loading along the edge. The problem under study consists in determining throughout the structural domain the optimum orientations and concentrations of the fiber fields in such a way as to maximize the integral stiffness of the composite disc or laminate under the seven loading. Minimization of the integral stiffness can also be performed. The optimization is performed subject to a prescribed bound on the total cost or weight of the composite that for given unit cost factors or specific weights determines the amounts of fiber and matrix materials in the structure. Examples are presented.

Synthesis of regional crust and upper-mantle structure from seismic and gravity data

NASA Technical Reports Server (NTRS)

Alexander, S. S.; Lavin, P. M.

1979-01-01

Available seismic and ground based gravity data are combined to infer the three dimensional crust and upper mantle structure in selected regions. This synthesis and interpretation proceeds from large-scale average models suitable for early comparison with high-altitude satellite potential field data to more detailed delineation of structural boundaries and other variations that may be significant in natural resource assessment. Seismic and ground based gravity data are the primary focal point, but other relevant information (e.g. magnetic field, heat flow, Landsat imagery, geodetic leveling, and natural resources maps) is used to constrain the structure inferred and to assist in defining structural domains and boundaries. The seismic data consists of regional refraction lines, limited reflection coverage, surface wave dispersion, teleseismic P and S wave delay times, anelastic absorption, and regional seismicity patterns. The gravity data base consists of available point gravity determinations for the areas considered.

Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution.

PubMed

Wu, Dongxia; Ran, Tinting; Wang, Weiwu; Xu, Dongqing

2016-01-01

Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated from Serratia sp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin from Serratia sp. FS14 was solved to a crystallographic R factor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189-198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution

PubMed Central

Wu, Dongxia; Ran, Tinting; Wang, Weiwu; Xu, Dongqing

2016-01-01

Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated from Serratia sp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin from Serratia sp. FS14 was solved to a crystallographic R factor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a ‘parallel β-roll’ C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189–198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the ‘parallel β-roll’ are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability. PMID:26750478

Fe–Ni solid solutions in nano-size dimensions: Effect of hydrogen annealing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Asheesh, E-mail: asheeshk@barc.gov.in; Meena, S.S.; Banerjee, S.

Highlights: • Fe–Ni solid solution with nano-size dimensions were prepared and characterized. • Both as prepared and hydrogenated solid solutions have FCC structure of Ni. • Paramagnetic and ferromagnetic domains coexist in these samples. - Abstract: Nanoparticles of Ni{sub 0.50}Fe{sub 0.50} and Ni{sub 0.75}Fe{sub 0.25} alloys were prepared by chemical reduction in ethylene glycol medium. XRD and {sup 57}Fe Mössbauer studies have confirmed the formation of Fe–Ni solid solution in nano-size dimensions with FCC structure. These samples consist of both ferromagnetic and paramagnetic domains which have been attributed to the coexistence of large and small particles as confirmed by atomicmore » force microscopic (AFM) and {sup 57}Fe Mössbauer spectroscopic studies. Improved extent of Fe–Fe exchange interaction existing in Ni{sub 0.50}Fe{sub 0.50} alloy compared to Ni{sub 0.75}Fe{sub 0.25} alloy explains the observed increase in the relative extent of ferromagnetic domains compared to paramagnetic domains in the former sample. Increase in the relative extent of ferromagnetic domains for hydrogenated alloys is due to increase in particle size brought about by the high temperature activation prior to hydrogenation.« less

Characteristics of biogenic calcite in the prismatic layer of a pearl oyster, Pinctada fucata.

PubMed

Okumura, Taiga; Suzuki, Michio; Nagasawa, Hiromichi; Kogure, Toshihiro

2010-10-01

The fine structure of the calcite prism in the outer layer of a pearl oyster, Pinctada fucata, has been investigated using various electron beam techniques, in order to understand its characteristics and growth mechanism including the role of intracrystalline organic substances. As the calcite prismatic layer grows thicker, sinuous boundaries develop to divide the prism into a number of domains. The crystal misorientation between the adjacent domains is several to more than ten degrees. The component of the misorientation is mainly the rotation about the c-axis. There is no continuous organic membrane at the boundaries. Furthermore, the crystal orientation inside the domains changes gradually, as indicated by the electron back-scattered diffraction (EBSD) in a scanning electron microscope (SEM). Transmission electron microscopy (TEM) examination revealed that the domain consists of sub-grains of a few hundred nanometers divided by small-angle grain boundaries, which are probably the origin of the gradual change of the crystal orientation inside the domains. Spherular Fresnel contrasts were often observed at the small-angle grain boundaries, in defocused TEM images. Electron energy-loss spectroscopy (EELS) indicated the spherules are organic macromolecules, suggesting that incorporation of organic macromolecules during the crystal growth forms the sub-grain structure of the calcite prism.

Identification of Scoliosis Research Society-22r Health-Related Quality of Life questionnaire domains using factor analysis methodology.

PubMed

Lai, Sue-Min; Asher, Marc A; Burton, Douglas C; Carlson, Brandon B

2010-05-20

Cross-sectional mail questionnaire. Examination of the underlying construct validity of the Scoliosis Research Society-22r (SRS-22r) Health-Related Quality of Life (HRQoL) Questionnaire using factor analysis. The original SRS-24 HRQoL questionnaire has undergone a series of modifications in an effort to further improve its psychometric properties and validate its use in patients from 10 years of age until well into adulthood. The SRS-22r questionnaire is the result of this effort. To date, the underlying construct validity of the original English version has not been analyzed by factor analysis. A questionnaire including all questions on the SRS-24, -23, -22, and -22r questionnaires (49 total questions) was mailed to a consecutive series of 235 patients who had received primary posterior or anterior instrumentation and arthrodesis. Domain structure of the SRS-22r questions was analyzed using iterated principal factor analysis with orthogonal rotation. One hundred twenty-one (51%) of the patients, age 23.34 +/- 4.52 years (range, 14.16-34.57 years), returned the questionnaire at 8.63 +/- 4.00 years (range, 2.32-15.94 years) following surgery. Factor analysis using all 22 questions resulted in 3 factors with many shared items because of significant collinearity of the satisfaction/dissatisfaction with management questions with the others. After 18 iterations, factor analysis using the 20 nonmanagement questions revealed 4 factors that explained 98% of the variance. These factors parallel the assigned domains of the SRS-22r questionnaire. Three questions (2 self-image and 1 function) were identified that had high loading in 2 factors. However, internal consistency was best when 2 of the questions (1 self-image and 1 function) were retained in their assigned SRS-22r domains and the third decreased self-image internal consistency by only 0.01%. The internal consistencies (Cronbach alpha) of the assigned SRS-22r nonmanagement domains were excellent or very good: function 0.83, pain 0.87, self-image 0.80, and mental health 0.90. For the management domain it was good: 0.73. Factor analysis of the SRS-22r HRQoL confirms placement of the 20 nonmanagement domain questions in the assigned 4 domains, all with excellent or very good internal consistency.

Development and validation of a new questionnaire assessing quality of life in adults with hypopituitarism: Adult Hypopituitarism Questionnaire (AHQ).

PubMed

Ishii, Hitoshi; Shimatsu, Akira; Okimura, Yasuhiko; Tanaka, Toshiaki; Hizuka, Naomi; Kaji, Hidesuke; Hanew, Kunihiko; Oki, Yutaka; Yamashiro, Sayuri; Takano, Koji; Chihara, Kazuo

2012-01-01

To develop and validate the Adult Hypopituitarism Questionnaire (AHQ) as a disease-specific, self-administered questionnaire for evaluation of quality of life (QOL) in adult patients with hypopituitarism. We developed and validated this new questionnaire, using a standardized procedure which included item development, pilot-testing and psychometric validation. Of the patients who participated in psychometric validation, those whose clinical conditions were judged to be stable were asked to answer the survey questionnaire twice, in order to assess test-retest reliability. Content validity of the initial questionnaire was evaluated via two pilot tests. After these tests, we made minor revisions and finalized the initial version of the questionnaire. The questionnaire was constructed with two domains, one psycho-social and the other physical. For psychometric assessment, analyses were performed on the responses of 192 adult patients with various types of hypopituitarism. The intraclass correlations of the respective domains were 0.91 and 0.95, and the Cronbach's alpha coefficients were 0.96 and 0.95, indicating adequate test-retest reliability and internal consistency for each domain. For known-group validity, patients with hypopituitarism due to hypothalamic disorder showed significantly lower scores in 11 out of 13 sub-domains compared to those who had hypopituitarism due to pituitary disorder. Regarding construct validity, the domain structure was found to be almost the same as that initially hypothesized. Exploratory factor analysis (n = 228) demonstrated that each domain consisted of six and seven sub-domains. The AHQ showed good reliability and validity for evaluating QOL in adult patients with hypopituitarism.

Analyzing Individual Differences in Vocational, Leisure, and Family Interests: A Multitrait-Multimethod Approach

ERIC Educational Resources Information Center

Gaudron, Jean-Philippe; Vautier, Stephane

2007-01-01

This study investigates the latent structure of individual differences in vocational, leisure, and family interests. The participants consisted of 302 French adults who rated their like or dislike for activities based on Holland's RIASEC typology and presented in three life domains: work, family and leisure. The multitrait-multimethod model…

The Subjective Well-Being Construct: A Test of Its Convergent, Discriminant, and Factorial Validity

ERIC Educational Resources Information Center

Arthaud-day, Marne L.; Rode, Joseph C.; Mooney, Christine H.; Near, Janet P.

2005-01-01

Using structural equation modeling, we found empirical support for the prevailing theory that subjective well-being consists of three domains: (1) cognitive evaluations of one's life (i.e., life satisfaction or happiness); (2) positive affect; and (3) negative affect. Multiple indicators of satisfaction/happiness were shown to have strong…

Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins.

PubMed Central

Cook, W B; Walker, J C

1992-01-01

A cDNA encoding a nuclear-encoded chloroplast nucleic acid-binding protein (NBP) has been isolated from maize. Identified as an in vitro DNA-binding activity, NBP belongs to a family of nuclear-encoded chloroplast proteins which share a common domain structure and are thought to be involved in posttranscriptional regulation of chloroplast gene expression. NBP contains an N-terminal chloroplast transit peptide, a highly acidic domain and a pair of ribonucleoprotein consensus sequence domains. NBP is expressed in a light-dependent, organ-specific manner which is consistent with its involvement in chloroplast biogenesis. The relationship of NBP to the other members of this protein family and their possible regulatory functions are discussed. Images PMID:1346929

A low energy electron microscopy study of the initial growth, structure, and thermal stability of 4,4'-biphenyldicarboxylic acid domains on Cu(001)

NASA Astrophysics Data System (ADS)

Khokhar, Fawad S.; van Gastel, Raoul; Schwarz, Daniel; Zandvliet, Harold J. W.; Poelsema, Bene

2011-09-01

The growth of 4,4'-biphenyldicarboxylic acid (BDA) on Cu(001) has been studied using low energy electron microscopy and selective area low energy electron diffraction. The emergence of large islands and hydrogen bonding to perpendicularly oriented, adjacent molecules is confirmed. The two benzene rings of adsorbed BDA are twisted along the molecular axis. Unconventional growth of the domains, followed by a second nucleation stage, is observed at room temperature. This unanticipated feature is attributed to the accumulation of stress in the islands. Ostwald ripening in the films and the decay of BDA domains at 448 K exhibits features that are consistent with diffusion limited behavior.

Hydroxycinnamic acid-derived polymers constitute the polyaromatic domain of suberin

NASA Technical Reports Server (NTRS)

Bernards, M. A.; Lopez, M. L.; Zajicek, J.; Lewis, N. G.

1995-01-01

Suberin is an abundant, complex, intractable, plant cell wall polymeric network that forms both protective and wound-healing layers. Its function is, therefore, critical to the survival of all vascular plants. Its chemical structure and biosynthesis are poorly defined, although it is known to consist of both aromatic and aliphatic domains. While the composition of the aliphatic component has been fairly well characterized, that of the phenolic component has not. Using a combination of specific carbon-13 labeling techniques, and in situ solid state 13C NMR spectroscopic analysis, we now provide the first direct evidence for the nature of the phenolic domain of suberin and report here that it is almost exclusively comprised of a covalently linked, hydroxycinnamic acid-derived polymeric matrix.

Development of terminology for mammographic techniques for radiological technologists.

PubMed

Yagahara, Ayako; Yokooka, Yuki; Tsuji, Shintaro; Nishimoto, Naoki; Uesugi, Masahito; Muto, Hiroshi; Ohba, Hisateru; Kurowarabi, Kunio; Ogasawara, Katsuhiko

2011-07-01

We are developing a mammographic ontology to share knowledge of the mammographic domain for radiologic technologists, with the aim of improving mammographic techniques. As a first step in constructing the ontology, we used mammography reference books to establish mammographic terminology for identifying currently available knowledge. This study proceeded in three steps: (1) determination of the domain and scope of the terminology, (2) lexical extraction, and (3) construction of hierarchical structures. We extracted terms mainly from three reference books and constructed the hierarchical structures manually. We compared features of the terms extracted from the three reference books. We constructed a terminology consisting of 440 subclasses grouped into 19 top-level classes: anatomic entity, image quality factor, findings, material, risk, breast, histological classification of breast tumors, role, foreign body, mammographic technique, physics, purpose of mammography examination, explanation of mammography examination, image development, abbreviation, quality control, equipment, interpretation, and evaluation of clinical imaging. The number of terms that occurred in the subclasses varied depending on which reference book was used. We developed a terminology of mammographic techniques for radiologic technologists consisting of 440 terms.

Importance of the lid and cap domains for the catalytic activity of gastric lipases.

PubMed

Miled, N; Bussetta, C; De caro, A; Rivière, M; Berti, L; Canaan, S

2003-09-01

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

Structure and Orientation of T4 Lysozyme Bound to the Small Heat Shock Protein α-Crystallin

PubMed Central

Claxton, Derek P.; Zou, Ping; Mchaourab, Hassane S.

2008-01-01

Summary We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat-shock protein α-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active site cleft. Furthermore, increased distances between i and i+4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, EPR spectral lineshapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and α-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two-modes of substrate binding originate from two discrete binding sites on the chaperone. PMID:18062989

Inverse scattering pre-stack depth imaging and it's comparison to some depth migration methods for imaging rich fault complex structure

NASA Astrophysics Data System (ADS)

Nurhandoko, Bagus Endar B.; Sukmana, Indriani; Mubarok, Syahrul; Deny, Agus; Widowati, Sri; Kurniadi, Rizal

2012-06-01

Migration is important issue for seismic imaging in complex structure. In this decade, depth imaging becomes important tools for producing accurate image in depth imaging instead of time domain imaging. The challenge of depth migration method, however, is in revealing the complex structure of subsurface. There are many methods of depth migration with their advantages and weaknesses. In this paper, we show our propose method of pre-stack depth migration based on time domain inverse scattering wave equation. Hopefully this method can be as solution for imaging complex structure in Indonesia, especially in rich thrusting fault zones. In this research, we develop a recent advance wave equation migration based on time domain inverse scattering wave which use more natural wave propagation using scattering wave. This wave equation pre-stack depth migration use time domain inverse scattering wave equation based on Helmholtz equation. To provide true amplitude recovery, an inverse of divergence procedure and recovering transmission loss are considered of pre-stack migration. Benchmarking the propose inverse scattering pre-stack depth migration with the other migration methods are also presented, i.e.: wave equation pre-stack depth migration, waveequation depth migration, and pre-stack time migration method. This inverse scattering pre-stack depth migration could image successfully the rich fault zone which consist extremely dip and resulting superior quality of seismic image. The image quality of inverse scattering migration is much better than the others migration methods.

Crystal Structure of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete chrysosporium*S⃞

PubMed Central

Ishida, Takuya; Fushinobu, Shinya; Kawai, Rie; Kitaoka, Motomitsu; Igarashi, Kiyohiko; Samejima, Masahiro

2009-01-01

Glycoside hydrolase family 55 consists of β-1,3-glucanases mainly from filamentous fungi. A β-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes β-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from β-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two β-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two β-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various β-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two β-helical domains, demonstrating an unexpected variety of carbohydrate binding modes. PMID:19193645

An efficient depth map preprocessing method based on structure-aided domain transform smoothing for 3D view generation

PubMed Central

Ma, Liyan; Qiu, Bo; Cui, Mingyue; Ding, Jianwei

2017-01-01

Depth image-based rendering (DIBR), which is used to render virtual views with a color image and the corresponding depth map, is one of the key techniques in the 2D to 3D conversion process. Due to the absence of knowledge about the 3D structure of a scene and its corresponding texture, DIBR in the 2D to 3D conversion process, inevitably leads to holes in the resulting 3D image as a result of newly-exposed areas. In this paper, we proposed a structure-aided depth map preprocessing framework in the transformed domain, which is inspired by recently proposed domain transform for its low complexity and high efficiency. Firstly, our framework integrates hybrid constraints including scene structure, edge consistency and visual saliency information in the transformed domain to improve the performance of depth map preprocess in an implicit way. Then, adaptive smooth localization is cooperated and realized in the proposed framework to further reduce over-smoothness and enhance optimization in the non-hole regions. Different from the other similar methods, the proposed method can simultaneously achieve the effects of hole filling, edge correction and local smoothing for typical depth maps in a united framework. Thanks to these advantages, it can yield visually satisfactory results with less computational complexity for high quality 2D to 3D conversion. Numerical experimental results demonstrate the excellent performances of the proposed method. PMID:28407027

Structure of C3b reveals conformational changes that underlie complement activity.

PubMed

Janssen, Bert J C; Christodoulidou, Agni; McCarthy, Andrew; Lambris, John D; Gros, Piet

2006-11-09

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Crystal structure refinement of ReSi1.75 with an ordered arrangement of silicon vacancies

NASA Astrophysics Data System (ADS)

Harada, Shunta; Hoshikawa, Hiroaki; Kuwabara, Kosuke; Tanaka, Katsushi; Okunishi, Eiji; Inui, Haruyuki

2011-08-01

The crystal structure and microstructure of ReSi1.75 were investigated by synchrotron X-ray diffraction combined with scanning transmission electron microscopy. ReSi1.75 contains an ordered arrangement of vacancies in Si sites in the underlying tetragonal C11b lattice of the MoSi2-type and the crystal structure is monoclinic with the space group Cm. Atomic positions of Si atoms near vacancies are considerably displaced from the corresponding positions in the parent C11b structure, and they exhibit anomalously large local thermal vibration accompanied by large values of atomic displacement parameter. There are four differently-oriented domains with two of them being related to each other by the 90° rotation about the c-axis of the underlying C11b lattice and the other two being their respective twins. The habit planes for domain boundaries observed experimentally are consistent with those predicted with ferroelastic theory.

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

PubMed

Goss, Reimund; Greifenhagen, Anne; Bergner, Juliane; Volke, Daniela; Hoffmann, Ralf; Wilhelm, Christian; Schaller-Laudel, Susann

2017-04-01

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

A Neuro-Oncology Workstation for Structuring, Modeling, and Visualizing Patient Records

PubMed Central

Hsu, William; Arnold, Corey W.; Taira, Ricky K.

2016-01-01

The patient medical record contains a wealth of information consisting of prior observations, interpretations, and interventions that need to be interpreted and applied towards decisions regarding current patient care. Given the time constraints and the large—often extraneous—amount of data available, clinicians are tasked with the challenge of performing a comprehensive review of how a disease progresses in individual patients. To facilitate this process, we demonstrate a neuro-oncology workstation that assists in structuring and visualizing medical data to promote an evidence-based approach for understanding a patient’s record. The workstation consists of three components: 1) a structuring tool that incorporates natural language processing to assist with the extraction of problems, findings, and attributes for structuring observations, events, and inferences stated within medical reports; 2) a data modeling tool that provides a comprehensive and consistent representation of concepts for the disease-specific domain; and 3) a visual workbench for visualizing, navigating, and querying the structured data to enable retrieval of relevant portions of the patient record. We discuss this workstation in the context of reviewing cases of glioblastoma multiforme patients. PMID:27583308

A Neuro-Oncology Workstation for Structuring, Modeling, and Visualizing Patient Records.

PubMed

Hsu, William; Arnold, Corey W; Taira, Ricky K

2010-11-01

The patient medical record contains a wealth of information consisting of prior observations, interpretations, and interventions that need to be interpreted and applied towards decisions regarding current patient care. Given the time constraints and the large-often extraneous-amount of data available, clinicians are tasked with the challenge of performing a comprehensive review of how a disease progresses in individual patients. To facilitate this process, we demonstrate a neuro-oncology workstation that assists in structuring and visualizing medical data to promote an evidence-based approach for understanding a patient's record. The workstation consists of three components: 1) a structuring tool that incorporates natural language processing to assist with the extraction of problems, findings, and attributes for structuring observations, events, and inferences stated within medical reports; 2) a data modeling tool that provides a comprehensive and consistent representation of concepts for the disease-specific domain; and 3) a visual workbench for visualizing, navigating, and querying the structured data to enable retrieval of relevant portions of the patient record. We discuss this workstation in the context of reviewing cases of glioblastoma multiforme patients.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.

PubMed

Horton, John R; Borgaro, Janine G; Griggs, Rose M; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong

2014-07-01

AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.

2014-07-03

AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves DNA containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises anmore » N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~ 70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~ 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.« less

Conformational heterogeneity in the C-terminal zinc fingers of human MTF-1: an NMR and zinc-binding study.

PubMed

Giedroc, D P; Chen, X; Pennella, M A; LiWang, A C

2001-11-09

The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys(2)-His(2) motifs, each of which was projected to form well-structured betabetaalpha domains upon Zn(II) binding. In this report, the structure and backbone dynamics of a fragment containing the unusual C-terminal fingers F4-F6 has been investigated. (15)N heteronuclear single quantum coherence (HSQC) spectra of uniformly (15)N-labeled hMTF-zf46 show that Zn(II) induces the folding of hMTF-zf46. Analysis of the secondary structure of Zn(3) hMTF-zf46 determined by (13)Calpha chemical shift indexing and the magnitude of (3)J(Halpha-HN) clearly reveal that zinc fingers F4 and F6 adopt typical betabetaalpha structures. An analysis of the heteronuclear backbone (15)N relaxation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution. Titration of apo-MTF-zf46 with Zn(II) reveals that the F4 domain binds Zn(II) significantly more tightly than do the other two finger domains. In contrast to fingers F4 and F6, the betabetaalpha fold of finger F5 is unstable and only partially populated at substoichiometric Zn(II); a slight molar excess of zinc results in severe conformational exchange broadening of all F5 NH cross-peaks. Finally, although Cd(II) binds to apo-hMTF-zf46 as revealed by intense S(-)-->Cd(II) absorption, a non-native structure results; addition of stoichiometric Zn(II) to the Cd(II) complex results in quantitative refolding of the betabetaalpha structure in F4 and F6. The functional implications of these results are discussed.

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auperin,T.; Bolduc, G.; Baron, M.

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} ofmore » the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.« less

Cellulose synthase (CesA) genes in the green alga Mesotaenium caldariorum.

PubMed

Roberts, Alison W; Roberts, Eric M; Delmer, Deborah P

2002-12-01

Cellulose, a microfibrillar polysaccharide consisting of bundles of beta-1,4-glucan chains, is a major component of plant and most algal cell walls and is also synthesized by some prokaryotes. Seed plants and bacteria differ in the structures of their membrane terminal complexes that make cellulose and, in turn, control the dimensions of the microfibrils produced. They also differ in the domain structures of their CesA gene products (the catalytic subunit of cellulose synthase), which have been localized to terminal complexes and appear to help maintain terminal complex structure. Terminal complex structures in algae range from rosettes (plant-like) to linear forms (bacterium-like). Thus, algal CesA genes may reveal domains that control terminal complex assembly and microfibril structure. The CesA genes from the alga Mesotaenium caldariorum, a member of the order Zygnematales, which have rosette terminal complexes, are remarkably similar to seed plant CesAs, with deduced amino acid sequence identities of up to 59%. In addition to the putative transmembrane helices and the D-D-D-QXXRW motif shared by all known CesA gene products, M. caldariorum and seed plant CesAs share a region conserved among plants, an N-terminal zinc-binding domain, and a variable or class-specific region. This indicates that the domains that characterize seed plant CesAs arose prior to the evolution of land plants and may play a role in maintaining the structures of rosette terminal complexes. The CesA genes identified in M. caldariorum are the first reported for any eukaryotic alga and will provide a basis for analyzing the CesA genes of algae with different types of terminal complexes.

Structural refinement of the hERG1 pore and voltage-sensing domains with ROSETTA-membrane and molecular dynamics simulations.

PubMed

Subbotina, Julia; Yarov-Yarovoy, Vladimir; Lees-Miller, James; Durdagi, Serdar; Guo, Jiqing; Duff, Henry J; Noskov, Sergei Yu

2010-11-01

The hERG1 gene (Kv11.1) encodes a voltage-gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template-driven de-novo design with ROSETTA-membrane modeling using side-chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2-Kv2.1 chimera channels, the missing parts are modeled de-novo. The impact of several alignments on the structure of the S4 helix in the voltage-sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage-sensor domain; and the topology of the extracellular S5-pore linker compared with that established by toxin foot-printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. © 2010 Wiley-Liss, Inc.

Structure and Interactions of the CS Domain of Human H/ACA RNP Assembly Protein Shq1

DOE PAGES

Singh, Mahavir; Wang, Zhonghua; Cascio, Duilio; ...

2014-12-29

Shq1 is an essential protein involved in the early steps of biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). Shq1 binds to dyskerin (Cbf5 in yeast) at an early step of H/ACA RNP assembly and is subsequently displaced by the H/ACA RNA. Shq1 contains an N-terminal CS and a C-terminal Shq1-specific domain (SSD). Dyskerin harbors many mutations associated with dyskeratosis congenita. Structures of yeast Shq1 SSD bound to Cbf5 revealed that only a subset of these mutations is in the SSD binding site, implicating another subset in the putative CS binding site. Here in this paper, we present the crystalmore » structure of human Shq1 CS (hCS) and the nuclear magnetic resonance (NMR) and crystal structures of hCS containing a serine substitution for proline 22 that is associated with some prostate cancers. The structure of hCS is similar to yeast Shq1 CS domain (yCS) and consists of two β-sheets that form an immunoglobulin-like β-sandwich fold. The N-terminal affinity tag sequence AHHHHHH associates with a neighboring protein in the crystal lattice to form an extra β-strand. Deletion of this tag was required to get spectra suitable for NMR structure determination, while the tag was required for crystallization. NMR chemical shift perturbation (CSP) experiments with peptides derived from putative CS binding sites on dyskerin and Cbf5 revealed a conserved surface on CS important for Cbf5/dyskerin binding. A HADDOCK (high-ambiguity-driven protein-protein docking) model of a Shq1-Cbf5 complex that defines the position of CS domain in the pre-H/ACA RNP was calculated using the CSP data.« less

Reliability, convergent validity and factor structure of the DASS-21 in a sample of Vietnamese adolescents.

PubMed

Le, Minh Thi Hong; Tran, Thach Duc; Holton, Sara; Nguyen, Huong Thanh; Wolfe, Rory; Fisher, Jane

2017-01-01

To assess the internal consistency, latent structure and convergent validity of the Depression, Anxiety and Stress Scale-21 (DASS-21) among adolescents in Vietnam. An anonymous, self-completed questionnaire was conducted among 1,745 high school students in Hanoi, Vietnam between October, 2013 and January, 2014. Confirmatory factor analyses were performed to assess the latent structure of the DASS-21. Factorial invariance between girls and boys was examined. Cronbach alphas and correlation coefficients between DASS-21 factor scores and the domain scores of the Duke Health Profile Adolescent Vietnamese validated version (ADHP-V) were calculated to assess DASS-21 internal consistency and convergent validity. A total of 1,606/ 1,745 (92.6%) students returned the questionnaire. Of those, 1,387 students provided complete DASS-21 data. The scale demonstrated adequate internal consistency (Cronbach α: 0.761 to 0.906). A four-factor model showed the best fit to the data. Items loaded significantly on a common general distress factor, the depression, and the anxiety factors, but few on the stress factor (p<0.05). DASS-21 convergent validity was confirmed with moderate correlation coefficients (-0.47 to -0.66) between its factor scores and the ADHP-V mental health related domains. The DASS-21 is reliable and suitable for use to assess symptoms of common mental health problems, especially depression and anxiety among Vietnamese adolescents. However, its ability in detecting stress among these adolescents may be limited. Further research is warrant to explore these results.

Crystal Structures of the Glutamate Receptor Ion Channel GluK3 and GluK5 Amino-Terminal Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Janesh; Mayer, Mark L.

2010-11-30

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 andmore » GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10{sup o}, in contrast to the 50{sup o} difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.« less

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding.

PubMed

Newcomer, Rebecca L; Fraser, LaTasha C R; Teschke, Carolyn M; Alexandrescu, Andrei T

2015-12-15

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining (3)JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of (1)HN and (15)N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and (3)JNC' H-bond couplings, are identified with an accuracy of 90% by (1)HN temperature coefficients. The accuracy is improved to 95% when (15)N temperature coefficients are also included. In contrast, the urea dependence of (1)HN and (15)N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke

Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and therebymore » suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.« less

Molecular dynamics simulations reveal a disorder-to-order transition on phosphorylation of smooth muscle myosin.

PubMed

Espinoza-Fonseca, L Michel; Kast, David; Thomas, David D

2007-09-15

We have performed molecular dynamics simulations of the phosphorylated (at S-19) and the unphosphorylated 25-residue N-terminal phosphorylation domain of the regulatory light chain (RLC) of smooth muscle myosin to provide insight into the structural basis of regulation. This domain does not appear in any crystal structure, so these simulations were combined with site-directed spin labeling to define its structure and dynamics. Simulations were carried out in explicit water at 310 K, starting with an ideal alpha-helix. In the absence of phosphorylation, large portions of the domain (residues S-2 to K-11 and R-16 through Y-21) were metastable throughout the simulation, undergoing rapid transitions among alpha-helix, pi-helix, and turn, whereas residues K-12 to Q-15 remained highly disordered, displaying a turn motif from 1 to 22.5 ns and a random coil pattern from 22.5 to 50 ns. Phosphorylation increased alpha-helical order dramatically in residues K-11 to A-17 but caused relatively little change in the immediate vicinity of the phosphorylation site (S-19). Phosphorylation also increased the overall dynamic stability, as evidenced by smaller temporal fluctuations in the root mean-square deviation. These results on the isolated phosphorylation domain, predicting a disorder-to-order transition induced by phosphorylation, are remarkably consistent with published experimental data involving site-directed spin labeling of the intact RLC bound to the two-headed heavy meromyosin. The simulations provide new insight into structural details not revealed by experiment, allowing us to propose a refined model for the mechanism by which phosphorylation affects the N-terminal domain of the RLC of smooth muscle myosin.

Cooperative Allosteric Ligand Binding in Calmodulin

NASA Astrophysics Data System (ADS)

Nandigrami, Prithviraj

Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (micro). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of micro. First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM. Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops. Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM. (Abstract shortened by ProQuest.).

Protein domain assignment from the recurrence of locally similar structures

PubMed Central

Tai, Chin-Hsien; Sam, Vichetra; Gibrat, Jean-Francois; Garnier, Jean; Munson, Peter J.

2010-01-01

Domains are basic units of protein structure and essential for exploring protein fold space and structure evolution. With the structural genomics initiative, the number of protein structures in the Protein Databank (PDB) is increasing dramatically and domain assignments need to be done automatically. Most existing structural domain assignment programs define domains using the compactness of the domains and/or the number and strength of intra-domain versus inter-domain contacts. Here we present a different approach based on the recurrence of locally similar structural pieces (LSSPs) found by one-against-all structure comparisons with a dataset of 6,373 protein chains from the PDB. Residues of the query protein are clustered using LSSPs via three different procedures to define domains. This approach gives results that are comparable to several existing programs that use geometrical and other structural information explicitly. Remarkably, most of the proteins that contribute the LSSPs defining a domain do not themselves contain the domain of interest. This study shows that domains can be defined by a collection of relatively small locally similar structural pieces containing, on average, four secondary structure elements. In addition, it indicates that domains are indeed made of recurrent small structural pieces that are used to build protein structures of many different folds as suggested by recent studies. PMID:21287617

New Tricks for “Old” Domains: How Novel Architectures and Promiscuous Hubs Contributed to the Organization and Evolution of the ECM

PubMed Central

Cromar, Graham; Wong, Ka-Chun; Loughran, Noeleen; On, Tuan; Song, Hongyan; Xiong, Xuejian; Zhang, Zhaolei; Parkinson, John

2014-01-01

The extracellular matrix (ECM) is a defining characteristic of metazoans and consists of a meshwork of self-assembling, fibrous proteins, and their functionally related neighbours. Previous studies, focusing on a limited number of gene families, suggest that vertebrate complexity predominantly arose through the duplication and subsequent modification of retained, preexisting ECM genes. These genes provided the structural underpinnings to support a variety of specialized tissues, as well as a platform for the organization of spatio-temporal signaling and cell migration. However, the relative contributions of ancient versus novel domains to ECM evolution have not been quantified across the full range of ECM proteins. Here, utilizing a high quality list comprising 324 ECM genes, we reveal general and clade-specific domain combinations, identifying domains of eukaryotic and metazoan origin recruited into new roles in approximately two-third of the ECM proteins in humans representing novel vertebrate proteins. We show that, rather than acquiring new domains, sampling of new domain combinations has been key to the innovation of paralogous ECM genes during vertebrate evolution. Applying a novel framework for identifying potentially important, noncontiguous, conserved arrangements of domains, we find that the distinct biological characteristics of the ECM have arisen through unique evolutionary processes. These include the preferential recruitment of novel domains to existing architectures and the utilization of high promiscuity domains in organizing the ECM network around a connected array of structural hubs. Our focus on ECM proteins reveals that distinct types of proteins and/or the biological systems in which they operate have influenced the types of evolutionary forces that drive protein innovation. This emphasizes the need for rigorously defined systems to address questions of evolution that focus on specific systems of interacting proteins. PMID:25323955

A comprehensive computational study on pathogenic mis-sense mutations spanning the RING2 and REP domains of Parkin protein.

PubMed

Biswas, Ria; Bagchi, Angshuman

2017-04-30

Various mutations in PARK2 gene, which encodes the protein parkin, are significantly associated with the onset of autosomal recessive juvenile Parkinson (ARJP) in neuronal cells. Parkin is a multi domain protein, the N-terminal part contains the Ubl and the C-terminal part consists of four zinc coordinating domains, viz., RING0, RING1, in between ring (IBR) and RING2. Disease mutations are spread over all the domains of Parkin, although mutations in some regions may affect the functionality of Parkin more adversely. The mutations in the RING2 domain are seen to abolish the neuroprotective E3 ligase activity of Parkin. In this current work, we carried out detailed in silico analysis to study the extent of pathogenicity of mutations spanning the Parkin RING2 domain and the adjoining REP region by SIFT, Mutation Accessor, PolyPhen2, SNPs and GO, GV/GD and I-mutant. To study the structural and functional implications of these mutations on RING2-REP domain of Parkin, we studied the solvent accessibility (SASA/RSA), hydrophobicity, intra-molecular hydrogen bonding profile and domain analysis by various computational tools. Finally, we analysed the interaction energy profiles of the mutants and compared them to the wild type protein using Discovery studio 2.5. By comparing the various analyses it could be safely concluded that except P437L and A379V mutations, all other mutations were potentially deleterious affecting various structural aspects of RING2 domain architecture. This study is based purely on computational approach which has the potential to identify disease mutations and the information could further be used in treatment of diseases and prognosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Single-channel kinetics of BK (Slo1) channels

PubMed Central

Geng, Yanyan; Magleby, Karl L.

2014-01-01

Single-channel kinetics has proven a powerful tool to reveal information about the gating mechanisms that control the opening and closing of ion channels. This introductory review focuses on the gating of large conductance Ca2+- and voltage-activated K+ (BK or Slo1) channels at the single-channel level. It starts with single-channel current records and progresses to presentation and analysis of single-channel data and the development of gating mechanisms in terms of discrete state Markov (DSM) models. The DSM models are formulated in terms of the tetrameric modular structure of BK channels, consisting of a central transmembrane pore-gate domain (PGD) attached to four surrounding transmembrane voltage sensing domains (VSD) and a large intracellular cytosolic domain (CTD), also referred to as the gating ring. The modular structure and data analysis shows that the Ca2+ and voltage dependent gating considered separately can each be approximated by 10-state two-tiered models with five closed states on the upper tier and five open states on the lower tier. The modular structure and joint Ca2+ and voltage dependent gating are consistent with a 50 state two-tiered model with 25 closed states on the upper tier and 25 open states on the lower tier. Adding an additional tier of brief closed (flicker states) to the 10-state or 50-state models improved the description of the gating. For fixed experimental conditions a channel would gate in only a subset of the potential number of states. The detected number of states and the correlations between adjacent interval durations are consistent with the tiered models. The examined models can account for the single-channel kinetics and the bursting behavior of gating. Ca2+ and voltage activate BK channels by predominantly increasing the effective opening rate of the channel with a smaller decrease in the effective closing rate. Ca2+ and depolarization thus activate by mainly destabilizing the closed states. PMID:25653620

Role of the constant region domain in the structural diversity of human antibody light chains.

PubMed

Hifumi, Emi; Taguchi, Hiroaki; Kato, Ryuichi; Uda, Taizo

2017-04-01

Issues regarding the structural diversity (heterogeneity) of an antibody molecule have been the subject of discussion along with the development of antibody drugs. Research on heterogeneity has been extensive in recent years, but no clear solution has been reached. Heterogeneity is also observed in catalytic antibody κ light chains (CLs). In this study, we investigated how the constant region domain of CLs concerns structural diversity because it is a simple and good example for elucidating heterogeneity. By means of cation-exchange chromatography, SDS-PAGE, and 2-dimensional electrophoresis for the CL, multimolecular forms consisting of different electrical charges and molecular sizes coexisted in the solution, resulting in the similar heterogeneity of the full length of CLs. The addition of copper ion could cause the multimolecular forms to change to monomolecular forms. Copper ion contributed greatly to the enrichment of the dimer form of CL and the homogenization of the differently charged CLs. Two molecules of the CL protein bound one copper ion. The binding affinity of the ion was 48.0 μM -1 Several divalent metal ions were examined, but only zinc showed a similar effect.-Hifumi, E., Taguchi, H., Kato, R., Uda, T. Role of the constant region domain in the structural diversity of human antibody light chains. © FASEB.

Coiled-coil intermediate filament stutter instability and molecular unfolding.

PubMed

Arslan, Melis; Qin, Zhao; Buehler, Markus J

2011-05-01

Intermediate filaments (IFs) are the key components of cytoskeleton in eukaryotic cells and are critical for cell mechanics. The building block of IFs is a coiled-coil alpha-helical dimer, consisting of several domains that include linkers and other structural discontinuities. One of the discontinuities in the dimer's coiled-coil region is the so-called 'stutter' region. The stutter is a region where a variation of the amino acid sequence pattern from other parts of the alpha-helical domains of the protein is found. It was suggested in earlier works that due to this sequence variation, the perfect coiled-coil arrangement ceases to exist. Here, we show using explicit water molecular dynamics and well-tempered metadynamics that for the coil2 domain of vimentin IFs the stutter is more stable in a non-alpha-helical, unfolded state. This causes a local structural disturbance in the alpha helix, which has a global effect on the nanomechanics of the structure. Our analysis suggests that the stutter features an enhanced tendency to unfolding even under the absence of external forces, implying a much greater structural instability than previously assumed. As a result it features a smaller local bending stiffness than other segments and presents a seed for the initiation of molecular bending and unfolding at large deformation.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hui; Klein, Michael G.; Snell, Gyorgy

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structuremore » reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.« less

System Specificity of the TpsB Transporters of Coexpressed Two-Partner Secretion Systems of Neisseria meningitidis

PubMed Central

ur Rahman, Sadeeq

2013-01-01

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure. PMID:23222722

Dynamic Self-Consistent Field Theories for Polymer Blends and Block Copolymers

NASA Astrophysics Data System (ADS)

Kawakatsu, Toshihiro

Understanding the behavior of the phase separated domain structures and rheological properties of multi-component polymeric systems require detailed information on the dynamics of domains and that of conformations of constituent polymer chains. Self-consistent field (SCF) theory is a useful tool to treat such a problem because the conformation entropy of polymer chains in inhomogeneous systems can be evaluated quantitatively using this theory. However, when we turn our attention to the dynamic properties in a non-equilibrium state, the basic assumption of the SCF theory, i.e. the assumption of equilibrium chain conformation, breaks down. In order to avoid such a difficulty, dynamic SCF theories were developed. In this chapter, we give a brief review of the recent developments of dynamic SCF theories, and discuss where the cutting-edge of this theory is.

A mixed formulation for interlaminar stresses in dropped-ply laminates

NASA Technical Reports Server (NTRS)

Harrison, Peter N.; Johnson, Eric R.

1993-01-01

A structural model is developed for the linear elastic response of structures consisting of multiple layers of varying thickness such as laminated composites containing internal ply drop-offs. The assumption of generalized plane deformation is used to reduce the solution domain to two dimensions while still allowing some out-of-plane deformation. The Hellinger-Reissner variational principle is applied to a layerwise assumed stress distribution with the resulting governing equations solved using finite differences.

The Crystal Structures of the Open and Catalytically Competent Closed Conformation of Escherichia coli Glycogen Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheng, Fang; Jia, Xiaofei; Yep, Alejandra

2009-07-06

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl groupmore » of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site« less

Analysis of proton wires in the enzyme active site suggests a mechanism of c-di-GMP hydrolysis by the EAL domain phosphodiesterases.

PubMed

Grigorenko, Bella L; Knyazeva, Marina A; Nemukhin, Alexander V

2016-11-01

We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Folding pathway of a multidomain protein depends on its topology of domain connectivity

PubMed Central

Inanami, Takashi; Terada, Tomoki P.; Sasai, Masaki

2014-01-01

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant. PMID:25267632

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

PubMed Central

2009-01-01

Background Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. Results Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. Conclusion The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family. PMID:19930663

Analysis of the structure and dynamics of human serum albumin.

PubMed

Guizado, T R Cuya

2014-10-01

Human serum albumin (HSA) is a biologically relevant protein that binds a variety of drugs and other small molecules. No less than 50 structures are deposited in the RCSB Protein Data Bank (PDB). Based on these structures, we first performed a clustering analysis. Despite the diversity of ligands, only two well defined conformations are detected, with a deviation of 0.46 nm between the average structures of the two clusters, while deviations within each cluster are smaller than 0.08 nm. Those two conformations are representative of the apoprotein and the HSA-myristate complex already identified in previous literature. Considering the structures within each cluster as a representative sample of the dynamical states of the corresponding conformation, we scrutinize the structural and dynamical differences between both conformations. Analysis of the fluctuations within each cluster set reveals that domain II is the most rigid one and better matches both structures. Then, taking this domain as reference, we show that the structural difference between both conformations can be expressed in terms of twist and hinge motions of domains I and III, respectively. We also characterize the dynamical difference between conformations by computing correlations and principal components for each set of dynamical states. The two conformations display different collective motions. The results are compared with those obtained from the trajectories of short molecular dynamics simulations, giving consistent outcomes. Let us remark that, beyond the relevance of the results for the structural and dynamical characterization of HAS conformations, the present methodology could be extended to other proteins in the PDB archive.

Autotransporter-based cell surface display in Gram-negative bacteria.

PubMed

Nicolay, Toon; Vanderleyden, Jos; Spaepen, Stijn

2015-02-01

Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

Conservation and variability in the structure and function of the Cas5d endoribonuclease in the CRISPR-mediated microbial immune system.

PubMed

Koo, Yoon; Ka, Donghyun; Kim, Eun-Jin; Suh, Nayoung; Bae, Euiyoung

2013-10-23

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR-Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event. Copyright © 2013 Elsevier Ltd. All rights reserved.

Boundaries, kinetic properties, and final domain structure of plane discrete uniform Poisson-Voronoi tessellations with von Neumann neighborhoods.

PubMed

Korobov, A

2009-03-01

Discrete random tessellations appear not infrequently in describing nucleation and growth transformations. Generally, several non-Euclidean metrics are possible in this case. Previously [A. Korobov, Phys. Rev. B 76, 085430 (2007)] continual analogs of such tessellations have been studied. Here one of the simplest discrete varieties of the Kolmogorov-Johnson-Mehl-Avrami model, namely, the model with von Neumann neighborhoods, has been examined per se, i.e., without continualization. The tessellation is uniform in the sense that domain boundaries consist of tiles. Similarities and distinctions between discrete and continual models are discussed.

Structural and functional aspects of C1-inhibitor.

PubMed

Bos, Ineke G A; Hack, C Erik; Abrahams, Jan Pieter

2002-09-01

C1-Inh is a serpin that inhibits serine proteases from the complement and the coagulation pathway. C1-Inh consists of a serpin domain and a unique N-terminal domain and is heavily glycosylated. Non-functional mutants of C1-Inh can give insight into the inhibitory mechanism of C1-Inh. This review describes a novel 3D model of C1-Inh, based on a newly developed homology modelling method. This model gives insight into a possible potentiation mechanism of C1-Inh and based on this model the essential residues for efficient inhibition by C1-Inh are discussed.

Defining popular iconic metaphor.

PubMed

Columbus, Peter J; Boerger, Michael A

2002-04-01

Popular Iconic Metaphor is added to the cognitive linguistic lexicon of figurative language. Popular Iconic Metaphors employ real or fictional celebrities of popular culture as source domains in figurative discourse. Some borders of Popular Iconic Metaphor are identified, and Elvis Presley is offered as a prototype example of a popular iconic source domain, due to his ubiquity in American popular culture, which affords his figurative usage in ways consistent with decision heuristics in everyday life. Further study of Popular Iconic Metaphors may serve to illuminate how figurative expressions emerge in their localized contexts, structure conduct and experience, and affect mediation of cultural and personal meanings.

The Thiamin Pyrophosphate-Motif

NASA Technical Reports Server (NTRS)

Dominiak, P.; Ciszak, E.

2003-01-01

Using databases the authors have identified a common thiamin pyrophosphate (TPP)-motif in the family of functionally diverse TPP-dependent enzymes. This common motif consists of multimeric organization of subunits and two catalytic centers. Each catalytic center (PP:PYR) is formed at the interface of the PP-domain binding the magnesium ion, pyrophosphate and amhopyrimidine ring of TPP, and the PYR-domain binding the aminopyrimidine ring of that cofactor. A pair of these catalytic centers constitutes the catalytic core (PP:PYR)(sub 2) within these enzymes. Analysis of the structural elements of this catalytic core reveals novel definition of the common amino acid sequences, which are GXPhiX(sub 4)(G)PhiXXGQ and GDGX(sub 25-30)NN in the PP-domain, and the EX(sub 4)(G)PhiXXGPhi in the PYR-domain, where Phi corresponds to a hydrophobic amino acid. This TPP-motif provides a novel tool for annotation of TPP-dependent enzymes useful in advancing functional proteomics.

Crystallization and Preliminary X-ray Analysis of the Human Long Myosin Light-Chain Kinase 1-Specific Domain IgCAM3

DOE Office of Scientific and Technical Information (OSTI.GOV)

W Vallen Graham; A Magis; K Bailey

2011-12-31

Myosin light-chain kinase-dependent tight junction regulation is a critical event in inflammatory cytokine-induced increases in epithelial paracellular permeability. MLCK is expressed in human intestinal epithelium as two isoforms, long MLCK1 and long MLCK2, and MLCK1 is specifically localized to the tight junction, where it regulates paracellular permeability. The sole difference between these long MLCK splice variants is the presence of an immunoglobulin-like cell-adhesion molecule domain, IgCAM3, in MLCK1. To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to 2.0 {angstrom} resolution andmore » were consistent with the primitive trigonal space group P2{sub 1}2{sub 1}2{sub 1}.« less

Conformational Flexibility of Metazoan Fatty Acid Synthase Enables Catalysis

PubMed Central

Brignole, Edward J.; Smith, Stuart; Asturias, Francisco J.

2008-01-01

The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers. While the 3D architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains. Using single-particle electron microscopy we have identified a near continuum of conformations consistent with remarkable flexibility of FAS. The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations suggesting a direct correlation between conformation and specific enzymatic activities. 3D reconstructions were interpreted by docking high-resolution structures of individual domains and illustrate that the substrate loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber. Concomitant rearrangement of the β-carbon processing domains synchronizes acyl-chain reduction in one chamber with acyl-chain elongation in the other. PMID:19151726

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC

PubMed Central

Reboul, Cyril F.; Cowieson, Nathan P.; Costa, Mauricio G. S.; Kass, Itamar; Jackson, Colin; Perahia, David; Buckle, Ashley M.; McGowan, Sheena

2015-01-01

The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency. PMID:26470022

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

PubMed Central

Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

2010-01-01

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:20072699

Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

PubMed

Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

2010-01-11

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Drawing on Experience: How Domain Knowledge Is Reflected in Sketches of Scientific Structures and Processes

NASA Astrophysics Data System (ADS)

Jee, Benjamin D.; Gentner, Dedre; Uttal, David H.; Sageman, Bradley; Forbus, Kenneth; Manduca, Cathryn A.; Ormand, Carol J.; Shipley, Thomas F.; Tikoff, Basil

2014-12-01

Capturing the nature of students' mental representations and how they change with learning is a primary goal in science education research. This can be challenging in spatially intense domains, such as geoscience, architecture, and engineering. In this research, we test whether sketching can be used to gauge level of expertise in geoscience, using new technology designed to facilitate this process. We asked participants with differing levels of geoscience experience to copy two kinds of geoscience images—photographs of rock formations and causal diagrams. To permit studying the process of sketching as well as the structure and content of the sketches, we used the CogSketch system (Forbus et al. 2011, Topics in Cognitive Science 3:648-666) to record the time course of sketching and analyze the sketches themselves. Relative to novices, geoscience students included more geological structures and relational symbols in their sketches of geoscience materials and were more likely to construct their sketches in a sequence consistent with the order of causal events. These differences appear to stem from differences in domain knowledge, because they did not show up in participants' sketches of materials from other fields. The findings and methods of this research suggest new ways to promote and assess science learning, which are well suited to the visual-spatial demands of many domains.

Predicting Health Care Utilization in Marginalized Populations: Black, Female, Street-based Sex Workers

PubMed Central

Varga, Leah M.; Surratt, Hilary L.

2014-01-01

Background Patterns of social and structural factors experienced by vulnerable populations may negatively affect willingness and ability to seek out health care services, and ultimately, their health. Methods The outcome variable was utilization of health care services in the previous 12 months. Using Andersen’s Behavioral Model for Vulnerable Populations, we examined self-reported data on utilization of health care services among a sample of 546 Black, street-based female sex workers in Miami, Florida. To evaluate the impact of each domain of the model on predicting health care utilization, domains were included in the logistic regression analysis by blocks using the traditional variables first and then adding the vulnerable domain variables. Findings The most consistent variables predicting health care utilization were having a regular source of care and self-rated health. The model that included only enabling variables was the most efficient model in predicting health care utilization. Conclusions Any type of resource, link, or connection to or with an institution, or any consistent point of care contributes significantly to health care utilization behaviors. A consistent and reliable source for health care may increase health care utilization and subsequently decrease health disparities among vulnerable and marginalized populations, as well as contribute to public health efforts that encourage preventive health. PMID:24657047

Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements.

PubMed

Kukic, Predrag; Lundström, Patrik; Camilloni, Carlo; Evenäs, Johan; Akke, Mikael; Vendruscolo, Michele

2016-01-12

Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca(2+)-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, (15)N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca(2+) by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca(2+)-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

Structural Effects of Two Camelid Nanobodies Directed to Distinct C-Terminal Epitopes on α-Synuclein.

PubMed

El-Turk, Farah; Newby, Francisco N; De Genst, Erwin; Guilliams, Tim; Sprules, Tara; Mittermaier, Anthony; Dobson, Christopher M; Vendruscolo, Michele

2016-06-07

α-Synuclein is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease and other related neurodegenerative disorders. Recently, two single-domain camelid antibodies (nanobodies) were shown to bind α-synuclein with high affinity. Herein, we investigated how these two nanobodies (NbSyn2 and NbSyn87), which are directed to two distinct epitopes within the C-terminal domain of α-synuclein, affect the conformational properties of this protein. Our results suggest that nanobody NbSyn2, which binds to the five C-terminal residues of α-synuclein (residues 136-140), does not disrupt the transient long-range interactions that generate a degree of compaction within the native structural ensemble of α-synuclein. In contrast, the data that we report indicate that NbSyn87, which targets a central region within the C-terminal domain (residues 118-128), has more substantial effects on the fluctuating secondary and tertiary structure of the protein. These results are consistent with the different effects that the two nanobodies have on the aggregation behavior of α-synuclein in vitro. Our findings thus provide new insights into the type of effects that nanobodies can have on the conformational ensemble of α-synuclein.

Relationship auditing of the FMA ontology

PubMed Central

Gu, Huanying (Helen); Wei, Duo; Mejino, Jose L.V.; Elhanan, Gai

2010-01-01

The Foundational Model of Anatomy (FMA) ontology is a domain reference ontology based on a disciplined modeling approach. Due to its large size, semantic complexity and manual data entry process, errors and inconsistencies are unavoidable and might remain within the FMA structure without detection. In this paper, we present computable methods to highlight candidate concepts for various relationship assignment errors. The process starts with locating structures formed by transitive structural relationships (part_of, tributary_of, branch_of) and examine their assignments in the context of the IS-A hierarchy. The algorithms were designed to detect five major categories of possible incorrect relationship assignments: circular, mutually exclusive, redundant, inconsistent, and missed entries. A domain expert reviewed samples of these presumptive errors to confirm the findings. Seven thousand and fifty-two presumptive errors were detected, the largest proportion related to part_of relationship assignments. The results highlight the fact that errors are unavoidable in complex ontologies and that well designed algorithms can help domain experts to focus on concepts with high likelihood of errors and maximize their effort to ensure consistency and reliability. In the future similar methods might be integrated with data entry processes to offer real-time error detection. PMID:19475727

Mapping disease-related missense mutations in the immunoglobulin-like fold domain of lamin A/C reveals novel genotype-phenotype associations for laminopathies.

PubMed

Scharner, Juergen; Lu, Hui-Chun; Fraternali, Franca; Ellis, Juliet A; Zammit, Peter S

2014-06-01

Mutations in A-type nuclear lamins cause laminopathies. However, genotype-phenotype correlations using the 340 missense mutations within the LMNA gene are unclear: partially due to the limited availability of three-dimensional structure. The immunoglobulin (Ig)-like fold domain has been solved, and using bioinformatics tools (including Polyphen-2, Fold X, Parameter OPtimized Surfaces, and PocketPicker) we characterized 56 missense mutations for position, surface exposure, change in charge and effect on Ig-like fold stability. We find that 21 of the 27 mutations associated with a skeletal muscle phenotype are distributed throughout the Ig-like fold, are nonsurface exposed and predicted to disrupt overall stability of the Ig-like fold domain. Intriguingly, the remaining 6 mutations clustered, had higher surface exposure, and did not affect stability. The majority of 9 lipodystrophy or 10 premature aging syndrome mutations also did not disrupt Ig-like fold domain stability and were surface exposed and clustered in distinct regions that overlap predicted binding pockets. Although buried, the 10 cardiac mutations had no other consistent properties. Finally, most lipodystrophy and premature aging mutations resulted in a -1 net charge change, whereas skeletal muscle mutations caused no consistent net charge changes. Since premature aging, lipodystrophy and the subset of 6 skeletal muscle mutations cluster tightly in distinct, charged regions, they likely affect lamin A/C -protein/DNA/RNA interactions: providing a consistent genotype-phenotype relationship for mutations in this domain. Thus, this subgroup of skeletal muscle laminopathies that we term the 'Skeletal muscle cluster', may have a distinct pathological mechanism. These novel associations refine the ability to predict clinical features caused by certain LMNA missense mutations. © 2013 Wiley Periodicals, Inc.

Structure of Bombyx mori Densovirus 1, a Silkworm Pathogen▿‡

PubMed Central

Kaufmann, Bärbel; El-Far, Mohamed; Plevka, Pavel; Bowman, Valorie D.; Li, Yi; Tijssen, Peter; Rossmann, Michael G.

2011-01-01

Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-Å resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel “jelly roll” fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a “domain-swapped” conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae. PMID:21367906

Spin Polarized Transport in Multilayer Structures with Complex Magnetic Configurations

NASA Astrophysics Data System (ADS)

Sahakyan, Avag; Poghosyan, Anahit; Movsesyan, Ruzan; Kocharian, Armen

The spin transport and spin polarization in a new class of multilayer structures are investigated for non-collinear and noncoplanar magnetic configurations containing repetitive magnetic layers. The magnetic configuration of the structure dictates the existence of certain degrees of freedom that determines magnetic transport and polarization properties. We consider magnetic structures in magnetic multilayers with canted spin configurations separated by non-magnetic quantum well so that the exchange interaction between the neighbor barriers can be ignored. Configurations of magnetizations in barriers include some structures consisting of two ''ferromagnetic'' or ''antiferromagnetic'' domains twisted relative to each other by a certain angle (angle noncollinearity). The similar system, formed from two noncollinear domains separated by canted ''magnetic defect'' is also considered. The above mentioned properties of these systems depend strongly on the type of magnetic configuration and variation of certain degrees of freedom. Simple theoretical approach with the transfer matrix method is carried out to understand and predict the magnetic properties of the multilayer systems. The work at California University Los Angeles was supported by the National Science Foundation-Partnerships for Research and Education in Materials under Grant DMR-1523588.

The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core.

PubMed

Bergal, Hans Thor; Hopkins, Alex Hunt; Metzner, Sandra Ines; Sousa, Marcelo Carlos

2016-02-02

The β-barrel assembly machine (BAM) mediates folding and insertion of integral β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermus marinus. The POTRA5 domain binds BamD between its tetratricopeptide repeats 3 and 4. The interface structural elements are conserved in the Escherichia coli proteins, which allowed structure validation by mutagenesis and disulfide crosslinking in E. coli. Furthermore, the interface is consistent with previously reported mutations that impair BamA-BamD binding. The structure serves as a linchpin to generate a BAM model where POTRA domains and BamD form an elongated periplasmic ring adjacent to the membrane with a central cavity approximately 30 × 60 Å wide. We propose that nascent OMPs bind this periplasmic ring prior to insertion and folding by BAM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure.

PubMed

Beyer, Eric C; Lipkind, Gregory M; Kyle, John W; Berthoud, Viviana M

2012-08-01

The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011. Published by Elsevier B.V.

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

PubMed

Pons, T; Hernández, L; Batista, F R; Chinea, G

2000-11-01

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

PubMed Central

Pons, T.; Hernández, L.; Batista, F. R.; Chinea, G.

2000-01-01

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family. PMID:11305239

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy.

PubMed

Mainprize, Iain L; Beniac, Daniel R; Falkovskaia, Elena; Cleverley, Robert M; Gierasch, Lila M; Ottensmeyer, F Peter; Andrews, David W

2006-12-01

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.

Global Organization of a Positive-strand RNA Virus Genome

PubMed Central

Wu, Baodong; Grigull, Jörg; Ore, Moriam O.; Morin, Sylvie; White, K. Andrew

2013-01-01

The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV) contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2′-hydroxyl acylation analysed by primer extension (i.e. SHAPE), which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context. PMID:23717202

Developing Learning Materials Using an Ontology of Mathematical Logic

ERIC Educational Resources Information Center

Boyatt, Russell; Joy, Mike

2012-01-01

Ontologies describe a body of knowledge and give formal structure to a domain by describing concepts and their relationships. The construction of an ontology provides an opportunity to develop a shared understanding and a consistent vocabulary to be used for a given activity. This paper describes the construction of an ontology for an area of…

Photonic Crystals on the Wing

DTIC Science & Technology

2009-04-30

The Green Hairstreak butterfly has ventrally scales consisting of 3D gyroid domains acting as photonic crystals, which can be easily...study on the structural coloration of the Green Hairstreak , Callophrys rubi. Extensive experimental measurements have been complemented with three...colorations, produced by purely dielectric materials are widely encountered [1-3]. Famous examples are the metallic- green buprestid and golden scarab

Contribution of Implicit Sequence Learning to Spoken Language Processing: Some Preliminary Findings with Hearing Adults

ERIC Educational Resources Information Center

Conway, Christopher M.; Karpicke, Jennifer; Pisoni, David B.

2007-01-01

Spoken language consists of a complex, sequentially arrayed signal that contains patterns that can be described in terms of statistical relations among language units. Previous research has suggested that a domain-general ability to learn structured sequential patterns may underlie language acquisition. To test this prediction, we examined the…

Hidden Structural Codes in Protein Intrinsic Disorder.

PubMed

Borkosky, Silvia S; Camporeale, Gabriela; Chemes, Lucía B; Risso, Marikena; Noval, María Gabriela; Sánchez, Ignacio E; Alonso, Leonardo G; de Prat Gay, Gonzalo

2017-10-17

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure-function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and β-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Application of Wavelet Transform for PDZ Domain Classification

PubMed Central

Daqrouq, Khaled; Alhmouz, Rami; Balamesh, Ahmed; Memic, Adnan

2015-01-01

PDZ domains have been identified as part of an array of signaling proteins that are often unrelated, except for the well-conserved structural PDZ domain they contain. These domains have been linked to many disease processes including common Avian influenza, as well as very rare conditions such as Fraser and Usher syndromes. Historically, based on the interactions and the nature of bonds they form, PDZ domains have most often been classified into one of three classes (class I, class II and others - class III), that is directly dependent on their binding partner. In this study, we report on three unique feature extraction approaches based on the bigram and trigram occurrence and existence rearrangements within the domain's primary amino acid sequences in assisting PDZ domain classification. Wavelet packet transform (WPT) and Shannon entropy denoted by wavelet entropy (WE) feature extraction methods were proposed. Using 115 unique human and mouse PDZ domains, the existence rearrangement approach yielded a high recognition rate (78.34%), which outperformed our occurrence rearrangements based method. The recognition rate was (81.41%) with validation technique. The method reported for PDZ domain classification from primary sequences proved to be an encouraging approach for obtaining consistent classification results. We anticipate that by increasing the database size, we can further improve feature extraction and correct classification. PMID:25860375

Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yang; Näsvall, Joakim; Wu, Shiying

The crystal structure of the aminoglycoside-adenylating enzyme AadA is reported together with functional experiments providing insights into its oligomeric state, ligand binding and catalysis. Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundlemore » domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.« less

Assessing implementation difficulties in tobacco use prevention and cessation counselling among dental providers

PubMed Central

2011-01-01

Background Tobacco use adversely affects oral health. Clinical guidelines recommend that dental providers promote tobacco abstinence and provide patients who use tobacco with brief tobacco use cessation counselling. Research shows that these guidelines are seldom implemented, however. To improve guideline adherence and to develop effective interventions, it is essential to understand provider behaviour and challenges to implementation. This study aimed to develop a theoretically informed measure for assessing among dental providers implementation difficulties related to tobacco use prevention and cessation (TUPAC) counselling guidelines, to evaluate those difficulties among a sample of dental providers, and to investigate a possible underlying structure of applied theoretical domains. Methods A 35-item questionnaire was developed based on key theoretical domains relevant to the implementation behaviours of healthcare providers. Specific items were drawn mostly from the literature on TUPAC counselling studies of healthcare providers. The data were collected from dentists (n = 73) and dental hygienists (n = 22) in 36 dental clinics in Finland using a web-based survey. Of 95 providers, 73 participated (76.8%). We used Cronbach's alpha to ascertain the internal consistency of the questionnaire. Mean domain scores were calculated to assess different aspects of implementation difficulties and exploratory factor analysis to assess the theoretical domain structure. The authors agreed on the labels assigned to the factors on the basis of their component domains and the broader behavioural and theoretical literature. Results Internal consistency values for theoretical domains varied from 0.50 ('emotion') to 0.71 ('environmental context and resources'). The domain environmental context and resources had the lowest mean score (21.3%; 95% confidence interval [CI], 17.2 to 25.4) and was identified as a potential implementation difficulty. The domain emotion provided the highest mean score (60%; 95% CI, 55.0 to 65.0). Three factors were extracted that explain 70.8% of the variance: motivation (47.6% of variance, α = 0.86), capability (13.3% of variance, α = 0.83), and opportunity (10.0% of variance, α = 0.71). Conclusions This study demonstrated a theoretically informed approach to identifying possible implementation difficulties in TUPAC counselling among dental providers. This approach provides a method for moving from diagnosing implementation difficulties to designing and evaluating interventions. PMID:21615948

Novel Mechanism of Hemin Capture by Hbp2, the Hemoglobin-binding Hemophore from Listeria monocytogenes*

PubMed Central

Malmirchegini, G. Reza; Sjodt, Megan; Shnitkind, Sergey; Sawaya, Michael R.; Rosinski, Justin; Newton, Salete M.; Klebba, Phillip E.; Clubb, Robert T.

2014-01-01

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2N2; residues 183–303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2N2 undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron. PMID:25315777

1H, 13C, and 15N resonance assignments of an enzymatically active domain from the catalytic component (CDTa, residues 216-420) of a binary toxin from Clostridium difficile.

PubMed

Roth, Braden M; Godoy-Ruiz, Raquel; Varney, Kristen M; Rustandi, Richard R; Weber, David J

2016-04-01

Clostridium difficile is a bacterial pathogen and is the most commonly reported source of nosocomial infection in industrialized nations. Symptoms of C. difficile infection (CDI) include antibiotic-associated diarrhea, pseudomembranous colitis, sepsis and death. Over the last decade, rates and severity of hospital infections in North America and Europe have increased dramatically and correlate with the emergence of a hypervirulent strain of C. difficile characterized by the presence of a binary toxin, CDT (C. difficile toxin). The binary toxin consists of an enzymatic component (CDTa) and a cellular binding component (CDTb) that together form the active binary toxin complex. CDTa harbors a pair of structurally similar but functionally distinct domains, an N-terminal domain (residues 1-215; (1-215)CDTa) that interacts with CDTb and a C-terminal domain (residues 216-420; (216-420)CDTa) that harbors the intact ADP-ribosyltransferase (ART) active site. Reported here are the (1)H, (13)C, and (15)N backbone resonance assignments of the 23 kDa, 205 amino acid C-terminal enzymatic domain of CDTa, termed (216-420)CDTa. These NMR resonance assignments for (216-420)CDTa represent the first for a family of ART binary toxins and provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.

Investigating the structure of the restricted, repetitive behaviours and interests domain of autism.

PubMed

Szatmari, Peter; Georgiades, Stelios; Bryson, Susan; Zwaigenbaum, Lonnie; Roberts, Wendy; Mahoney, William; Goldberg, Jeremy; Tuff, Lawrence

2006-06-01

The Restricted, Repetitive Behaviours and Interests (RRBIs) are represented in the DSM-IV and measured by the Autism Diagnostic Interview-Revised (ADI-R) as one of the three homogeneous symptom categories of Pervasive Developmental Disorders. Although this conceptualisation is well accepted in the field, the grouping of symptoms is based primarily on clinical judgment rather than on empirical evidence. The objective of this study was to examine the factor structure of the RRBI domain of autism. Eleven items from this domain of the ADI-R were used in a Principal Components Analysis (PCA). Our sample consisted of 339 individuals with a Best Estimate diagnosis of Pervasive Developmental Disorder (PDD). Findings indicate that the RRBI domain is composed of two distinct factors or dimensions: Insistence on Sameness (IS) and Repetitive Sensory and Motor Behaviours (RSMB). RSMB is negatively correlated with adaptive skills; that is, lower functioning individuals tend to have higher levels of repetitive sensory and motor behaviours. On the other hand, IS is positively correlated with autistic symptoms in the communication and language domain. Further analyses suggest moderate familial aggregation among affected sibling pairs within the IS but not the RSMB factor. These results provide evidence for the heterogeneity of the RRBI domain of the ADI-R in terms of both clinical presentation and other correlates. In addition, the IS factor seems to be under familial (presumably genetic) control, while RSMB appears to simply reflect variation in developmental level.

Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

PubMed

Horn, James V C; Ellena, Rachel A; Tran, Jesse J; Beck, Wendy H J; Narayanaswami, Vasanthy; Weers, Paul M M

2017-08-01

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Basis of the Heterodimer Formation between Cell Shape-Determining Proteins Csd1 and Csd2 from Helicobacter pylori.

PubMed

An, Doo Ri; Im, Ha Na; Jang, Jun Young; Kim, Hyoun Sook; Kim, Jieun; Yoon, Hye Jin; Hesek, Dusan; Lee, Mijoon; Mobashery, Shahriar; Kim, Soon-Jong; Suh, Se Won

2016-01-01

Colonization of the human gastric mucosa by Helicobacter pylori requires its high motility, which depends on the helical cell shape. In H. pylori, several genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5, and csd6) play key roles in determining the cell shape by alteration of cross-linking or by trimming of peptidoglycan stem peptides. H. pylori Csd1, Csd2, and Csd3/HdpA are M23B metallopeptidase family members and may act as d,d-endopeptidases to cleave the d-Ala4-mDAP3 peptide bond of cross-linked dimer muropeptides. Csd3 functions also as the d,d-carboxypeptidase to cleave the d-Ala4-d-Ala5 bond of the muramyl pentapeptide. To provide a basis for understanding molecular functions of Csd1 and Csd2, we have carried out their structural characterizations. We have discovered that (i) Csd2 exists in monomer-dimer equilibrium and (ii) Csd1 and Csd2 form a heterodimer. We have determined crystal structures of the Csd2121-308 homodimer and the heterodimer between Csd1125-312 and Csd2121-308. Overall structures of Csd1125-312 and Csd2121-308 monomers are similar to each other, consisting of a helical domain and a LytM domain. The helical domains of both Csd1 and Csd2 play a key role in the formation of homodimers or heterodimers. The Csd1 LytM domain contains a catalytic site with a Zn2+ ion, which is coordinated by three conserved ligands and two water molecules, whereas the Csd2 LytM domain has incomplete metal ligands and no metal ion is bound. Structural knowledge of these proteins sheds light on the events that regulate the cell wall in H. pylori.