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Sample records for 3clpro structure basis

  1. Potential Broad Spectrum Inhibitors of the Coronavirus 3CLpro: A Virtual Screening and Structure-Based Drug Design Study

    PubMed Central

    Berry, Michael; Fielding, Burtram C.; Gamieldien, Junaid

    2015-01-01

    Human coronaviruses represent a significant disease burden; however, there is currently no antiviral strategy to combat infection. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) less than 10 years later demonstrates the potential of coronaviruses to cross species boundaries and further highlights the importance of identifying novel lead compounds with broad spectrum activity. The coronavirus 3CLpro provides a highly validated drug target and as there is a high degree of sequence homology and conservation in main chain architecture the design of broad spectrum inhibitors is viable. The ZINC drugs-now library was screened in a consensus high-throughput pharmacophore modeling and molecular docking approach by Vina, Glide, GOLD and MM-GBSA. Molecular dynamics further confirmed results obtained from structure-based techniques. A highly defined hit-list of 19 compounds was identified by the structure-based drug design methodologies. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds is bioactive is excellent. Additionally, the compounds segregate into 15 significantly dissimilar (p < 0.05) clusters based on shape and features, which represent valuable scaffolds that can be used as a basis for future anti-coronaviral inhibitor discovery experiments. Importantly though, the enriched subset of 19 compounds identified from the larger library has to be validated experimentally. PMID:26694449

  2. Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro)

    PubMed Central

    Tomar, Sakshi; Johnston, Melanie L.; St. John, Sarah E.; Osswald, Heather L.; Nyalapatla, Prasanth R.; Paul, Lake N.; Ghosh, Arun K.; Denison, Mark R.; Mesecar, Andrew D.

    2015-01-01

    All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CLpro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CLpro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CLpro is less efficient at processing a peptide substrate due to MERS-CoV 3CLpro being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CLpro enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CLpro is a weakly associated dimer (Kd ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CLpro were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CLpro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CLpro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CLpro dimerization. Activation of MERS-CoV 3CLpro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CLpro inhibitors as antiviral agents. PMID:26055715

  3. X-ray structure and inhibition of 3C-like protease from porcine epidemic diarrhea virus

    DOE PAGES

    St. John, Sarah E.; Anson, Brandon J.; Mesecar, Andrew D.

    2016-05-13

    Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CLpro) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 angstrom X-ray structure of 3CLpro from PEDV. Analysis of the PEDV 3CLpro structure and comparison to other coronaviral 3CLpro's from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CLpro's are conserved, with the exception of a loop that comprises the proteasemore » S-2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro, (R)-16, to have inhibitor activity against PEDV 3CLpro, despite that SARS-3CLpro and PEDV 3CLpro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CLpro are conserved in PEDV-3CLpro; however, the sequence variation and positional difference in the loop forming the S-2 pocket may account for large observed difference in IC50 values. In conclusion, this work advances our understanding of the subtle, but important, differences in coronaviral 3CLpro architecture and contributes to the broader structural knowledge of coronaviral 3CLpro's.« less

  4. X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus

    PubMed Central

    St. John, Sarah E.; Anson, Brandon J.; Mesecar, Andrew D.

    2016-01-01

    Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CLpro) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CLpro from PEDV. Analysis of the PEDV 3CLpro structure and comparison to other coronaviral 3CLpro’s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CLpro’s are conserved, with the exception of a loop that comprises the protease S2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro, (R)-16, to have inhibitor activity against PEDV 3CLpro, despite that SARS-3CLpro and PEDV 3CLpro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CLpro are conserved in PEDV-3CLpro; however, the sequence variation and positional difference in the loop forming the S2 pocket may account for large observed difference in IC50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CLpro architecture and contributes to the broader structural knowledge of coronaviral 3CLpro’s. PMID:27173881

  5. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-02-01

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

  6. Structural and Inhibitor Studies of Norovirus 3C-like Proteases

    PubMed Central

    Takahashi, Daisuke; Kim, Yunjeong; Lovell, Scott; Prakash, Om; Groutas, William C; Chang, Kyeong-Ok

    2013-01-01

    Noroviruses have a single-stranded, positive sense 7–8 kb RNA genome, which encodes a polyprotein precursor processed by a virus-encoded 3C-like cysteine protease (3CLpro) to generate mature non-structural proteins. Because processing of the polyprotein is essential for virus replication, norovirus 3CLpro has been targeted for the discovery of anti-norovirus small molecule therapeutics. Thus, we performed functional, structural and inhibition studies of norovirus 3CLpro with fluorescence resonance energy transfer (FRET) assay, X-ray crystallography, and NMR spectroscopy with a synthetic protease inhibitor. Three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murine norovirus-1 (MNV-1, genogroup V) were optimized for a FRET assay, and compared for the inhibitory activities of a synthetic protease inhibitor (GC376). The apo 3D structures of NV 3CLpro determined with X-ray crystallography and NMR spectroscopy were further analyzed. In addition, the binding mode of NV 3CLpro-GC376 was compared with X-ray crystallography and NMR spectroscopy. The results of this report provide insight into the interaction of NV 3CLpro with substrate/inhibitor for better understanding of the enzyme and antiviral drug development. PMID:24055466

  7. Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity

    PubMed Central

    Needle, Danielle; Lountos, George T.; Waugh, David S.

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3CLpro). Since 3CLpro is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3CLpro inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3CLpro enzyme were determined. The aim was to co-crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C-terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme–product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS-CoV 3CLpro; however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure-based design of small-molecule inhibitors of the MERS-CoV 3CLpro enzyme. PMID:25945576

  8. Discovery, Synthesis, And Structure-Based Optimization of a Series of N-(tert-Butyl)-2-(N-arylamido)-2-(pyridin-3-yl) Acetamides (ML188) as Potent Noncovalent Small Molecule Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL Protease

    SciTech Connect

    Jacobs, Jon; Grum-Tokars, Valerie; Zhou, Ya; Turlington, Mark; Saldanha, S. Adrian; Chase, Peter; Eggler, Aimee; Dawson, Eric S.; Baez-Santos, Yahira M.; Tomar, Sakshi; Mielech, Anna M.; Baker, Susan C.; Lindsley, Craig W.; Hodder, Peter; Mesecar, Andrew; Stauffer, Shaun R.

    2012-12-11

    A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). But, unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure–activity relationships within S1', S1, and S2enzyme binding pockets. Moreover, the X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.

  9. Structural basis of spectrin elasticity

    SciTech Connect

    Shen, B.W.; Stevens, F.J.; Luthi, U.; Goldin, S.B.

    1991-10-17

    A new model of human erythrocyte {alpha}-spectrin is proposed. The secondary structure of human erythrocyte {alpha}-spectrin and its folding into a condensed structure that can convert reversibly in situ, into an elongated configuration is predicted from its deduced protein sequence. Results from conformational and amphipathicity analyses suggest that {alpha}-spectrin consists mainly of short amphipathicity helices interconnected by flexible turns and/or coils. The distribution of charges and amphipathicity of the helices can facilitate their folding into stable domains of 4 and 3 helices surrounding a hydrophobic core. The association between adjacent four- and three-helix domains further organize them into recurring seven-helix motifs that might constitute the basic structural units of the extended {alpha}-spectrin. The elongated spectrin molecule packs, in a sinusoidal fashion, through interactions between neighboring motifs into a compact structure. We suggest that the reversible extension and contraction of this sigmoidally packed structure is the molecular basis of the mechanism by which spectrin contributes to the elasticity of the red cell membrane.

  10. Structural basis of metal hypersensitivity

    PubMed Central

    Wang, Yang

    2014-01-01

    Metal hypersensitivity is a common immune disorder. Human immune systems mount the allergic attacks on metal ions through skin contacts, lung inhalation and metal-containing artificial body implants. The consequences can be simple annoyances to life-threatening systemic illness. Allergic hyper-reactivities to nickel (Ni) and beryllium (Be) are the best-studied human metal hypersensitivities. Ni-contact dermatitis affects 10 % of the human population, whereas Be compounds are the culprits of chronic Be disease (CBD). αβ T cells (T cells) play a crucial role in these hypersensitivity reactions. Metal ions work as haptens and bind to the surface of major histocompatibility complex (MHC) and peptide complex. This modifies the binding surface of MHC and triggers the immune response of T cells. Metal-specific αβ T cell receptors (TCRs) are usually MHC restricted, especially MHC class II (MHCII) restricted. Numerous models have been proposed, yet the mechanisms and molecular basis of metal hypersensitivity remain elusive. Recently, we determined the crystal structures of the Ni and Be presenting human MHCII molecules, HLA-DR52c (DRA*0101, DRB3*0301) and HLA-DP2 (DPA1*0103, DPB1*0201). These structures revealed unusual features of MHCII molecules and shed light on how metal ions are recognized by T cells. PMID:22983897

  11. Structural basis for selectin mechanochemistry.

    PubMed

    Springer, Timothy A

    2009-01-01

    Selectins are adhesion molecules that resist large tensile forces applied by hydrodynamic forces to leukocytes binding to vessel walls. In crystals, the liganded (high-affinity) and unliganded (low-affinity) conformations differ in orientation between their tandem lectin and EGF domains. I examine how tensile force exerted on a selectin-ligand complex in vivo could favor the more extended, high-affinity conformation. Allostery is transmitted from the EGF-lectin domain interface to the ligand-binding interface on the lectin domain, 30 A away. Trp-1 of the lectin domain and the long axis of the EGF domain form an L-shaped prybar that is welded together by hydrogen bonds to the Trp-1 alpha-amino group. Pivoting of the prybar induced by force demolishes an interface between the Trp-1 side chain and the lectin domain at a switch1 region. These changes are transmitted by rigid body movement of the switch2 region to rearrangements in the switch3 region at the ligand binding site. Another switch region corresponds to a single residue in the EGF domain with large effects on ligand binding and rolling adhesion. Allostery in selectins, and the alignment of tensile force on a selectin-ligand complex with the transition pathway for conformational change, explain much of the structural basis for selectin mechanochemistry.

  12. Structural basis of transcription activation.

    PubMed

    Feng, Yu; Zhang, Yu; Ebright, Richard H

    2016-06-10

    Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme. PMID:27284196

  13. NCI Scientists Solve Structure of Protein that Enables MERS Virus to Spread | Poster

    Cancer.gov

    Scientists at the Frederick National Lab have produced three crystal structures that reveal a specific part of a protein that can be targeted to fight the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an emerging viral respiratory illness. Senior Investigator David Waugh, Ph.D., Macromolecular Crystallography Laboratory, has solved the structure of an enzyme known as the 3C-like protease (3CLpro), which, if blocked, can prevent the virus from replicating...

  14. Structural basis of enzymatic benzene ring reduction.

    PubMed

    Weinert, Tobias; Huwiler, Simona G; Kung, Johannes W; Weidenweber, Sina; Hellwig, Petra; Stärk, Hans-Joachim; Biskup, Till; Weber, Stefan; Cotelesage, Julien J H; George, Graham N; Ermler, Ulrich; Boll, Matthias

    2015-08-01

    In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.

  15. The structural basis for cancer treatment decisions

    PubMed Central

    Nussinov, Ruth; Jang, Hyunbum; Tsai, Chung-Jung

    2014-01-01

    Cancer treatment decisions rely on genetics, large data screens and clinical pharmacology. Here we point out that genetic analysis and treatment decisions may overlook critical elements in cancer development, progression and drug resistance. Two critical structural elements are missing in genetics-based decision-making: the mechanisms of oncogenic mutations and the cellular network which is rewired in cancer. These lay the foundation for the structural basis for cancer treatment decisions, which is rooted in the physical principles of the molecular conformational behavior of single molecules and their interactions. Improved tumor mutational analysis platforms and knowledge of the redundant pathways which can take over in cancer, may not only supplement known actionable findings, but forecast possible cancer progression and resistance. Such forward-looking can be powerful, endowing the oncologist with mechanistic insight and cancer prognosis, and consequently more informed treatment options. Examples include redundant pathways taking over after inhibition of EGFR constitutive activation, mutations in PIK3CA p110α and p85, and the non-hotspot AKT1 mutants conferring constitutive membrane localization. PMID:25277176

  16. Structural Basis for Catalysis by Onconase

    PubMed Central

    Lee, J. Eugene; Bae, Euiyoung; Bingman, Craig A.; Phillips, George N.; Raines, Ronald T.

    2007-01-01

    Onconase (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC·nucleic acid complexes: a T89N/E91A ONC·5′-AMP complex at 1.65 Å resolution and a wild-type ONC·d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis was used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH–kcat/KM profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 102-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known kcat/KM value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC·d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance. PMID:18001769

  17. Amyloid Polymorphism: Structural Basis and Neurobiological Relevance

    PubMed Central

    Tycko, Robert

    2015-01-01

    Summary Our understanding of the molecular structures of amyloid fibrils that are associated with neurodegenerative diseases, of mechanisms by which disease-associated peptides and proteins aggregate into fibrils, and of structural properties of aggregation intermediates has advanced considerably in recent years. Detailed molecular structural models for certain fibrils and aggregation intermediates are now available. It is now well established that amyloid fibrils are generally polymorphic at the molecular level, with a given peptide or protein being capable of forming a variety of distinct, self-propagating fibril structures. Recent results from structural studies and from studies involving cell cultures, transgenic animals, and human tissue provide initial evidence that molecular structural variations in amyloid fibrils and related aggregates may correlate with or even produce variations in disease development. This article reviews our current knowledge of the structural and mechanistic aspects of amyloid formation, as well as current evidence for the biological relevance of structural variations. PMID:25950632

  18. Basis for Structural Diversity in Homologous RNAs

    SciTech Connect

    Krasilnikov, Andrey S.; Xiao, Yinghua; Pan, Tao; Mondragon, Alfonso

    2010-03-08

    Large RNA molecules, such as ribozymes, fold with well-defined tertiary structures that are important for their activity. There are many instances of ribozymes with identical function but differences in their secondary structures, suggesting alternative tertiary folds. Here, we report a crystal structure of the 161-nucleotide specificity domain of an A-type ribonuclease P that differs in secondary and tertiary structure from the specificity domain of a B-type molecule. Despite the differences, the cores of the domains have similar three-dimensional structure. Remarkably, the similar geometry of the cores is stabilized by a different set of interactions involving distinct auxiliary elements.

  19. Structural basis of photosynthetic water-splitting

    SciTech Connect

    Shen, Jian-Ren; Kawakami, Keisuke; Kamiya, Nobuo

    2013-12-10

    Photosynthetic water-splitting takes place in photosystem II (PSII), a membrane protein complex consisting of 20 subunits with an overall molecular mass of 350 kDa. The light-induced water-splitting reaction catalyzed by PSII not only converts light energy into biologically useful chemical energy, but also provides us with oxygen indispensible for sustaining oxygenic life on the earth. We have solved the structure of PSII at a 1.9 Å resolution, from which, the detailed structure of the Mn{sub 4}CaO{sub 5}-cluster, the catalytic center for water-splitting, became clear. Based on the structure of PSII at the atomic resolution, possible mechanism of light-induced water-splitting was discussed.

  20. Structural basis for phosphatidylinositol-phosphate biosynthesis

    NASA Astrophysics Data System (ADS)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  1. Structural basis for phosphatidylinositol-phosphate biosynthesis

    PubMed Central

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-01-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis. PMID:26510127

  2. Structural basis for retroviral integration into nucleosomes.

    PubMed

    Maskell, Daniel P; Renault, Ludovic; Serrao, Erik; Lesbats, Paul; Matadeen, Rishi; Hare, Stephen; Lindemann, Dirk; Engelman, Alan N; Costa, Alessandro; Cherepanov, Peter

    2015-07-16

    Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

  3. Structural basis for retroviral integration into nucleosomes

    PubMed Central

    Maskell, Daniel P.; Renault, Ludovic; Serrao, Erik; Lesbats, Paul; Matadeen, Rishi; Hare, Stephen; Lindemann, Dirk; Engelman, Alan N.; Costa, Alessandro; Cherepanov, Peter

    2015-01-01

    Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1,2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. PMID:26061770

  4. Structural basis for PECAM-1 homophilic binding.

    PubMed

    Paddock, Cathy; Zhou, Dongwen; Lertkiatmongkol, Panida; Newman, Peter J; Zhu, Jieqing

    2016-02-25

    Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a β-sandwich topology formed by 2 sheets of antiparallel β strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å(2). These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions. PMID:26702061

  5. Structural Basis of Dscam Isoform Specificity

    SciTech Connect

    Meijers,R.; Puettmann-Holgado, R.; Skiniotis, G.; Liu, J.; Walz, T.; Wang, J.; Schmucker, D.

    2007-01-01

    The Dscam gene gives rise to thousands of diverse cell surface receptors1 thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

  6. Structural basis of cohesin cleavage by separase

    PubMed Central

    Lin, Zhonghui; Luo, Xuelian; Yu, Hongtao

    2016-01-01

    Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man1,2. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin3–6, and by phosphorylation of both the enzyme and substrates7–12. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here, we report crystal structures of the separase protease domain from Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study13, mutating two securin residues in a conserved motif that partially matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin. PMID:27027290

  7. Structural Basis for microRNA Targeting

    PubMed Central

    Schirle, Nicole T.; Sheu-Gruttadauria, Jessica; MacRae, Ian J.

    2015-01-01

    Summary MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. We determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides 2–5 for initial target pairing. Pairing to nt 2–5 promotes conformational changes that expose nt 2–8 and 13–16 for further target recognition. Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, while an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. These results explain the conserved nucleotide pairing patterns in animal miRNA target sites first observed over two decades ago. PMID:25359968

  8. Acquired prosopagnosia: structural basis and processing impairments.

    PubMed

    Davies-Thompson, Jodie; Pancaroglu, Raika; Barton, Jason

    2014-01-01

    Cognitive models propose a hierarchy of parallel processing stages in face perception, and functional neuroimaging shows a network of regions involved in face processing. Reflecting this, acquired prosopagnosia is not a single entity but a family of disorders with different anatomic lesions and different functional deficits. One classic distinction is between an apperceptive variant, in which there is impaired perception of facial structure, and an associative/amnestic variant, in which perception is relatively intact, with subsequent problems matching perception to facial memories, because of either disconnection or loss of those memories. These disorders also have to be distinguished from people-specific amnesia, a multimodal impairment, and prosop-anomia, in which familiarity with faces is preserved but access to names is disrupted. These different disorders can be conceived as specific deficits at different processing stages in cognitive models, and suggests that these functional stages may have distinct neuroanatomic substrates. It remains to be seen whether a similar anatomic and functional variability is present in developmental prosopagnosia.

  9. Structural basis for transcription inhibition by tagetitoxin

    PubMed Central

    Vassylyev, Dmitry G.; Svetlov, Vladimir; Vassylyeva, Marina N.; Perederina, Anna; Igarashi, Noriyuki; Matsugaki, Naohiro; Wakatsuki, Soichi; Artsimovitch, Irina

    2005-01-01

    Tagetitoxin (Tgt) inhibits plastid-encoded, bacterial and some eukaryotic RNA polymerases (RNAPs) by an unknown mechanism. A 2.4Å-resolution structure of the Thermus thermophilus RNAP/Tgt complex revealed that Tgt-binding site within the RNAP secondary channel overlaps with that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the β and β′ residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg2+ ion distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg2+ ion plays a key role in stabilization of an inactive transcription intermediate. This and other recent studies suggest that Mg-mediated remodeling of the active site could be a common theme in the regulation of catalysis by nucleic acid enzymes. PMID:16273103

  10. A structural basis for cellular senescence

    PubMed Central

    Aranda-Anzaldo, Armando

    2009-01-01

    Replicative senescence (RS) that limits the proliferating potential of normal eukaryotic cells occurs either by a cell-division counting mechanism linked to telomere erosion or prematurely through induction by cell stressors such as oncogene hyper-activation. However, there is evidence that RS also occurs by a stochastic process that is independent of number of cell divisions or cellular stress and yet it leads to a highly-stable, non-reversible post-mitotic state that may be long-lasting and that such a process is widely represented among higher eukaryotes. Here I present and discuss evidence that the interactions between DNA and the nuclear substructure, commonly known as the nuclear matrix, define a higher-order structure within the cell nucleus that following thermodynamic constraints, stochastically evolves towards maximum stability, thus becoming limiting for mitosis to occur. It is suggested that this process is responsible for ultimate replicative senescence and yet it is compatible with long-term cell survival. PMID:20157542

  11. Structural basis of respiratory syncytial virus neutralization by motavizumab

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Kim, Albert; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2010-04-13

    Motavizumab is {approx}tenfold more potent than its predecessor, palivizumab (Synagis), the FDA-approved monoclonal antibody used to prevent respiratory syncytial virus (RSV) infection. The structure of motavizumab in complex with a 24-residue peptide corresponding to its epitope on the RSV fusion (F) glycoprotein reveals the structural basis for this greater potency. Modeling suggests that motavizumab recognizes a different quaternary configuration of the F glycoprotein than that observed in a homologous structure.

  12. Identifying Psychopathy Subtypes on the Basis of Personality Structure

    ERIC Educational Resources Information Center

    Hicks, Brian M.; Markon, Kristian E.; Patrick, Christopher J.; Krueger, Robert F.; Newman, Joseph P.

    2004-01-01

    The authors used model-based cluster analysis to identify subtypes of criminal psychopaths on the basis of differences in personality structure. Participants included 96 male prisoners diagnosed as psychopathic, using the Psychopathy Checklist Revised (PCL-R; R. D. Hare, 1991). Personality was assessed using the brief form of the Multidimensional…

  13. Structural basis for a lethal mutation in U6 RNA.

    PubMed

    Sashital, Dipali G; Allmann, Anne M; Van Doren, Steven R; Butcher, Samuel E

    2003-02-18

    U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3' intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67 x G80), and disrupts a protonated C67 x A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (Delta Delta G(fold) = -3.6 +/- 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4. PMID:12578359

  14. Design-Load Basis for LANL Structures, Systems, and Components

    SciTech Connect

    I. Cuesta

    2004-09-01

    This document supports the recommendations in the Los Alamos National Laboratory (LANL) Engineering Standard Manual (ESM), Chapter 5--Structural providing the basis for the loads, analysis procedures, and codes to be used in the ESM. It also provides the justification for eliminating the loads to be considered in design, and evidence that the design basis loads are appropriate and consistent with the graded approach required by the Department of Energy (DOE) Code of Federal Regulation Nuclear Safety Management, 10, Part 830. This document focuses on (1) the primary and secondary natural phenomena hazards listed in DOE-G-420.1-2, Appendix C, (2) additional loads not related to natural phenomena hazards, and (3) the design loads on structures during construction.

  15. Structural basis of substrate specificity in the serine proteases.

    PubMed Central

    Perona, J. J.; Craik, C. S.

    1995-01-01

    Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518

  16. Structural Basis of Cooperative Ligand Binding by the Glycine Riboswitch

    SciTech Connect

    E Butler; J Wang; Y Xiong; S Strobel

    2011-12-31

    The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 {angstrom} crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.

  17. Structural Basis for Recognition of S-adenosylhomocysteine by Riboswitches

    SciTech Connect

    A Edwards; F Reyes; A Heroux; R Batey

    2011-12-31

    S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.

  18. Structural Basis for Protein Phosphatase 1 Regulation and Specificity

    PubMed Central

    Peti, Wolfgang; Nairn, Angus C.; Page, Rebecca

    2012-01-01

    The ubiquitous Ser/Thr Protein Phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (≥200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity. PMID:22284538

  19. Structural Basis for Molecular Recognition at Serotonin Receptors

    PubMed Central

    Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D.; Gao, Xiang; Zhou, Edward X.; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.; Xu, H. Eric

    2013-01-01

    Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist anti-migraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared to the accompanying structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs. PMID:23519210

  20. Structural basis for the enhanced stability of highly fluorinated proteins

    PubMed Central

    Buer, Benjamin C.; Meagher, Jennifer L.; Stuckey, Jeanne A.; Marsh, E. Neil G.

    2012-01-01

    Noncanonical amino acids have proved extremely useful for modifying the properties of proteins. Among them, extensively fluorinated (fluorous) amino acids seem particularly effective in increasing protein stability; however, in the absence of structural data, the basis of this stabilizing effect remains poorly understood. To address this problem, we solved X-ray structures for three small proteins with hydrophobic cores that are packed with either fluorocarbon or hydrocarbon side chains and compared their stabilities. Although larger, the fluorinated residues are accommodated within the protein with minimal structural perturbation, because they closely match the shape of the hydrocarbon side chains that they replace. Thus, stability increases seem to be better explained by increases in buried hydrophobic surface area that accompany fluorination than by specific fluorous interactions between fluorinated side chains. This finding is illustrated by the design of a highly fluorinated protein that, by compensating for the larger volume and surface area of the fluorinated side chains, exhibits similar stability to its nonfluorinated counterpart. These structure-based observations should inform efforts to rationally modulate protein function using noncanonical amino acids. PMID:22411812

  1. Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases

    SciTech Connect

    V Parashar; N Mirouze; D Dubnau; M Neiditch

    2011-12-31

    Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic 'switch' residue to an internal position when the {beta}4-{alpha}4 loop adopts an active-site proximal conformation.

  2. Structural basis of transcobalamin recognition by human CD320 receptor

    PubMed Central

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  3. Structural basis of complement membrane attack complex formation

    NASA Astrophysics Data System (ADS)

    Serna, Marina; Giles, Joanna L.; Morgan, B. Paul; Bubeck, Doryen

    2016-02-01

    In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a `multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a `split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.

  4. Structural basis of transcobalamin recognition by human CD320 receptor.

    PubMed

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S; Zenobi, Renato; Locher, Kaspar P

    2016-07-14

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  5. Structural and Physical Basis for Anti-IgE Therapy

    NASA Astrophysics Data System (ADS)

    Wright, Jon D.; Chu, Hsing-Mao; Huang, Chun-Hsiang; Ma, Che; Wen Chang, Tse; Lim, Carmay

    2015-06-01

    Omalizumab, an anti-IgE antibody, used to treat severe allergic asthma and chronic idiopathic urticaria, binds to IgE in blood or membrane-bound on B lymphocytes but not to IgE bound to its high (FcɛRI) or low (CD23) affinity receptor. Mutagenesis studies indicate overlapping FcɛRI and omalizumab-binding sites in the Cɛ3 domain, but crystallographic studies show FcɛRI and CD23-binding sites that are far apart, so how can omalizumab block IgE from binding both receptors? We report a 2.42-Å omalizumab-Fab structure, a docked IgE-Fc/omalizumab-Fab structure consistent with available experimental data, and the free energy contributions of IgE residues to binding omalizumab, CD23, and FcɛRI. These results provide a structural and physical basis as to why omalizumab cannot bind receptor-bound IgE and why omalizumab-bound IgE cannot bind to CD23/FcɛRI. They reveal the key IgE residues and their roles in binding omalizumab, CD23, and FcɛRI.

  6. Structural basis of AMPK regulation by small molecule activators

    NASA Astrophysics Data System (ADS)

    Xiao, Bing; Sanders, Matthew J.; Carmena, David; Bright, Nicola J.; Haire, Lesley F.; Underwood, Elizabeth; Patel, Bhakti R.; Heath, Richard B.; Walker, Philip A.; Hallen, Stefan; Giordanetto, Fabrizio; Martin, Stephen R.; Carling, David; Gamblin, Steven J.

    2013-12-01

    AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

  7. Structural Basis of TLR5-Flagellin Recognition and Signaling

    SciTech Connect

    Yoon, Sung-il; Kurnasov, Oleg; Natarajan, Venkatesh; Hong, Minsun; Gudkov, Andrei V.; Osterman, Andrei L.; Wilson, Ian A.

    2012-03-01

    Toll-like receptor 5 (TLR5) binding to bacterial flagellin activates signaling through the transcription factor NF-{kappa}B and triggers an innate immune response to the invading pathogen. To elucidate the structural basis and mechanistic implications of TLR5-flagellin recognition, we determined the crystal structure of zebrafish TLR5 (as a variable lymphocyte receptor hybrid protein) in complex with the D1/D2/D3 fragment of Salmonella flagellin, FliC, at 2.47 angstrom resolution. TLR5 interacts primarily with the three helices of the FliC D1 domain using its lateral side. Two TLR5-FliC 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex that is stabilized by quaternary contacts of the FliC D1 domain with the convex surface of the opposing TLR5. The proposed signaling mechanism is supported by structure-guided mutagenesis and deletion analyses on CBLB502, a therapeutic protein derived from FliC.

  8. Structural basis of transcobalamin recognition by human CD320 receptor

    NASA Astrophysics Data System (ADS)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-07-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  9. Structural basis of transcobalamin recognition by human CD320 receptor.

    PubMed

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S; Zenobi, Renato; Locher, Kaspar P

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  10. Electronic structure basis for the extraordinary magnetoresistance in WTe2

    DOE PAGES

    Pletikosić, I.; Ali, Mazhar N.; Fedorov, A. V.; Cava, R. J.; Valla, T.

    2014-11-19

    The electronic structure basis of the extremely large magnetoresistance in layered non-magnetic tungsten ditelluride has been investigated by angle-resolved photoelectron spectroscopy. Hole and electron pockets of approximately the same size were found at the Fermi level, suggesting that carrier compensation should be considered the primary source of the effect. The material exhibits a highly anisotropic, quasi one-dimensional Fermi surface from which the pronounced anisotropy of the magnetoresistance follows. As a result, a change in the Fermi surface with temperature was found and a high-density-of-states band that may take over conduction at higher temperatures and cause the observed turn-on behavior ofmore » the magnetoresistance in WTe₂ was identified.« less

  11. Structural basis of antizyme-mediated regulation of polyamine homeostasis.

    PubMed

    Wu, Hsiang-Yi; Chen, Shin-Fu; Hsieh, Ju-Yi; Chou, Fang; Wang, Yu-Hsuan; Lin, Wan-Ting; Lee, Pei-Ying; Yu, Yu-Jen; Lin, Li-Ying; Lin, Te-Sheng; Lin, Chieh-Liang; Liu, Guang-Yaw; Tzeng, Shiou-Ru; Hung, Hui-Chih; Chan, Nei-Li

    2015-09-01

    Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

  12. Structural basis of antizyme-mediated regulation of polyamine homeostasis

    PubMed Central

    Wu, Hsiang-Yi; Chen, Shin-Fu; Hsieh, Ju-Yi; Chou, Fang; Wang, Yu-Hsuan; Lin, Wan-Ting; Lee, Pei-Ying; Yu, Yu-Jen; Lin, Li-Ying; Lin, Te-Sheng; Lin, Chieh-Liang; Liu, Guang-Yaw; Tzeng, Shiou-Ru; Hung, Hui-Chih; Chan, Nei-Li

    2015-01-01

    Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN–Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation. PMID:26305948

  13. Structural basis for the antibody neutralization of Herpes simplex virus

    SciTech Connect

    Lee, Cheng-Chung; Lin, Li-Ling; Chan, Woan-Eng; Ko, Tzu-Ping; Lai, Jiann-Shiun; Wang, Andrew H.-J.

    2013-10-01

    The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

  14. Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

    SciTech Connect

    Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won

    2010-09-22

    The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B{prime} helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

  15. Structural basis for recognition of Co2+ by RNA aptamers.

    PubMed

    Wrzesinski, Jan; Jóźwiakowski, Stanisław K

    2008-04-01

    Co(2+) binding RNA aptamers were chosen as research models to reveal the structural basis underlying the recognition of Co(2+) by RNA, with the application of two distinct methods. Using the nucleotide analog interference mapping assay, we found strong interference effects after incorporation of the 7-deaza guanosine phosphorotioate analog into the RNA chain at equivalent positions G27 and G28 in aptamer no. 18 and G25 and G26 in aptamer no. 20. The results obtained by nucleotide analog interference mapping suggest that these guanine bases are crucial for the creation of Co(2+) binding sites and that they appear to be involved in the coordination of the ion to the exposed N7 atom of the tandem guanines. Additionally, most 7-deaza guanosine phosphorotioate and 7-deaza adenosine phosphorotioate interferences were located in the common motifs: loop E-like in aptamer no. 18 and kissing dimer in aptamer no. 20. We also found that purine-rich stretches containing guanines with the highest interference values were the targets for hybridization of 6-mers, which are members of the semi-random oligodeoxyribonucleotide library in both aptamers. It transpired that DNA oligomer directed RNase H digestions are sensitive to Co(2+) and, at an elevated metal ion concentration, the hybridization of oligomers to aptamer targets is inhibited, probably due to higher stability and complexity of the RNA structure. PMID:18312410

  16. Structural basis of Rap phosphatase inhibition by Phr peptides.

    PubMed

    Gallego del Sol, Francisca; Marina, Alberto

    2013-01-01

    Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change.

  17. The Structural Basis of Antibody-Antigen Recognition

    PubMed Central

    Sela-Culang, Inbal; Kunik, Vered; Ofran, Yanay

    2013-01-01

    The function of antibodies (Abs) involves specific binding to antigens (Ags) and activation of other components of the immune system to fight pathogens. The six hypervariable loops within the variable domains of Abs, commonly termed complementarity determining regions (CDRs), are widely assumed to be responsible for Ag recognition, while the constant domains are believed to mediate effector activation. Recent studies and analyses of the growing number of available Ab structures, indicate that this clear functional separation between the two regions may be an oversimplification. Some positions within the CDRs have been shown to never participate in Ag binding and some off-CDRs residues often contribute critically to the interaction with the Ag. Moreover, there is now growing evidence for non-local and even allosteric effects in Ab-Ag interaction in which Ag binding affects the constant region and vice versa. This review summarizes and discusses the structural basis of Ag recognition, elaborating on the contribution of different structural determinants of the Ab to Ag binding and recognition. We discuss the CDRs, the different approaches for their identification and their relationship to the Ag interface. We also review what is currently known about the contribution of non-CDRs regions to Ag recognition, namely the framework regions (FRs) and the constant domains. The suggested mechanisms by which these regions contribute to Ag binding are discussed. On the Ag side of the interaction, we discuss attempts to predict B-cell epitopes and the suggested idea to incorporate Ab information into B-cell epitope prediction schemes. Beyond improving the understanding of immunity, characterization of the functional role of different parts of the Ab molecule may help in Ab engineering, design of CDR-derived peptides, and epitope prediction. PMID:24115948

  18. Structural basis for substrate discrimination and integrin binding by autotaxin

    PubMed Central

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis

    2010-01-01

    Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We identify a hydrophobic lipid-binding pocket and map key residues required for catalysis and selection between nucleotide and phospholipid substrates. We show that ATX interacts with cell-surface integrins via its N-terminal somatomedin-B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling, and enable new approaches to target ATX with small-molecule therapeutics. PMID:21240271

  19. Structural basis of substrate discrimination and integrin binding by autotaxin

    SciTech Connect

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna J.S.; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig W.; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

  20. Structural and biomechanical basis of mitochondrial movement in eukaryotic cells.

    PubMed

    Wu, Min; Kalyanasundaram, Aruna; Zhu, Jie

    2013-01-01

    Mitochondria serve as energy-producing organelles in eukaryotic cells. In addition to providing the energy supply for cells, the mitochondria are also involved in other processes, such as proliferation, differentiation, information transfer, and apoptosis, and play an important role in regulation of cell growth and the cell cycle. In order to achieve these functions, the mitochondria need to move to the corresponding location. Therefore, mitochondrial movement has a crucial role in normal physiologic activity, and any mitochondrial movement disorder will cause irreparable damage to the organism. For example, recent studies have shown that abnormal movement of the mitochondria is likely to be the reason for Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Parkinson's disease, and schizophrenia. So, in the cell, especially in the particular polarized cell, the appropriate distribution of mitochondria is crucial to the function and survival of the cell. Mitochondrial movement is mainly associated with the cytoskeleton and related proteins. However, those components play different roles according to cell type. In this paper, we summarize the structural basis of mitochondrial movement, including microtubules, actin filaments, motor proteins, and adaptin, and review studies of the biomechanical mechanisms of mitochondrial movement in different types of cells.

  1. Structural and biomechanical basis of mitochondrial movement in eukaryotic cells

    PubMed Central

    Wu, Min; Kalyanasundaram, Aruna; Zhu, Jie

    2013-01-01

    Mitochondria serve as energy-producing organelles in eukaryotic cells. In addition to providing the energy supply for cells, the mitochondria are also involved in other processes, such as proliferation, differentiation, information transfer, and apoptosis, and play an important role in regulation of cell growth and the cell cycle. In order to achieve these functions, the mitochondria need to move to the corresponding location. Therefore, mitochondrial movement has a crucial role in normal physiologic activity, and any mitochondrial movement disorder will cause irreparable damage to the organism. For example, recent studies have shown that abnormal movement of the mitochondria is likely to be the reason for Charcot–Marie–Tooth disease, amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and schizophrenia. So, in the cell, especially in the particular polarized cell, the appropriate distribution of mitochondria is crucial to the function and survival of the cell. Mitochondrial movement is mainly associated with the cytoskeleton and related proteins. However, those components play different roles according to cell type. In this paper, we summarize the structural basis of mitochondrial movement, including microtubules, actin filaments, motor proteins, and adaptin, and review studies of the biomechanical mechanisms of mitochondrial movement in different types of cells. PMID:24187495

  2. Structural Basis of Clostridium perfringens Toxin Complex Formation

    SciTech Connect

    Adams,J.; Gregg, K.; Bayer, E.; Boraston, A.; Smith, S.

    2008-01-01

    The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the {mu}-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (Ka = 1.44 x 1011 M-1) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The {mu}-toxin dockerin module in this complex is positioned {approx}180 relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.

  3. Structural basis for iron piracy by pathogenic Neisseria

    PubMed Central

    Noinaj, N.; Easley, N.C.; Oke, M.; Mizuno, N.; Gumbart, J.; Boura, E.; Steere, A.N.; Zak, O.; Aisen, P.; Tajkhorshid, E.; Evans, R.W.; Gorringe, A.R.; Mason, A.B.; Steven, A.C.; Buchanan, S.K.

    2012-01-01

    SUMMARY Neisseria are obligate human pathogens causing bacterial meningitis, septicemia, and gonorrhea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are: 1) how human transferrin is specifically targeted, and 2) how the bacteria liberate iron from transferrin at neutral pH. To address them, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Collectively, our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process. PMID:22327295

  4. Structural basis for iron piracy by pathogenic Neisseria.

    PubMed

    Noinaj, Nicholas; Easley, Nicole C; Oke, Muse; Mizuno, Naoko; Gumbart, James; Boura, Evzen; Steere, Ashley N; Zak, Olga; Aisen, Philip; Tajkhorshid, Emad; Evans, Robert W; Gorringe, Andrew R; Mason, Anne B; Steven, Alasdair C; Buchanan, Susan K

    2012-02-12

    Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

  5. Structural Basis for Antibody Recognition of Lipid A

    PubMed Central

    Haji-Ghassemi, Omid; Müller-Loennies, Sven; Rodriguez, Teresa; Brade, Lore; Kosma, Paul; Brade, Helmut; Evans, Stephen V.

    2015-01-01

    Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1–15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1–15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1–15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1–15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. PMID:26085093

  6. Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3.

    PubMed

    Jensen, Maria Risager; Bajic, Goran; Zhang, Xianwei; Laustsen, Anne Kjær; Koldsø, Heidi; Skeby, Katrine Kirkeby; Schiøtt, Birgit; Andersen, Gregers R; Vorup-Jensen, Thomas

    2016-08-12

    The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists.

  7. Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3.

    PubMed

    Jensen, Maria Risager; Bajic, Goran; Zhang, Xianwei; Laustsen, Anne Kjær; Koldsø, Heidi; Skeby, Katrine Kirkeby; Schiøtt, Birgit; Andersen, Gregers R; Vorup-Jensen, Thomas

    2016-08-12

    The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists. PMID:27339893

  8. Exploring continuum structures with a pseudo-state basis

    SciTech Connect

    Lay, J. A.; Moro, A. M.; Arias, J. M.; Gomez-Camacho, J.

    2010-08-15

    The ability of a recently developed square-integrable discrete basis to represent the properties of the continuum of a two-body system is investigated. The basis is obtained performing a simple analytic local scale transformation to the harmonic oscillator basis. Scattering phase-shifts and the electric transition probabilities B(E1) and B(E2) have been evaluated for several potentials using the proposed basis. Both quantities are found to be in excellent agreement with the exact values calculated from the true scattering states. The basis has been applied to describe the projectile continuum in the {sup 6}He scattering by {sup 12}C and {sup 208}Pb targets at 240 MeV/nucleon and the {sup 11}Be scattering by {sup 12}C at 67 MeV/nucleon. The calculated breakup differential cross sections are found to be in very good agreement with the available experimental data for these reactions.

  9. Structural basis for stop codon recognition in eukaryotes

    PubMed Central

    Murray, Jason; Hegde, Ramanujan S.; Ramakrishnan, V.

    2015-01-01

    Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UGA, UAA, or UAG. Release factors recognise stop codons in the ribosomal A site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases1. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognise all three stop codons2. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here, we present electron cryo-microscopy (cryo-EM) structures at 3.5 – 3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A site. Binding of eRF1 flips nucleotide A1825 of 18S rRNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A site, where it is stabilised by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during tRNA selection to drive mRNA compaction. Stop codons are favoured in this compacted mRNA conformation by a hydrogen-bonding network with essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination3,4. PMID:26245381

  10. Structural basis for stop codon recognition in eukaryotes.

    PubMed

    Brown, Alan; Shao, Sichen; Murray, Jason; Hegde, Ramanujan S; Ramakrishnan, V

    2015-08-27

    Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5-3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination.

  11. A Structural Basis for Regulation of Actin Polymerization by Pectenotoxins

    PubMed Central

    Allingham, John S.; Miles, Christopher O.; Rayment, Ivan

    2007-01-01

    Pectenotoxins (PTXs) are polyether macrolides found in certain dinoflagellates, sponges and shellfish, and have been associated with diarrhetic shellfish poisoning. In addition to their in vivo toxicity, some PTXs are potently cytotoxic in human cancer cell lines. Recent studies have demonstrated that disruption of the actin cytoskeleton may be a key function of these compounds, although no clarification their mechanism of action at a molecular level was available. We have obtained an X-ray crystal structure of PTX-2 bound to actin which, in combination with analyses of the effect of PTX-2 on purified actin filament dynamics, provides a molecular explanation for its effects on actin. PTX-2 formed a 1:1 complex with actin and engaged a novel site between subdomains 1 and 3. Based on models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby capping the barbed-end. The location of this binding position within the interior of the filament indicates that it may not be accessible once polymerization has occurred, a hypothesis supported by our observation that PTX-2 caused filament capping without inducing filament severing. This mode of action is unique, as other actin filament destabilizing toxins appear to exclusively disrupt longitudinal monomer contacts allowing many of them to sever filaments in addition to capping them. Examination of the PTX-binding site on actin provides a rationalization for the structure–activity relationships observed in vivo and in vitro, and may provide a basis for predicting toxicity of PTX analogues. PMID:17599353

  12. Demonstrating Structural Adequacy of Nuclear Power Plant Containment Structures for Beyond Design-Basis Pressure Loadings

    SciTech Connect

    Braverman, J.I.; Morante, R.

    2010-07-18

    ABSTRACT Demonstrating the structural integrity of U.S. nuclear power plant (NPP) containment structures, for beyond design-basis internal pressure loadings, is necessary to satisfy Nuclear Regulatory Commission (NRC) requirements and performance goals. This paper discusses methods for demonstrating the structural adequacy of the containment for beyond design-basis pressure loadings. Three distinct evaluations are addressed: (1) estimating the ultimate pressure capacity of the containment structure (10 CFR 50 and US NRC Standard Review Plan, Section 3.8) ; (2) demonstrating the structural adequacy of the containment subjected to pressure loadings associated with combustible gas generation (10 CFR 52 and 10 CFR 50); and (3) demonstrating the containment structural integrity for severe accidents (10 CFR 52 as well as SECY 90-016, SECY 93-087, and related NRC staff requirements memoranda (SRMs)). The paper describes the technical basis for specific aspects of the methods presented. It also presents examples of past issues identified in licensing activities related to these evaluations.

  13. Structural basis for exon recognition by a group II intron

    SciTech Connect

    Toor, Navtej; Rajashankar, Kanagalaghatta; Keating, Kevin S.; Pyle, Anna Marie

    2008-11-18

    Free group II introns are infectious retroelements that can bind and insert themselves into RNA and DNA molecules via reverse splicing. Here we report the 3.4-A crystal structure of a complex between an oligonucleotide target substrate and a group IIC intron, as well as the refined free intron structure. The structure of the complex reveals the conformation of motifs involved in exon recognition by group II introns.

  14. The E-state as the basis for molecular structure space definition and structure similarity

    PubMed

    Hall; Kier

    2000-05-01

    The electrotopological state (E-state) is presented as a representation of molecular structure useful for definition of a space for chemical structures. This E-state representation provides the basis for chemical database management. The E-state formalism is presented along with its extension to the atom-type E-state. An approach to database organization, using polychlorobiphenyls (PCBs) as examples, reveals the descriptive power of the E-state paradigm. A well-organized chemical database, as described here, may be searched to find structures similar to a target structure with the expectation that such structures may exhibit properties similar to the target. Searches using the atom-type E-state indices are demonstrated with two example drug molecules.

  15. Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

    SciTech Connect

    Liu, Wei; Chun, Eugene; Thompson, Aaron A.; Chubukov, Pavel; Xu, Fei; Katritch, Vsevolod; Han, Gye Won; Roth, Christopher B.; Heitman, Laura H.; IJzerman, Adriaan P.; Cherezov, Vadim; Stevens, Raymond C.

    2012-08-31

    Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A{sub 2A} adenosine receptor by replacing its third intracellular loop with apocytochrome b{sub 562}RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp{sup 2.50}. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

  16. Structural basis of selective inhibition of human tankyrases.

    PubMed

    Narwal, Mohit; Venkannagari, Harikanth; Lehtiö, Lari

    2012-02-01

    Tankyrases are poly(ADP-ribose) polymerases that have many cellular functions. They play pharmaceutically important roles, at least in telomere homeostasis and Wnt signaling, by covalently ADP-ribosylating target proteins and consequently regulating their functions. These features make tankyrases potential targets for treatment of cancer. We report here crystal structures of human tankyrase 2 catalytic fragment in complex with a byproduct, nicotinamide, and with selective inhibitors of tankyrases (IWR-1) and PARPs 1 and 2 (olaparib). Binding of these inhibitors to tankyrase 2 induces specific conformational changes. The crystal structures explain the selectivity of the inhibitors, reveal the flexibility of a substrate binding loop, and explain existing structure-activity relationship data. The first crystal structure of a PARP enzyme in complex with a potent inhibitor, IWR-1, that does not bind to the widely utilized nicotinamide-binding site makes the structure valuable for development of PARP inhibitors in general. PMID:22233320

  17. The Three-Dimensional Structural Basis of Type II Hyperprolinemia

    SciTech Connect

    Srivastava, Dhiraj; Singh, Ranjan K.; Moxley, Michael A.; Henzl, Michael T.; Becker, Donald F.; Tanner, John J.

    2012-08-31

    Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in {Delta}{sup 1}-pyrroline-5-carboxylate dehydrogenase (P5CDH; also known as ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here, we report the first structure of human P5CDH (HsP5CDH) and investigate the impact of the hyperprolinemia-associated mutation of Ser352 to Leu on the structure and catalytic properties of the enzyme. The 2. 5-{angstrom}-resolution crystal structure of HsP5CDH was determined using experimental phasing. Structures of the mutant enzymes S352A (2.4 {angstrom}) and S352L (2.85 {angstrom}) were determined to elucidate the structural consequences of altering Ser352. Structures of the 93% identical mouse P5CDH complexed with sulfate ion (1.3 {angstrom} resolution), glutamate (1.5 {angstrom}), and NAD{sup +} (1.5 {angstrom}) were determined to obtain high-resolution views of the active site. Together, the structures show that Ser352 occupies a hydrophilic pocket and is connected via water-mediated hydrogen bonds to catalytic Cys348. Mutation of Ser352 to Leu is shown to abolish catalytic activity and eliminate NAD{sup +} binding. Analysis of the S352A mutant shows that these functional defects are caused by the introduction of the nonpolar Leu352 side chain rather than the removal of the Ser352 hydroxyl. The S352L structure shows that the mutation induces a dramatic 8-{angstrom} rearrangement of the catalytic loop. Because of this conformational change, Ser349 is not positioned to interact with the aldehyde substrate, conserved Glu447 is no longer poised to bind NAD{sup +}, and Cys348 faces the wrong direction for nucleophilic attack. These structural alterations render the enzyme inactive.

  18. Structural basis for the inhibition of the eukaryotic ribosome.

    PubMed

    Garreau de Loubresse, Nicolas; Prokhorova, Irina; Holtkamp, Wolf; Rodnina, Marina V; Yusupova, Gulnara; Yusupov, Marat

    2014-09-25

    The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.

  19. Structural basis for EGFR ligand sequestration by Argos

    SciTech Connect

    Klein, Daryl E.; Stayrook, Steven E.; Shi, Fumin; Narayan, Kartik; Lemmon, Mark A.

    2008-06-26

    Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR5. Here we describe the 1.6-{angstrom} resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-{beta} family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.

  20. The structural basis of pregnane X receptor binding promiscuity

    PubMed Central

    Ngan, Chi-Ho; Beglov, Dmitri; Rudnitskaya, Aleksandra N.; Kozakov, Dima; Waxman, David J.; Vajda, Sandor

    2009-01-01

    The steroid and xenobiotic-responsive human pregnane X receptor (PXR) binds a broad range of structurally diverse compounds. The structures of the apo and ligand-bound forms of PXR are very similar, in contrast to most promiscuous proteins that generally adapt their shape to different ligands. We investigated the structural origins of PXR's recognition promiscuity using computational solvent mapping, a technique developed for the identification and characterization of hot spots, i.e., regions of the protein surface that are major contributors to the binding free energy. Results reveal that the smooth and nearly spherical binding site of PXR has a well-defined hot spot structure, with four hot spots located on four different sides of the pocket and a fifth close to its center. Three of these hot spots are already present in the ligand-free protein. The most important hot spot is defined by three structurally and sequentially conserved residues, W299, F288, and Y306. This largely hydrophobic site is not very specific, and interacts with all known PXR ligands. Depending on their sizes and shapes, individual PXR ligands extend into 2, 3, or 4 more hot spot regions. The large number of potential arrangements within the binding site explains why PXR is able to accommodate a large variety of compounds. All five hot spots include at least one important residue, which is conserved in all mammalian PXRs, suggesting that the hot spot locations have remained largely invariant during mammalian evolution. The same side chains also show a high level of structural conservation across hPXR structures. However, each of the hPXR hot spots also includes residues with moveable side chains, further increasing the size variation in ligands that PXR can bind. Results also suggest a unique signal transduction mechanism between the PXR homodimerization interface and its co-activator binding site. PMID:19856963

  1. Structural and functional basis of protein phosphatase 5 substrate specificity

    PubMed Central

    Oberoi, Jasmeen; Dunn, Diana M.; Woodford, Mark R.; Mariotti, Laura; Schulman, Jacqualyn; Bourboulia, Dimitra; Mollapour, Mehdi

    2016-01-01

    The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP–substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors. PMID:27466404

  2. Structural basis for mammalian vitamin B12 transport by transcobalamin

    PubMed Central

    Wuerges, Jochen; Garau, Gianpiero; Geremia, Silvano; Fedosov, Sergey N.; Petersen, Torben E.; Randaccio, Lucio

    2006-01-01

    Cobalamin (Cbl, vitamin B12) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal α6-α6 barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H2O at the upper axial side of added H2O-Cbl is displaced by a histidine residue of the α6-α6 barrel. Analysis of amino acid conservation on TC’s surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the α6-α6 barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery. PMID:16537422

  3. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    NASA Astrophysics Data System (ADS)

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-07-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  4. The structural basis of light-harvesting in purple bacteria.

    PubMed

    Cogdell, Richard J; Isaacs, Neil W; Freer, Andrew A; Howard, Tina D; Gardiner, Alastair T; Prince, Steve M; Papiz, Miroslavr Z

    2003-11-27

    A typical purple bacterial photosynthetic unit consists of two types of light-harvesting complex (LH1 and LH2) together with a reaction centre. This short review presents a description of the structure of the LH2 complex from Rhodopseudomonas acidophila, which has recently been improved to a resolution of 2.0 A [Papiz et al., J. Mol. Biol. 326 (2003) 1523-1538]. We show how this structure has helped to reveal the details of the various excitation energy transfer events in which it is involved.

  5. Structural basis for cytokinin production by LOG from Corynebacterium glutamicum.

    PubMed

    Seo, Hogyun; Kim, Sangwoo; Sagong, Hye-Young; Son, Hyeoncheol Francis; Jin, Kyeong Sik; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-08-10

    "Lonely guy" (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a "PGGXGTXXE" motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms.

  6. Structural basis for the blockade of MATE multidrug efflux pumps

    DOE PAGES

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-06

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystalmore » structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.« less

  7. Structural basis for the blockade of MATE multidrug efflux pumps

    SciTech Connect

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-06

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

  8. Structural Basis for Translation Termination on a Pseudouridylated Stop Codon.

    PubMed

    Svidritskiy, Egor; Madireddy, Rohini; Korostelev, Andrei A

    2016-05-22

    Pseudouridylation of messenger RNA emerges as an abundant modification involved in gene expression regulation. Pseudouridylation of stop codons in eukaryotic and bacterial cells results in stop-codon read through. The structural mechanism of this phenomenon is not known. Here we present a 3.1-Å crystal structure of Escherichia coli release factor 1 (RF1) bound to the 70S ribosome in response to the ΨAA codon. The structure reveals that recognition of a modified stop codon does not differ from that of a canonical stop codon. Our in vitro biochemical results support this finding by yielding nearly identical rates for peptide release from E. coli ribosomes programmed with pseudouridylated and canonical stop codons. The crystal structure also brings insight into E. coli RF1-specific interactions and suggests involvement of L27 in bacterial translation termination. Our results are consistent with a mechanism in which read through of a pseudouridylated stop codon in bacteria results from increased decoding by near-cognate tRNAs (miscoding) rather than from decreased efficiency of termination. PMID:27107638

  9. Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

    SciTech Connect

    Strushkevich, Natallia; MacKenzie, Farrell; Cherkesova, Tatyana; Grabovec, Irina; Usanov, Sergey; Park, Hee-Won

    2011-09-06

    In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1 - the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 {angstrom} away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

  10. Structural basis for adaptation of lactobacilli to gastrointestinal mucus.

    PubMed

    Etzold, Sabrina; Kober, Olivia I; Mackenzie, Donald A; Tailford, Louise E; Gunning, A Patrick; Walshaw, John; Hemmings, Andrew M; Juge, Nathalie

    2014-03-01

    The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the underpinning mechanisms of these interactions are not understood. Here, we provide structural and functional insights into the canonical mucus-binding protein (MUB), a multi-repeat cell-surface adhesin found in Lactobacillus inhabitants of the GI tract. X-ray crystallography together with small-angle X-ray scattering demonstrated a 'beads on a string' arrangement of repeats, generating 174 nm long protein fibrils, as shown by atomic force microscopy. Each repeat consists of tandemly arranged Ig- and mucin-binding protein (MucBP) modules. The binding of full-length MUB was confined to mucus via multiple interactions involving terminal sialylated mucin glycans. While individual MUB domains showed structural similarity to fimbrial proteins from Gram-positive pathogens, the particular organization of MUB provides a structural explanation for the mechanisms in which lactobacilli have adapted to their host niche by maximizing interactions with the mucus receptors, potentiating the retention of bacteria within the mucus layer. Together, this study reveals functional and structural features which may affect tropism of microbes across mucus and along the GI tract, providing unique insights into the mechanisms adopted by commensals and probiotics to adapt to the mucosal environment. PMID:24373178

  11. Structural basis for the blockade of MATE multidrug efflux pumps

    NASA Astrophysics Data System (ADS)

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-01

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

  12. Structural Basis of Substrate Specificity in Geobacter metallireducens SMUG1.

    PubMed

    Zhang, Zhemin; Shen, Jiemin; Yang, Ye; Li, Jing; Cao, Weiguo; Xie, Wei

    2016-06-17

    Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are critical to maintain genome integrity, in which the base excision repair pathway plays an essential role. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for removing the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Geobacter metallireducens SMUG1 (GmeSMUG1) is an interesting family 3 enzyme in the UDG superfamily, with dual substrate specificities for DNA with uracil or xanthine. In contrast, the mutant G63P of GmeSMUG1 has exclusive activity for uracil, while N58D is inactive for both substrates, as we have reported previously. However, the structural bases for these substrate specificities are not well understood. In this study, we solved a series of crystal structures of WT and mutants of GmeSMUG1 at relatively high resolutions. These structures provide insight on the molecular mechanism of xanthine recognition for GmeSMUG1 and indicate that H210 plays a key role in xanthine recognition, which is in good agreement with the results of our EMSA and activity assays. More importantly, our mutant structures allow us to build models to rationalize our previous experimental observations of altered substrate activities of these mutants. PMID:27071000

  13. Structural Basis for Catalytic Activation of a Serine Recombinase

    SciTech Connect

    Keenholtz, Ross A.; Rowland, Sally-J.; Boocock, Martin R.; Stark, W. Marshall; Rice, Phoebe A.

    2014-10-02

    Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 {angstrom} crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.

  14. Structural basis of human γ-secretase assembly

    PubMed Central

    Sun, Linfeng; Zhao, Lingyun; Yang, Guanghui; Yan, Chuangye; Zhou, Rui; Zhou, Xiaoyuan; Xie, Tian; Zhao, Yanyu; Wu, Shenjie; Li, Xueming; Shi, Yigong

    2015-01-01

    The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer’s disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase. PMID:25918421

  15. Structural basis for inhibition of DNA replication by aphidicolin

    SciTech Connect

    Baranovskiy, A. G.; Babayeva, N. D.; Suwa, Y.; Gu, J.; Pavlov, Y. I.; Tahirov, T. H.

    2014-11-27

    Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

  16. Structural basis for inhibition of DNA replication by aphidicolin

    DOE PAGES

    Baranovskiy, A. G.; Babayeva, N. D.; Suwa, Y.; Gu, J.; Pavlov, Y. I.; Tahirov, T. H.

    2014-11-27

    Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with anmore » RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.« less

  17. Structural basis for cytokinin production by LOG from Corynebacterium glutamicum

    PubMed Central

    Seo, Hogyun; Kim, Sangwoo; Sagong, Hye-Young; Son, Hyeoncheol Francis; Jin, Kyeong Sik; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-01-01

    “Lonely guy” (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a “PGGXGTXXE” motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms. PMID:27507425

  18. Structural basis of substrate selectivity of E. coli prolidase.

    PubMed

    Weaver, Jeremy; Watts, Tylan; Li, Pingwei; Rye, Hays S

    2014-01-01

    Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site. PMID:25354344

  19. Structural Basis of Substrate Selectivity of E. coli Prolidase

    PubMed Central

    Weaver, Jeremy; Watts, Tylan; Li, Pingwei; Rye, Hays S.

    2014-01-01

    Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site. PMID:25354344

  20. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

    SciTech Connect

    Wang, Huanchen; Falck, J.R.; Hall, Traci M. Tanaka; Shears, Stephen B.

    2012-01-11

    Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF{sub 3}{sup -} transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.

  1. Structural Basis of Histone H4 Recognition by p55

    SciTech Connect

    Song,J.; Garlick, J.; Kingston, R.

    2008-01-01

    p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a ?-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones.

  2. Structural basis of template-boundary definition in Tetrahymena telomerase.

    PubMed

    Jansson, Linnea I; Akiyama, Ben M; Ooms, Alexandra; Lu, Cheng; Rubin, Seth M; Stone, Michael D

    2015-11-01

    Telomerase is required to maintain repetitive G-rich telomeric DNA sequences at chromosome ends. To do so, the telomerase reverse transcriptase (TERT) subunit reiteratively uses a small region of the integral telomerase RNA (TER) as a template. An essential feature of telomerase catalysis is the strict definition of the template boundary to determine the precise TER nucleotides to be reverse transcribed by TERT. We report the 3-Å crystal structure of the Tetrahymena TERT RNA-binding domain (tTRBD) bound to the template boundary element (TBE) of TER. tTRBD is wedged into the base of the TBE RNA stem-loop, and each of the flanking RNA strands wraps around opposite sides of the protein domain. The structure illustrates how the tTRBD establishes the template boundary by positioning the TBE at the correct distance from the TERT active site to prohibit copying of nontemplate nucleotides.

  3. Structural Basis for Methyl Transfer by a Radical SAM Enzyme

    SciTech Connect

    Boal, Amie K.; Grove, Tyler L.; McLaughlin, Monica I.; Yennawar, Neela H.; Booker, Squire J.; Rosenzweig, Amy C.

    2014-10-02

    The radical S-adenosyl-l-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys{sup 355}) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys{sup 355} is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.

  4. Structural basis for Klf4 recognition of methylated DNA.

    PubMed

    Liu, Yiwei; Olanrewaju, Yusuf Olatunde; Zheng, Yu; Hashimoto, Hideharu; Blumenthal, Robert M; Zhang, Xing; Cheng, Xiaodong

    2014-04-01

    Transcription factor Krüppel-like factor 4 (Klf4), one of the factors directing cellular reprogramming, recognizes the CpG dinucleotide (whether methylated or unmodified) within a specific G/C-rich sequence. The binding affinity of the mouse Klf4 DNA-binding domain for methylated DNA is only slightly stronger than that for an unmodified oligonucleotide. The structure of the C-terminal three Krüppel-like zinc fingers (ZnFs) of mouse Klf4, in complex with fully methylated DNA, was determined at 1.85 Å resolution. An arginine and a glutamate interact with the methyl group. By comparison with two other recently characterized structures of ZnF protein complexes with methylated DNA, we propose a common principle of recognition of methylated CpG by C2H2 ZnF proteins, which involves a spatially conserved Arg-Glu pair. PMID:24520114

  5. The structural basis for receptor recognition of human interleukin-18

    SciTech Connect

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.

  6. The structural basis for receptor recognition of human interleukin-18

    DOE PAGES

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; et al

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is uniquemore » among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.« less

  7. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    SciTech Connect

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; Melcher, Karsten

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

  8. Structural basis of diverse membrane target recognitions by ankyrins.

    PubMed

    Wang, Chao; Wei, Zhiyi; Chen, Keyu; Ye, Fei; Yu, Cong; Bennett, Vann; Zhang, Mingjie

    2014-01-01

    Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins' membrane targets, with implications for other proteins containing extended ANK repeat domains. PMID:25383926

  9. The structural basis for receptor recognition of human interleukin-18

    PubMed Central

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-01-01

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity. PMID:25500532

  10. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    DOE PAGES

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; et al

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

  11. Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase

    PubMed Central

    Rajakannan, Venkatachalam; Lee, Hui-Sun; Chong, Seon-Ha; Ryu, Han-Bong; Bae, Ji-Young; Whang, Eun-Young; Huh, Jae-Wan; Cho, Sung-Woo; Kang, Lin-Woo; Choe, Han; Robinson, Robert C.

    2011-01-01

    Background UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics. Methodology Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle. Conclusion In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme. PMID:21984906

  12. Structural Basis of Subtilase Cytotoxin SubAB Assembly*

    PubMed Central

    Le Nours, Jérôme; Paton, Adrienne W.; Byres, Emma; Troy, Sally; Herdman, Brock P.; Johnson, Matthew D.; Paton, James C.; Rossjohn, Jamie; Beddoe, Travis

    2013-01-01

    Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin. PMID:23921389

  13. The structural basis of non-photochemical quenching is revealed?

    PubMed

    Cogdell, Richard J

    2006-02-01

    Light-harvesting complex II (LHCII, the major plant light-harvesting pigment-protein complex, efficiently harvests light-energy. However, if the incident light intensity is too high and photosynthesis becomes saturated, LHCII can switch into a quenching state that prevents photodamage. This important process is called non-photochemical quenching, or NPQ, and represents feedback control. Andrew Pascal et al. have recently proposed a detailed model of NPQ based upon the crystal structure of LHCII from spinach.

  14. The structural basis for specificity in lipoxygenase catalysis.

    PubMed

    Newcomer, Marcia E; Brash, Alan R

    2015-03-01

    Many intriguing facets of lipoxygenase (LOX) catalysis are open to a detailed structural analysis. Polyunsaturated fatty acids with two to six double bonds are oxygenated precisely on a particular carbon, typically forming a single chiral fatty acid hydroperoxide product. Molecular oxygen is not bound or liganded during catalysis, yet it is directed precisely to one position and one stereo configuration on the reacting fatty acid. The transformations proceed upon exposure of substrate to enzyme in the presence of O2 (RH + O2 → ROOH), so it has proved challenging to capture the precise mode of substrate binding in the LOX active site. Beginning with crystal structures with bound inhibitors or surrogate substrates, and most recently arachidonic acid bound under anaerobic conditions, a picture is consolidating of catalysis in a U-shaped fatty acid binding channel in which individual LOX enzymes use distinct amino acids to control the head-to-tail orientation of the fatty acid and register of the selected pentadiene opposite the non-heme iron, suitably positioned for the initial stereoselective hydrogen abstraction and subsequent reaction with O2 . Drawing on the crystal structures available currently, this review features the roles of the N-terminal β-barrel (C2-like, or PLAT domain) in substrate acquisition and sensitivity to cellular calcium, and the α-helical catalytic domain in fatty acid binding and reactions with O2 that produce hydroperoxide products with regio and stereospecificity. LOX structures combine to explain how similar enzymes with conserved catalytic machinery differ in product, but not substrate, specificities.

  15. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    PubMed Central

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization. PMID:26151670

  16. Structural basis of Smoothened regulation by its extracellular domains

    NASA Astrophysics Data System (ADS)

    Byrne, Eamon F. X.; Sircar, Ria; Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Newstead, Simon; Rohatgi, Rajat; Siebold, Christian

    2016-07-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

  17. Structural basis of interprotein electron transfer in bacterial sulfite oxidation

    PubMed Central

    McGrath, Aaron P; Laming, Elise L; Casas Garcia, G Patricia; Kvansakul, Marc; Guss, J Mitchell; Trewhella, Jill; Calmes, Benoit; Bernhardt, Paul V; Kappler, Ulrike; Maher, Megan J

    2015-01-01

    Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly. DOI: http://dx.doi.org/10.7554/eLife.09066.001 PMID:26687009

  18. Structural basis of arginine asymmetrical dimethylation by PRMT6.

    PubMed

    Wu, Hong; Zheng, Weihong; Eram, Mohammad S; Vhuiyan, Mynol; Dong, Aiping; Zeng, Hong; He, Hao; Brown, Peter; Frankel, Adam; Vedadi, Masoud; Luo, Minkui; Min, Jinrong

    2016-10-01

    PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here, we report the synthesis of a potent bisubstrate inhibitor GMS [6'-methyleneamine sinefungin, an analog of sinefungin (SNF)], and the crystal structures of human PRMT6 in complex, respectively, with S-adenosyl-L-homocysteine (SAH) and the bisubstrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared with SAH and an S-adenosyl-L-methionine competitive methyltransferase inhibitor SNF. In addition, we also crystallized PRMT6 in complex with SAH and a short arginine-containing peptide. Based on the structural information here and available in the PDB database, we proposed a mechanism that can rationalize the distinctive arginine methylation product specificity of different types of arginine methyltransferases and pinpoint the structural determinant of such a specificity. PMID:27480107

  19. Structural basis for angiopoietin-1–mediated signaling initiation

    SciTech Connect

    Yu, Xuehong; Seegar, Tom C. M.; Dalton, Annamarie C.; Tzvetkova-Robev, Dorothea; Goldgur, Yehuda; Rajashankar, Kanagalaghatta R.; Nikolov, Dimitar B.; Barton, William A.

    2013-04-30

    Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

  20. Structural basis of Smoothened regulation by its extracellular domains

    NASA Astrophysics Data System (ADS)

    Byrne, Eamon F. X.; Sircar, Ria; Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Newstead, Simon; Rohatgi, Rajat; Siebold, Christian

    2016-07-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

  1. Structural basis of Zika virus helicase in recognizing its substrates.

    PubMed

    Tian, Hongliang; Ji, Xiaoyun; Yang, Xiaoyun; Zhang, Zhongxin; Lu, Zuokun; Yang, Kailin; Chen, Cheng; Zhao, Qi; Chi, Heng; Mu, Zhongyu; Xie, Wei; Wang, Zefang; Lou, Huiqiang; Yang, Haitao; Rao, Zihe

    2016-08-01

    The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication. PMID:27430951

  2. Structural basis of Zika virus helicase in recognizing its substrates.

    PubMed

    Tian, Hongliang; Ji, Xiaoyun; Yang, Xiaoyun; Zhang, Zhongxin; Lu, Zuokun; Yang, Kailin; Chen, Cheng; Zhao, Qi; Chi, Heng; Mu, Zhongyu; Xie, Wei; Wang, Zefang; Lou, Huiqiang; Yang, Haitao; Rao, Zihe

    2016-08-01

    The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

  3. The structural basis of Erwinia rhapontici isomaltulose synthase.

    PubMed

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

  4. Structural basis of Smoothened regulation by its extracellular domains.

    PubMed

    Byrne, Eamon F X; Sircar, Ria; Miller, Paul S; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D; Mydock-McGrane, Laurel; Covey, Douglas F; Rambo, Robert P; Sansom, Mark S P; Newstead, Simon; Rohatgi, Rajat

    2016-07-28

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains. PMID:27437577

  5. Structural basis of interprotein electron transfer in bacterial sulfite oxidation.

    PubMed

    McGrath, Aaron P; Laming, Elise L; Casas Garcia, G Patricia; Kvansakul, Marc; Guss, J Mitchell; Trewhella, Jill; Calmes, Benoit; Bernhardt, Paul V; Hanson, Graeme R; Kappler, Ulrike; Maher, Megan J

    2015-12-19

    Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly.

  6. A neurodevelopmental basis for BECTS: Evidence from structural MRI

    PubMed Central

    Pardoe, Heath R.; Berg, Anne T.; Archer, John S.; Fulbright, Robert K.; Jackson, Graeme D.

    2013-01-01

    Summary Purpose BECTS (benign epilepsy with centro-temporal spikes) is one of the most common childhood-onset epilepsy syndromes. We investigated quantitative evidence for brain morphological variation associated with BECTS to provide insights into the neuroanatomical basis of this disorder. Methods Three independent BECTS groups were imaged at different stages: (a) near onset (n = 16, mean age 9.3 ± 1.6 years), (b) ~9 years after onset (n = 9, mean age 15.8 ± 2.3 years), and (c) ~15 years after onset (n = 10, mean age 22.7 ± 2.7 years). Age-matched controls were imaged with each group. Whole brain T1-weighted MRI was acquired. Voxel-based morphometry (groups a–c) and cortical thickness analyses (groups b and c) were undertaken within each group and for the groups combined. The relationship between cortical morphology and age was investigated. Key findings The voxel-based morphometry analysis indicated increased bilateral grey matter volume in the superior frontal gyrus, insula and right inferior frontal gyrus regions in BECTS. The magnitude of the increase lessened with age of the cases. Cortical thickness analysis revealed thicker cortex in BECTS along middle and inferior frontal gyri bilaterally, left insula and bilateral supramarginal gyrus in the 9-year-after-onset group, that normalised with age. The rate of cortical thickness changes with age were greater in BECTS cases than in controls. Significance Increased cortical gray matter associated with BECTS was found. The decreasing magnitude of the effect with increasing age parallels the natural history of the disorder. The areas affected are consistent with neurocognitive dysfunction in BECTS. PMID:23375559

  7. Structural basis for the inhibition of bacterial multidrug exporters.

    PubMed

    Nakashima, Ryosuke; Sakurai, Keisuke; Yamasaki, Seiji; Hayashi, Katsuhiko; Nagata, Chikahiro; Hoshino, Kazuki; Onodera, Yoshikuni; Nishino, Kunihiko; Yamaguchi, Akihito

    2013-08-01

    The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.

  8. Structural Basis for Metallic-Like Conductivity in Microbial Nanowires

    PubMed Central

    Malvankar, Nikhil S.; Vargas, Madeline; Nevin, Kelly; Tremblay, Pier-Luc; Evans-Lutterodt, Kenneth; Nykypanchuk, Dmytro; Martz, Eric; Tuominen, Mark T.

    2015-01-01

    ABSTRACT Direct measurement of multiple physical properties of Geobacter sulfurreducens pili have demonstrated that they possess metallic-like conductivity, but several studies have suggested that metallic-like conductivity is unlikely based on the structures of the G. sulfurreducens pilus predicted from homology models. In order to further evaluate this discrepancy, pili were examined with synchrotron X-ray microdiffraction and rocking-curve X-ray diffraction. Both techniques revealed a periodic 3.2-Å spacing in conductive, wild-type G. sulfurreducens pili that was missing in the nonconductive pili of strain Aro5, which lack key aromatic acids required for conductivity. The intensity of the 3.2-Å peak increased 100-fold when the pH was shifted from 10.5 to 2, corresponding with a previously reported 100-fold increase in pilus conductivity with this pH change. These results suggest a clear structure-function correlation for metallic-like conductivity that can be attributed to overlapping π-orbitals of aromatic amino acids. A homology model of the G. sulfurreducens pilus was constructed with a Pseudomonas aeruginosa pilus model as a template as an alternative to previous models, which were based on a Neisseria gonorrhoeae pilus structure. This alternative model predicted that aromatic amino acids in G. sulfurreducens pili are packed within 3 to 4 Å, consistent with the experimental results. Thus, the predictions of homology modeling are highly sensitive to assumptions inherent in the model construction. The experimental results reported here further support the concept that the pili of G. sulfurreducens represent a novel class of electronically functional proteins in which aromatic amino acids promote long-distance electron transport. PMID:25736881

  9. Structural Basis for Metallic-Like Conductivity in Microbial Nanowires

    SciTech Connect

    Malvankar, Nikhil S.; Vargas, Madeline; Nevin, Kelly; Tremblay, Pier-Luc; Evans-Lutterodt, Kenneth; Nykypanchuk, Dmytro; Martz, Eric; Tuominen, Mark T.; Lovley, Derek R.

    2015-03-03

    Direct measurement of multiple physical properties of Geobacter sulfurreducens pili have demonstrated that they possess metallic-like conductivity, but several studies have suggested that metallic-like conductivity is unlikely based on the structures of the G. sulfurreducens pilus predicted from homology models. In order to further evaluate this discrepancy, pili were examined with synchrotron X-ray microdiffraction and rocking-curve X-ray diffraction. Both techniques revealed a periodic 3.2-Å spacing in conductive, wild-type G. sulfurreducens pili that was missing in the nonconductive pili of strain Aro5, which lack key aromatic acids required for conductivity. The intensity of the 3.2-Å peak increased 100-fold when the pH was shifted from 10.5 to 2, corresponding with a previously reported 100-fold increase in pilus conductivity with this pH change. These results suggest a clear structure-function correlation for metallic-like conductivity that can be attributed to overlapping π-orbitals of aromatic amino acids. A homology model of the G. sulfurreducens pilus was constructed with a Pseudomonas aeruginosa pilus model as a template as an alternative to previous models, which were based on a Neisseria gonorrhoeae pilus structure. This alternative model predicted that aromatic amino acids in G. sulfurreducens pili are packed within 3 to 4 Å, consistent with the experimental results. Thus, the predictions of homology modeling are highly sensitive to assumptions inherent in the model construction. Finally, the experimental results reported here further support the concept that the pili of G. sulfurreducens represent a novel class of electronically functional proteins in which aromatic amino acids promote long-distance electron transport.

  10. Structural Basis for Metallic-Like Conductivity in Microbial Nanowires

    DOE PAGES

    Malvankar, Nikhil S.; Vargas, Madeline; Nevin, Kelly; Tremblay, Pier-Luc; Evans-Lutterodt, Kenneth; Nykypanchuk, Dmytro; Martz, Eric; Tuominen, Mark T.; Lovley, Derek R.

    2015-03-03

    Direct measurement of multiple physical properties of Geobacter sulfurreducens pili have demonstrated that they possess metallic-like conductivity, but several studies have suggested that metallic-like conductivity is unlikely based on the structures of the G. sulfurreducens pilus predicted from homology models. In order to further evaluate this discrepancy, pili were examined with synchrotron X-ray microdiffraction and rocking-curve X-ray diffraction. Both techniques revealed a periodic 3.2-Å spacing in conductive, wild-type G. sulfurreducens pili that was missing in the nonconductive pili of strain Aro5, which lack key aromatic acids required for conductivity. The intensity of the 3.2-Å peak increased 100-fold when the pHmore » was shifted from 10.5 to 2, corresponding with a previously reported 100-fold increase in pilus conductivity with this pH change. These results suggest a clear structure-function correlation for metallic-like conductivity that can be attributed to overlapping π-orbitals of aromatic amino acids. A homology model of the G. sulfurreducens pilus was constructed with a Pseudomonas aeruginosa pilus model as a template as an alternative to previous models, which were based on a Neisseria gonorrhoeae pilus structure. This alternative model predicted that aromatic amino acids in G. sulfurreducens pili are packed within 3 to 4 Å, consistent with the experimental results. Thus, the predictions of homology modeling are highly sensitive to assumptions inherent in the model construction. Finally, the experimental results reported here further support the concept that the pili of G. sulfurreducens represent a novel class of electronically functional proteins in which aromatic amino acids promote long-distance electron transport.« less

  11. Structural Basis for the Reduced Toxicity of Dinophysistoxin-2

    SciTech Connect

    Huhn, J.; Jeffrey, F; Larsen, K; Rundberget, T; Rise, F; Cox, N; Arcus, V; Shi, Y; Miles, C

    2009-01-01

    Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic {pi}-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, the axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140{sup o} so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized.

  12. Structural basis of diverse membrane target recognitions by ankyrins

    PubMed Central

    Wang, Chao; Wei, Zhiyi; Chen, Keyu; Ye, Fei; Yu, Cong; Bennett, Vann; Zhang, Mingjie

    2014-01-01

    Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins' membrane targets, with implications for other proteins containing extended ANK repeat domains. DOI: http://dx.doi.org/10.7554/eLife.04353.001 PMID:25383926

  13. Structural basis for the transcriptional regulation of membrane lipid homeostasis

    SciTech Connect

    Miller, Darcie J.; Zhang, Yong-Mei; Subramanian, Chitra; Rock, Charles O.; White, Stephen W.

    2010-11-09

    DesT is a transcriptional repressor that regulates the genes that control the unsaturated:saturated fatty acid ratio available for membrane lipid synthesis. DesT bound to unsaturated acyl-CoA has a high affinity for its cognate palindromic DNA-binding site, whereas DesT bound to saturated acyl-CoA does not bind this site. Structural analyses of the DesT-oleoyl-CoA-DNA and DesT-palmitoyl-CoA complexes reveal that acyl chain shape directly influences the packing of hydrophobic core residues within the DesT ligand-binding domain. These changes are propagated to the paired DNA-binding domains via conformational changes to modulate DNA binding. These structural interpretations are supported by the in vitro and in vivo characterization of site-directed mutants. The regulation of DesT by the unsaturated:saturated ratio of acyl chains rather than the concentration of a single ligand is a paradigm for understanding transcriptional regulation of membrane lipid homeostasis.

  14. The Structural Basis of Cholesterol Activity in Membranes

    SciTech Connect

    Olsen, Brett N.; Bielska, Agata; Lee, Tiffany; Daily, Michael D.; Covey, Douglas F.; Schlesinger, Paul H.; Baker, Nathan A.; Ory, Daniel S.

    2013-10-15

    Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.

  15. Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

    PubMed

    Prota, Andrea E; Magiera, Maria M; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C; Kammerer, Richard A; Janke, Carsten; Steinmetz, Michel O

    2013-02-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  16. Structural Basis for Cofactor-Independent Dioxygenation in Vancomycin Biosynthesis

    SciTech Connect

    Widboom,P.; Fielding, E.; Liu, Y.; Bruner, S.

    2007-01-01

    Enzyme-catalyzed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion1. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.

  17. Structural Basis for Activation of Class Ib Ribonucleotide Reductase

    SciTech Connect

    Boal, Amie K.; Cotruvo, Jr., Joseph A.; Stubbe, JoAnne; Rosenzweig, Amy C.

    2010-12-03

    The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn{sub 2}{sup III}-tyrosyl radical (Y{sm_bullet}) or a Fe{sub 2}{sup III}-Y{sm_bullet} cofactor in the NrdF subunit. Whereas Fe{sub 2}{sup III}-Y{sm_bullet} can self-assemble from Fe{sub 2}{sup II}-NrdF and O{sub 2}, activation of Mn{sub 2}{sup II}-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O{sub 2}. The crystal structures reported here of E. coli Mn{sub 2}{sup II}-NrdF and Fe{sub 2}{sup II}-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn{sub 2}{sup II}-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn{sub 2}{sup II} active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn{sub 2}{sup III}-Y{sm_bullet} cofactor assembly.

  18. Structural basis for tandem L27 domain-mediated polymerization

    SciTech Connect

    Yang, Xue; Xie, Xingqiao; Chen, Liu; Zhou, Hao; Wang, Zheng; Zhao, Weijing; Tian, Ran; Zhang, Rongguang; Tian, Changlin; Long, Jiafu; Shen, Yuequan

    2010-12-01

    The establishment of epithelial cell polarity requires the assembly of multiprotein complexes and is crucial during epithelial morphogenesis. Three scaffolding proteins, Dlg1, MPP7, and Mals3, can be assembled to form a complex that functions in the establishment and maintenance of apicobasal polarity in epithelial tissues through their L27 domains. Here we report the crystal structure of a 4-L27-domain complex derived from the human tripartite complex Dlg1-MPP7-Mals3 in combination with paramagnetic relaxation enhancement measurements. The heterotrimer consists of 2 pairs of heterodimeric L27 domains. These 2 dimers are asymmetric due to the large difference between the N- and C-terminal tandem L27 domain of MPP7. Structural analysis combined with biochemical experiments further reveals that the loop {alpha}A-{alpha}B and helix {alpha}B of the C-terminal L27 domain of MPP7 play a critical role in assembling the entire tripartite complex, suggesting a synergistic tandem L27-mediated assembling event.

  19. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    PubMed

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-04-18

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

  20. Structural basis for germline antibody recognition of HIV-1 immunogens

    PubMed Central

    Scharf, Louise; West, Anthony P; Sievers, Stuart A; Chen, Courtney; Jiang, Siduo; Gao, Han; Gray, Matthew D; McGuire, Andrew T; Scheid, Johannes F; Nussenzweig, Michel C; Stamatatos, Leonidas; Bjorkman, Pamela J

    2016-01-01

    Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design. DOI: http://dx.doi.org/10.7554/eLife.13783.001 PMID:26997349

  1. Dissecting the structural basis of MEIG1 interaction with PACRG

    PubMed Central

    Li, Wei; Walavalkar, Ninad M.; Buchwald, William A.; Teves, Maria E.; Zhang, Ling; Liu, Hong; Bilinovich, Stephanie; Peterson, Darrell L.; Strauss III, Jerome F.; Williams Jr, David C.; Zhang, Zhibing

    2016-01-01

    The product of the meiosis-expressed gene 1 (MEIG1) is found in the cell bodies of spermatocytes and recruited to the manchette, a structure unique to elongating spermatids, by Parkin co-regulated gene (PACRG). This complex is essential for targeting cargo to the manchette during sperm flagellum assembly. Here we show that MEIG1 adopts a unique fold that provides a large surface for interacting with other proteins. We mutated 12 exposed and conserved amino acids and show that four of these mutations (W50A, K57E, F66A, Y68A) dramatically reduce binding to PACRG. These four amino acids form a contiguous hydrophobic patch on one end of the protein. Furthermore, each of these four mutations diminishes the ability of MEIG1 to stabilize PACRG when expressed in bacteria. Together these studies establish the unique structure and key interaction surface of MEIG1 and provide a framework to explore how MEIG1 recruits proteins to build the sperm tail. PMID:26726850

  2. Structural basis of ultraviolet-B perception by UVR8.

    PubMed

    Wu, Di; Hu, Qi; Yan, Zhen; Chen, Wen; Yan, Chuangye; Huang, Xi; Zhang, Jing; Yang, Panyu; Deng, Haiteng; Wang, Jiawei; Deng, XingWang; Shi, Yigong

    2012-04-12

    The Arabidopsis thaliana protein UVR8 is a photoreceptor for ultraviolet-B. Upon ultraviolet-B irradiation, UVR8 undergoes an immediate switch from homodimer to monomer, which triggers a signalling pathway for ultraviolet protection. The mechanism by which UVR8 senses ultraviolet-B remains largely unknown. Here we report the crystal structure of UVR8 at 1.8 Å resolution, revealing a symmetric homodimer of seven-bladed β-propeller that is devoid of any external cofactor as the chromophore. Arginine residues that stabilize the homodimeric interface, principally Arg 286 and Arg 338, make elaborate intramolecular cation-π interactions with surrounding tryptophan amino acids. Two of these tryptophans, Trp 285 and Trp 233, collectively serve as the ultraviolet-B chromophore. Our structural and biochemical analyses identify the molecular mechanism for UVR8-mediated ultraviolet-B perception, in which ultraviolet-B radiation results in destabilization of the intramolecular cation-π interactions, causing disruption of the critical intermolecular hydrogen bonds mediated by Arg 286 and Arg 338 and subsequent dissociation of the UVR8 homodimer.

  3. Structural basis of HIV-1 resistance to AZT by excision.

    PubMed

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D; Clark, Arthur D; Hou, Xiaorong; Frenkel, Yulia V; Gaffney, Barbara L; Jones, Roger A; Boyer, Paul L; Hughes, Stephen H; Sarafianos, Stefan G; Arnold, Eddy

    2010-10-01

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  4. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  5. Structural Basis of Biological NO Generation by Octaheme Oxidoreductases*

    PubMed Central

    Maalcke, Wouter J.; Dietl, Andreas; Marritt, Sophie J.; Butt, Julea N.; Jetten, Mike S. M.; Keltjens, Jan T.; Barends, Thomas R. M.; Kartal, Boran

    2014-01-01

    Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have. PMID:24302732

  6. Structural basis of protein phosphatase 2A stable latency

    PubMed Central

    Jiang, Li; Stanevich, Vitali; Satyshur, Kenneth A; Kong, Mei; Watkins, Guy R.; Wadzinski, Brian E.; Sengupta, Rituparna; Xing, Yongna

    2013-01-01

    The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac–α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes. PMID:23591866

  7. Structural basis of pharmacological chaperoning for human β-galactosidase.

    PubMed

    Suzuki, Hironori; Ohto, Umeharu; Higaki, Katsumi; Mena-Barragán, Teresa; Aguilar-Moncayo, Matilde; Ortiz Mellet, Carmen; Nanba, Eiji; Garcia Fernandez, Jose M; Suzuki, Yoshiyuki; Shimizu, Toshiyuki

    2014-05-23

    GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, β-Gal(R201C) and β-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.

  8. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.

  9. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

    PubMed Central

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C.

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  10. Structural basis for DNA binding by replication initiator Mcm10

    SciTech Connect

    Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin; Greer, Briana; Bielinsky, Anja-Katrin; Chazin, Walter J.; Eichman, Brandt F.

    2009-06-30

    Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.

  11. Understanding the structural basis for controlling chromosome division

    PubMed Central

    Barford, David

    2015-01-01

    The process of chromosome division, termed mitosis, involves a complex sequence of events that is tightly controlled to ensure that the faithful segregation of duplicated chromosomes is coordinated with each cell division cycle. The large macromolecular complex responsible for regulating this process is the anaphase-promoting complex or cyclosome (APC/C). In humans, the APC/C is assembled from 20 subunits derived from 15 different proteins. The APC/C functions to ubiquitinate cell cycle regulatory proteins, thereby targeting them for destruction by the proteasome. This review describes our research aimed at understanding the structure and mechanism of the APC/C. We have determined the crystal structures of individual subunits and subcomplexes that provide atomic models to interpret density maps of the whole complex derived from single particle cryo-electron microscopy. With this information, we are generating pseudo-atomic models of functional states of the APC/C that provide insights into its overall architecture and mechanisms of substrate recognition, catalysis and regulation by inhibitory complexes. PMID:25624511

  12. Structural basis for cyclodextrins' suppression of human growth hormone aggregation

    PubMed Central

    Otzen, Daniel Erik; Knudsen, Benjamin Raerup; Aachmann, Finn; Larsen, Kim Lambertsen; Wimmer, Reinhard

    2002-01-01

    Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various β-CD derivatives is sufficient to completely avoid this. α- and γ-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of β-CD are analyzed according to a minimalist association model to yield an apparent hGH-β-CD dissociation constant of ∼6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with β-CD. Nuclear magnetic resonance studies indicate that β-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation. PMID:12070330

  13. Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

    PubMed

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  14. The Structural Basis of Cholesterol Accessibility in Membranes

    PubMed Central

    Olsen, Brett N.; Bielska, Agata A.; Lee, Tiffany; Daily, Michael D.; Covey, Douglas F.; Schlesinger, Paul H.; Baker, Nathan A.; Ory, Daniel S.

    2013-01-01

    Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity. PMID:24138860

  15. Caspar carboxylates: the structural basis of tobamovirus disassembly.

    PubMed Central

    Wang, H; Planchart, A; Stubbs, G

    1998-01-01

    Carboxylate groups have been known for many years to drive the disassembly of simple viruses, including tobacco mosaic virus (TMV). The identities of the carboxylate groups involved and the mechanism by which they initiate disassembly have not, however, been clear. Structures have been determined at resolutions between 2.9 and 3.5 A for five tobamoviruses by fiber diffraction methods. Site-directed mutagenesis has also been used to change numerous carboxylate side chains in TMV to the corresponding amides. Comparison of the stabilities of the various mutant viruses shows that disassembly is driven by a much more complex set of carboxylate interactions than had previously been postulated. Despite the importance of the carboxylate interactions, they are not conserved during viral evolution. Instead, it appears that during evolution, patches of electrostatic interaction drift across viral subunit interfaces. The flexibility of these interactions confers a considerable advantage on the virus, enabling it to change its surface structure rapidly and thus evade host defenses. PMID:9449364

  16. Structural basis of HIV-1 resistance to AZT by excision

    SciTech Connect

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D.; Clark, Jr., Arthur D.; Hou, Xiaorong; Frenkel, Yulia V.; Gaffney, Barbara L.; Jones, Roger A.; Boyer, Paul L.; Hughes, Stephen H.; Sarafianos, Stefan G.; Arnold, Eddy

    2011-11-23

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  17. Structural Basis for Myosin V Discrimination Between Distinct Cargoes

    SciTech Connect

    Pashkova,N.; Jin, Y.; Ramaswamy, S.; Weisman, L.

    2006-01-01

    Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo-specific receptors. Here we report the crystal structure at 2.2 {angstrom} of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo-specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong-shaped cargo-binding domain, and moreover are offset by 180{sup o}. The fact that the cargo-binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle-specific myosin V receptors.

  18. Structural basis of RND-type multidrug exporters

    PubMed Central

    Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

    2015-01-01

    Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient

  19. Structural basis for membrane anchoring of HIV-1 envelope spike.

    PubMed

    Dev, Jyoti; Park, Donghyun; Fu, Qingshan; Chen, Jia; Ha, Heather Jiwon; Ghantous, Fadi; Herrmann, Tobias; Chang, Weiting; Liu, Zhijun; Frey, Gary; Seaman, Michael S; Chen, Bing; Chou, James J

    2016-07-01

    HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We used nuclear magnetic resonance to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An amino-terminal coiled-coil and a carboxyl-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes, and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design. PMID:27338706

  20. A Structural Basis for How Motile Cilia Beat

    PubMed Central

    Satir, Peter; Heuser, Thomas; Sale, Winfield S.

    2014-01-01

    The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke–central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation. PMID:26955066

  1. Structural Basis of Substrate Conversion in a New Aromatic Peroxygenase

    PubMed Central

    Piontek, Klaus; Strittmatter, Eric; Ullrich, René; Gröbe, Glenn; Pecyna, Marek J.; Kluge, Martin; Scheibner, Katrin; Hofrichter, Martin; Plattner, Dietmar A.

    2013-01-01

    Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO. PMID:24126915

  2. Structural basis for therapeutic inhibition of complement C5.

    PubMed

    Jore, Matthijs M; Johnson, Steven; Sheppard, Devon; Barber, Natalie M; Li, Yang I; Nunn, Miles A; Elmlund, Hans; Lea, Susan M

    2016-05-01

    Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation. PMID:27018802

  3. Structural Basis for Translation Termination on the 70S Ribosome

    SciTech Connect

    Laurberg, M.; Asahara, H.; Korostelev, A.; Zhu, J.; Trakhanov, S.; Noller, H.F.

    2009-05-20

    At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 {angstrom} resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.

  4. Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

    PubMed

    Hagen, Christoph; Dent, Kyle C; Zeev-Ben-Mordehai, Tzviya; Grange, Michael; Bosse, Jens B; Whittle, Cathy; Klupp, Barbara G; Siebert, C Alistair; Vasishtan, Daven; Bäuerlein, Felix J B; Cheleski, Juliana; Werner, Stephan; Guttmann, Peter; Rehbein, Stefan; Henzler, Katja; Demmerle, Justin; Adler, Barbara; Koszinowski, Ulrich; Schermelleh, Lothar; Schneider, Gerd; Enquist, Lynn W; Plitzko, Jürgen M; Mettenleiter, Thomas C; Grünewald, Kay

    2015-12-17

    Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

  5. Structural basis for selective activation of ABA receptors

    SciTech Connect

    Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F.

    2010-11-01

    Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

  6. Structural basis of rifampin inactivation by rifampin phosphotransferase

    PubMed Central

    Qi, Xiaofeng; Lin, Wei; Ma, Miaolian; Wang, Chengyuan; He, Yang; He, Nisha; Gao, Jing; Zhou, Hu; Xiao, Youli; Wang, Yong

    2016-01-01

    Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins. PMID:27001859

  7. Structural basis of transport of lysophospholipids by human serum albumin

    SciTech Connect

    Guo, Shihui; Shi, Xiaoli; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Bian, Chuanbing; Huang, Mingdong

    2010-10-08

    Lysophospholipids play important roles in cellular signal transduction and are implicated in many biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, cancer and neuronal survival. The intracellular transport of lysophospholipids is through FA (fatty acid)-binding protein. Lysophospholipids are also found in the extracellular space. However, the transport mechanism of lysophospholipids in the extracellular space is unknown. HSA (human serum albumin) is the most abundant carrier protein in blood plasma and plays an important role in determining the absorption, distribution, metabolism and excretion of drugs. In the present study, LPE (lysophosphatidylethanolamine) was used as the ligand to analyse the interaction of lysophospholipids with HSA by fluorescence quenching and crystallography. Fluorescence measurement showed that LPE binds to HSA with a K{sub d} (dissociation constant) of 5.6 {micro}M. The presence of FA (myristate) decreases this binding affinity (K{sub d} of 12.9 {micro}M). Moreover, we determined the crystal structure of HSA in complex with both myristate and LPE and showed that LPE binds at Sudlow site I located in subdomain IIA. LPE occupies two of the three subsites in Sudlow site I, with the LPE acyl chain occupying the hydrophobic bottom of Sudlow site I and the polar head group located at Sudlow site I entrance region pointing to the solvent. This orientation of LPE in HSA suggests that HSA is capable of accommodating other lysophospholipids and phospholipids. The study provides structural information on HSA-lysophospholipid interaction and may facilitate our understanding of the transport and distribution of lysophospholipids.

  8. Lamprey hemoglobin. Structural basis of the bohr effect.

    PubMed

    Qiu, Y; Maillett, D H; Knapp, J; Olson, J S; Riggs, A F

    2000-05-01

    Lampreys, among the most primitive living vertebrates, have hemoglobins (Hbs) with self-association and ligand-binding properties very different from those that characterize the alpha(2)beta(2) tetrameric Hbs of higher vertebrates. Monomeric, ligated lamprey Hb self-associates to dimers and tetramers upon deoxygenation. Dissociation to monomers upon oxygenation accounts for the cooperative binding of O(2) and its pH dependence. Honzatko and Hendrickson (Honzatko, R. B., and Hendrickson, W. A. (1986) Proc. Natl. Acad. Sci. U. S. A 83, 8487-8491) proposed that the dimeric interface of the Hb resembles either the alpha(1)beta(2) interface of mammalian Hbs or the contacts in clam Hb where the E and F helices form the interface. Perutz (Perutz, M. F. (1989) Quart. Rev. Biophys. 2, 139- 236) proposed a version of the clam model in which the distal histidine swings out of the heme pocket upon deoxygenation to form a bond with a carboxyl group of a second monomer. The sedimentation behavior and oxygen equilibria of nine mutants of the major Hb component, PMII, from Petromyzon marinus have been measured to test these models. The results strongly support a critical role of the E helix and the AB corner in forming the subunit interface in the dimer and rule out the alpha(1)beta(2) model. The pH dependence of both the sedimentation equilibrium and the oxygen binding of the mutant E75Q indicate that Glu(75) is one of two groups responsible for the Bohr effect. Changing the distal histidine 73 to glutamine almost completely abolishes the self-association of the deoxy-Hb and causes a large increase in O(2) affinity. The recent x-ray crystallographic determination of the structure of deoxy lamprey Hb, reported after the completion of this work (Heaslet, H. A., and Royer, W. E. (1999) Structure 7, 517-526), shows that the dimer interface does involve the E helix and the AB corner, supporting the measurements and interpretations reported here.

  9. Structural basis and functions of abscisic acid receptors PYLs

    PubMed Central

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

    2015-01-01

    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

  10. Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

    SciTech Connect

    J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

    2011-12-31

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  11. Structural basis for alginate secretion across the bacterial outer membrane

    SciTech Connect

    Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

    2011-08-09

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  12. The Structural Basis for Matrix Metalloproteinase 1 Catalyzed Collagenolysis

    PubMed Central

    Bertini, Ivano; Fragai, Marco; Luchinat, Claudio; Melikian, Maxime; Toccafondi, Mirco; Lauer, Janelle L.; Fields, Gregg B.

    2012-01-01

    The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782–785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray “closed” conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally-derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function. PMID:22239621

  13. Structural basis for glucose-6-phosphate activation of glycogen synthase

    SciTech Connect

    Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.

    2010-11-22

    Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

  14. Structural Basis for Viral Late-Domain Binding to Alix

    SciTech Connect

    Lee,S.; Joshi, A.; Nagashima, K.; Freed, E.; Hurley, J.

    2007-01-01

    The modular protein Alix is a central node in endosomal-lysosomal trafficking and the budding of human immunodeficiency virus (HIV)-1. The Gag p6 protein of HIV-1 contains a LYPx{sub n}LxxL motif that is required for Alix-mediated budding and binds a region of Alix spanning residues 360-702. The structure of this fragment of Alix has the shape of the letter 'V' and is termed the V domain. The V domain has a topologically complex arrangement of 11 {alpha}-helices, with connecting loops that cross three times between the two arms of the V. The conserved residue Phe676 is at the center of a large hydrophobic pocket and is crucial for binding to a peptide model of HIV-1 p6. Overexpression of the V domain inhibits HIV-1 release from cells. This inhibition of release is reversed by mutations that block binding of the Alix V domain to p6.

  15. Structural Basis of Glycogen Biosynthesis Regulation in Bacteria.

    PubMed

    Cifuente, Javier O; Comino, Natalia; Madariaga-Marcos, Julene; López-Fernández, Sonia; García-Alija, Mikel; Agirre, Jon; Albesa-Jové, David; Guerin, Marcelo E

    2016-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.

  16. Structural basis for the mechanism of ABC transporters.

    PubMed

    Beis, Konstantinos

    2015-10-01

    The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

  17. A structural basis for electron transfer in bacterial photosynthesis

    SciTech Connect

    Norris, J.R.; DiMagno, T.J.; Angerhofer, A.; Chang, C.H.; El-Kabbani, O.; Schiffer, M.

    1989-01-01

    Triplet data for the primary donor in single crystals of bacterial reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis are interpreted in terms of the corresponding x-ray structures. The analysis of electron paramagnetic resonance data from single crystals (triplet zero field splitting and cation and triplet linewidth of the primary special pair donor of bacterial reaction centers) is extended to systems of a non-crystalline nature. A unified interpretation based on frontier molecular orbitals concludes that the special pair behaves like a supermolecule in all wild-type bacteria investigated here. However, in heterodimers of Rb. capsulatus (His/sup M200/ changed to Leu or Phe with the result that the M-half of the special pair is converted to bacteriopheophytin) the special pair possesses the EPR properties more appropriately described in terms of a monomer. In all cases the triplet state and cation EPR properties appear to be dominated by the highest occupied molecular orbitals. These conclusions derived from EPR experiments are supplemented by data from Stark spectroscopy of reaction centers from Rb. capsulatus. 41 refs., 3 tabs.

  18. Structural Basis for Norovirus Inhibition and Fucose Mimicry by Citrate

    SciTech Connect

    Hansman, Grant S.; Shahzad-ul-Hussan, Syed; McLellan, Jason S.; Chuang, Gwo-Yu; Georgiev, Ivelin; Shimoike, Takashi; Katayama, Kazuhiko; Bewley, Carole A.; Kwong, Peter D.

    2012-01-20

    Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 {angstrom} and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 {mu}M). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 {mu}M) and H type 2 trisaccharide (390 {mu}M), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.

  19. Structural basis for norovirus inhibition and fucose mimicry by citrate.

    PubMed

    Hansman, Grant S; Shahzad-Ul-Hussan, Syed; McLellan, Jason S; Chuang, Gwo-Yu; Georgiev, Ivelin; Shimoike, Takashi; Katayama, Kazuhiko; Bewley, Carole A; Kwong, Peter D

    2012-01-01

    Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.

  20. Structural basis underlying viral hijacking of a histone chaperone complex

    PubMed Central

    Huang, Hongda; Deng, Zhong; Vladimirova, Olga; Wiedmer, Andreas; Lu, Fang; Lieberman, Paul M.; Patel, Dinshaw J.

    2016-01-01

    The histone H3.3 chaperone DAXX is implicated in formation of heterochromatin and transcription silencing, especially for newly infecting DNA virus genomes entering the nucleus. Epstein-Barr virus (EBV) can efficiently establish stable latent infection as a chromatinized episome in the nucleus of infected cells. The EBV tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency. Here we report the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4. BNRF1 DID contacts DAXX HBD and histones through non-conserved loops. The BNRF1-DAXX interface is responsible for BNRF1 localization to PML-nuclear bodies typically associated with host-antiviral resistance and transcriptional repression. Paradoxically, the interface is also required for selective transcription activation of viral latent cycle genes required for driving B-cell proliferation. These findings reveal molecular details of virus reprogramming of an antiviral histone chaperone to promote viral latency and cellular immortalization. PMID:27581705

  1. Structural Basis of Glycogen Biosynthesis Regulation in Bacteria.

    PubMed

    Cifuente, Javier O; Comino, Natalia; Madariaga-Marcos, Julene; López-Fernández, Sonia; García-Alija, Mikel; Agirre, Jon; Albesa-Jové, David; Guerin, Marcelo E

    2016-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role. PMID:27545622

  2. Structural basis of glycan interaction in gastroenteric viral pathogens

    PubMed Central

    Prasad, B.V. Venkataram; Shanker, Sreejesh; Hu, Liya; Choi, Jae-Mun; Crawford, Sue E; Ramani, Sasirekha; Czako, Rita; Atmar, Robert L; Estes, Mary K

    2014-01-01

    A critical event in the life cycle of a virus is its initial attachment to host cells. This involves recognition by the viruses of specific receptors on the cell surface, including glycans. Viruses typically exhibit strain-dependent variations in recognizing specific glycan receptors, a feature that contributes significantly to cell tropism, host specificity, host adaptation and interspecies transmission. Examples include influenza viruses, noroviruses, rotaviruses, and parvoviruses. Both rotaviruses and noroviruses are well known gastroenteric pathogens that are of significant global health concern. While rotaviruses, in the family Reoviridae, are the major causative agents of life-threatening diarrhea in children, noroviruses, which belong to Caliciviridae family, cause epidemic and sporadic cases of acute gastroenteritis across all age groups. Both exhibit enormous genotypic and serotypic diversity. Consistent with this diversity each exhibits strain-dependent variations in the types of glycans they recognize for cell attachment. This chapter reviews current status of the structural biology of such strain-dependent glycan specificities in these two families of viruses. PMID:25073118

  3. Structural basis of haem-iron acquisition by fungal pathogens.

    PubMed

    Nasser, Lena; Weissman, Ziva; Pinsky, Mariel; Amartely, Hadar; Dvir, Hay; Kornitzer, Daniel

    2016-01-01

    Pathogenic microorganisms must cope with extremely low free-iron concentrations in the host's tissues. Some fungal pathogens rely on secreted haemophores that belong to the Common in Fungal Extracellular Membrane (CFEM) protein family, to extract haem from haemoglobin and to transfer it to the cell's interior, where it can serve as a source of iron. Here we report the first three-dimensional structure of a CFEM protein, the haemophore Csa2 secreted by Candida albicans. The CFEM domain adopts a novel helical-basket fold that consists of six α-helices, and is uniquely stabilized by four disulfide bonds formed by its eight signature cysteines. The planar haem molecule is bound between a flat hydrophobic platform located on top of the helical basket and a peripheral N-terminal 'handle' extension. Exceptionally, an aspartic residue serves as the CFEM axial ligand, and so confers coordination of Fe(3+) haem, but not of Fe(2+) haem. Histidine substitution mutants of this conserved Asp acquired Fe(2+) haem binding and retained the capacity to extract haem from haemoglobin. However, His-substituted CFEM proteins were not functional in vivo and showed disturbed haem exchange in vitro, which suggests a role for the oxidation-state-specific Asp coordination in haem acquisition by CFEM proteins. PMID:27617569

  4. Structural basis for the antifolding activity of a molecular chaperone

    NASA Astrophysics Data System (ADS)

    Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.

    2016-09-01

    Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.

  5. Structural basis for reduced glomerular filtration capacity in nephrotic humans.

    PubMed Central

    Drumond, M C; Kristal, B; Myers, B D; Deen, W M

    1994-01-01

    Previous studies have established that in a variety of human glomerulopathies the reduced glomerular filtration rate (GFR) is due to a marked lowering of the ultrafiltration coefficient (Kf). To identify the factors which lower Kf, we measured the filtering surface area per glomerulus, filtration slit frequency, basement membrane thickness, and GFR and its determinants in patients with minimal change and membraneous nephropathies and in age-matched healthy controls. Overall values of Kf for the two kidneys were calculated from GFR, renal plasma flow rate, systemic colloid osmotic pressure, and three assumed values for the transcapillary pressure difference. "Experimental" values of the glomerular hydraulic permeability (kexp) were then calculated from Kf, glomerular filtering surface area, and estimates of the total number of nephrons of the two kidneys. Independent estimates of the glomerular hydraulic permeability (kmodel) were obtained using a recent mathematical model that is based on analyses of viscous flow through the various structural components of the glomerular capillary wall. Individual values of basement membrane thickness and filtration slit frequency were used as inputs in this model. The results indicate that the reductions of Kf in both nephropathies can be attributed entirely to reduced glomerular hydraulic permeability. The mean values of kexp and kmodel were very similar in both disorders and much smaller in the nephrotic groups than in healthy controls. There was good agreement between kexp and kmodel for any given group of subjects. It was shown that, in both groups of nephrotics, filtration slit frequency was a more important determinant of the water flow resistance than was basement membrane thickness. The decrease in filtration slit frequency observed in both disorders caused the average path length for the filtrate to increase, thereby explaining the decreased hydraulic permeability. Images PMID:8083359

  6. 26 CFR 1.1502-31 - Stock basis after a group structure change.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... transaction is a group structure change under § 1.1502-33(f)(1), and the earnings and profits of P and S are... the 26 CFR part 1 edition revised as of April 1, 2003. For group structure changes that occur in... 26 Internal Revenue 12 2010-04-01 2010-04-01 false Stock basis after a group structure change....

  7. 26 CFR 1.1502-31 - Stock basis after a group structure change.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... See § 1.1502-33(f)(1) for the definition of group structure change. For example, if P owns all of the... the 26 CFR part 1 edition revised as of April 1, 2003. For group structure changes that occur in... 26 Internal Revenue 12 2012-04-01 2012-04-01 false Stock basis after a group structure change....

  8. A novel Gaussian-Sinc mixed basis set for electronic structure calculations

    NASA Astrophysics Data System (ADS)

    Jerke, Jonathan L.; Lee, Young; Tymczak, C. J.

    2015-08-01

    A Gaussian-Sinc basis set methodology is presented for the calculation of the electronic structure of atoms and molecules at the Hartree-Fock level of theory. This methodology has several advantages over previous methods. The all-electron electronic structure in a Gaussian-Sinc mixed basis spans both the "localized" and "delocalized" regions. A basis set for each region is combined to make a new basis methodology—a lattice of orthonormal sinc functions is used to represent the "delocalized" regions and the atom-centered Gaussian functions are used to represent the "localized" regions to any desired accuracy. For this mixed basis, all the Coulomb integrals are definable and can be computed in a dimensional separated methodology. Additionally, the Sinc basis is translationally invariant, which allows for the Coulomb singularity to be placed anywhere including on lattice sites. Finally, boundary conditions are always satisfied with this basis. To demonstrate the utility of this method, we calculated the ground state Hartree-Fock energies for atoms up to neon, the diatomic systems H2, O2, and N2, and the multi-atom system benzene. Together, it is shown that the Gaussian-Sinc mixed basis set is a flexible and accurate method for solving the electronic structure of atomic and molecular species.

  9. A novel Gaussian-Sinc mixed basis set for electronic structure calculations

    SciTech Connect

    Jerke, Jonathan L.; Lee, Young; Tymczak, C. J.

    2015-08-14

    A Gaussian-Sinc basis set methodology is presented for the calculation of the electronic structure of atoms and molecules at the Hartree–Fock level of theory. This methodology has several advantages over previous methods. The all-electron electronic structure in a Gaussian-Sinc mixed basis spans both the “localized” and “delocalized” regions. A basis set for each region is combined to make a new basis methodology—a lattice of orthonormal sinc functions is used to represent the “delocalized” regions and the atom-centered Gaussian functions are used to represent the “localized” regions to any desired accuracy. For this mixed basis, all the Coulomb integrals are definable and can be computed in a dimensional separated methodology. Additionally, the Sinc basis is translationally invariant, which allows for the Coulomb singularity to be placed anywhere including on lattice sites. Finally, boundary conditions are always satisfied with this basis. To demonstrate the utility of this method, we calculated the ground state Hartree–Fock energies for atoms up to neon, the diatomic systems H{sub 2}, O{sub 2}, and N{sub 2}, and the multi-atom system benzene. Together, it is shown that the Gaussian-Sinc mixed basis set is a flexible and accurate method for solving the electronic structure of atomic and molecular species.

  10. A novel Gaussian-Sinc mixed basis set for electronic structure calculations.

    PubMed

    Jerke, Jonathan L; Lee, Young; Tymczak, C J

    2015-08-14

    A Gaussian-Sinc basis set methodology is presented for the calculation of the electronic structure of atoms and molecules at the Hartree-Fock level of theory. This methodology has several advantages over previous methods. The all-electron electronic structure in a Gaussian-Sinc mixed basis spans both the "localized" and "delocalized" regions. A basis set for each region is combined to make a new basis methodology-a lattice of orthonormal sinc functions is used to represent the "delocalized" regions and the atom-centered Gaussian functions are used to represent the "localized" regions to any desired accuracy. For this mixed basis, all the Coulomb integrals are definable and can be computed in a dimensional separated methodology. Additionally, the Sinc basis is translationally invariant, which allows for the Coulomb singularity to be placed anywhere including on lattice sites. Finally, boundary conditions are always satisfied with this basis. To demonstrate the utility of this method, we calculated the ground state Hartree-Fock energies for atoms up to neon, the diatomic systems H2, O2, and N2, and the multi-atom system benzene. Together, it is shown that the Gaussian-Sinc mixed basis set is a flexible and accurate method for solving the electronic structure of atomic and molecular species. PMID:26277128

  11. Crystal structure of human persulfide dioxygenase: structural basis of ethylmalonic encephalopathy

    PubMed Central

    Pettinati, Ilaria; Brem, Jürgen; McDonough, Michael A.; Schofield, Christopher J.

    2015-01-01

    The ethylmalonic encephalopathy protein 1 (ETHE1) catalyses the oxygen-dependent oxidation of glutathione persulfide (GSSH) to give persulfite and glutathione. Mutations to the hETHE1 gene compromise sulfide metabolism leading to the genetic disease ethylmalonic encephalopathy. hETHE1 is a mono-iron binding member of the metallo-β-lactamase (MBL) fold superfamily. We report crystallographic analysis of hETHE1 in complex with iron to 2.6 Å resolution. hETHE1 contains an αββα MBL-fold, which supports metal-binding by the side chains of an aspartate and two histidine residues; three water molecules complete octahedral coordination of the iron. The iron binding hETHE1 enzyme is related to the ‘classical’ di-zinc binding MBL hydrolases involved in antibiotic resistance, but has distinctive features. The histidine and aspartate residues involved in iron-binding in ETHE1, occupy similar positions to those observed across both the zinc 1 and zinc 2 binding sites in classical MBLs. The active site of hETHE1 is very similar to an ETHE1-like enzyme from Arabidopsis thaliana (60% sequence identity). A channel leading to the active site is sufficiently large to accommodate a GSSH substrate. Some of the observed hETHE1 clinical mutations cluster in the active site region. The structure will serve as a basis for detailed functional and mechanistic studies on ETHE1 and will be useful in the development of selective MBL inhibitors. PMID:25596185

  12. Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

    SciTech Connect

    Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

    2007-01-01

    The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

  13. Structural Basis of Disease-Causing Mutations in Hepatocyte Nuclear Factor 1β†,‡

    PubMed Central

    Lu, Peng; Rha, Geun Bae; Chi, Young-In

    2008-01-01

    HNF1β is an atypical POU transcription factor that participates in a hierarchical network of transcription factors controlling the development and proper function of vital organs such as liver, pancreas, and kidney. Many inheritable mutations on HNF1β are the monogenic causes of diabetes and several kidney diseases. To elucidate the molecular mechanism of its function and the structural basis of mutations, we have determined the crystal structure of human HNF1β DNA binding domain in complex with a high-affinity promoter. Disease-causing mutations have been mapped to our structure, and their predicted effects have been tested by a set of biochemical/ functional studies. These findings together with earlier findings with a homologous protein HNF1α, help us to understand the structural basis of promoter recognition by these atypical POU transcription factors and the site-specific functional disruption by disease-causing mutations. PMID:17924661

  14. Technical basis for the aboveground structure failure and associated represented hazardous conditions

    SciTech Connect

    MANGAN, D.

    2003-03-20

    The purpose of the Technical Basis Document is to determine the consequences and frequency of aboveground structure failures. These failures include drops of contained equipment, such as a pump, from a SST or DST, a crane failure resulting in a load drop onto a HEPA filter. These failures can result in an uncontrolled release of radiological and toxicological material.

  15. Electrostriction in field-structured composites: Basis for a fast artificial muscle?

    SciTech Connect

    Martin, J.E.; Anderson, R.A.

    1999-09-01

    The electrostriction of composites consisting of dielectric particles embedded in a gel or elastomer is discussed. It is shown that when these particles are organized by a uniaxial field before gelation, the resulting {ital field-structured} composites are expected to exhibit enhanced electrostriction in a uniform field applied along the same axis as the structuring field. The associated stresses might be large enough to form the basis of a polymer-based fast artificial muscle. {copyright} {ital 1999 American Institute of Physics.}

  16. Structural Basis for the Coevolution of a Viral RNA-Protein Complex

    SciTech Connect

    Chao,J.; Patskovsky, Y.; Almo, S.; Singer, R.

    2008-01-01

    The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved beta-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.

  17. A Novel Gaussian-Sinc mixed Basis Set for Electronic Structure calculations

    NASA Astrophysics Data System (ADS)

    Jerke, Jonathan; Lee, Young; Tymczak, C. J.

    2015-03-01

    A Gaussian-Sinc mixed basis set for the computation of the electronic structure of atoms and molecules is presented. Excellent bases functions are known for ``core'' and ``valence'' separately, such as Gaussians for the ``core'' wave functions and Plane-waves for ``valance'' wave functions, but as yet no method is known that can accurately deal with both regimes in a single basis. A Gaussian-Sinc mixed basis can do both. This method resolves several issues such as: i) the Sincs basis spans the same space as the plane-waves basis, yet are semi-local enough to define all interaction elements including Exchange; ii) the Gaussians span the spherically symmetric core states and can be mixed with the Sinc functions in a computationally efficient methodology; iii) together, this mixed basis set is a flexible, computationally efficient and a highly accurate method for solving atomic and molecular problems. This methodology has been implemented within the Hartree-Fock level of theory within ultra-strong magnetic fields. To demonstrate the utility of this new method, we calculated the ground state Hartree-Fock energies to five digits accuracy in ultra strong magnetic fields for Helium to Neon, Molecular Hydrogen, Water, Carbon dioxide and Benzene. Welch Foundation (Grant J-1675), the ARO (Grant W911Nf-13-1-0162), the Texas Southern University High Performance Computing Center (http:/hpcc.tsu.edu/; Grant PHY-1126251) and NSF-CREST CRCN project (Grant HRD-1137732).

  18. Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase

    SciTech Connect

    Starks, C.M.; Noel, J.P. |; Back, K.; Chappell, J.

    1997-09-19

    Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of there natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diposphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases.

  19. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    SciTech Connect

    MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth

    2010-03-29

    Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

  20. Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

    SciTech Connect

    T Nguyen; S Chang; I Evnouchidou; I York; C Zikos; K Rock; A Goldberg; E Stratikos; L Stern

    2011-12-31

    ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

  1. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

    NASA Technical Reports Server (NTRS)

    Swingle, M. R.; Honkanen, R.; Ciszak, E. M.

    2004-01-01

    Serinehhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we resolved the mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a con served Aspn-271-M(sub 1):M(sub 2)-W(sup 1)-His-427-His-304-Asp-274 catalytic motif. The structure of PPSc provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  2. Structural basis for amino acid export by DMT superfamily transporter YddG.

    PubMed

    Tsuchiya, Hirotoshi; Doki, Shintaro; Takemoto, Mizuki; Ikuta, Tatsuya; Higuchi, Takashi; Fukui, Keita; Usuda, Yoshihiro; Tabuchi, Eri; Nagatoishi, Satoru; Tsumoto, Kouhei; Nishizawa, Tomohiro; Ito, Koichi; Dohmae, Naoshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-05-30

    The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.

  3. Structural basis for amino acid export by DMT superfamily transporter YddG.

    PubMed

    Tsuchiya, Hirotoshi; Doki, Shintaro; Takemoto, Mizuki; Ikuta, Tatsuya; Higuchi, Takashi; Fukui, Keita; Usuda, Yoshihiro; Tabuchi, Eri; Nagatoishi, Satoru; Tsumoto, Kouhei; Nishizawa, Tomohiro; Ito, Koichi; Dohmae, Naoshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-06-16

    The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins. PMID:27281193

  4. Development and comparison of advanced reduced-basis methods for the transient structural analysis of unconstrained structures

    NASA Technical Reports Server (NTRS)

    Mcgowan, David M.; Bostic, Susan W.; Camarda, Charles J.

    1993-01-01

    The development of two advanced reduced-basis methods, the force derivative method and the Lanczos method, and two widely used modal methods, the mode displacement method and the mode acceleration method, for transient structural analysis of unconstrained structures is presented. Two example structural problems are studied: an undamped, unconstrained beam subject to a uniformly distributed load which varies as a sinusoidal function of time and an undamped high-speed civil transport aircraft subject to a normal wing tip load which varies as a sinusoidal function of time. These example problems are used to verify the methods and to compare the relative effectiveness of each of the four reduced-basis methods for performing transient structural analyses on unconstrained structures. The methods are verified with a solution obtained by integrating directly the full system of equations of motion, and they are compared using the number of basis vectors required to obtain a desired level of accuracy and the associated computational times as comparison criteria.

  5. Assessing the utility of phase-space-localized basis functions: Exploiting direct product structure and a new basis function selection procedure

    NASA Astrophysics Data System (ADS)

    Brown, James; Carrington, Tucker

    2016-06-01

    In this paper we show that it is possible to use an iterative eigensolver in conjunction with Halverson and Poirier's symmetrized Gaussian (SG) basis [T. Halverson and B. Poirier, J. Chem. Phys. 137, 224101 (2012)] to compute accurate vibrational energy levels of molecules with as many as five atoms. This is done, without storing and manipulating large matrices, by solving a regular eigenvalue problem that makes it possible to exploit direct-product structure. These ideas are combined with a new procedure for selecting which basis functions to use. The SG basis we work with is orders of magnitude smaller than the basis made by using a classical energy criterion. We find significant convergence errors in previous calculations with SG bases. For sum-of-product Hamiltonians, SG bases large enough to compute accurate levels are orders of magnitude larger than even simple pruned bases composed of products of harmonic oscillator functions.

  6. Assessing the utility of phase-space-localized basis functions: Exploiting direct product structure and a new basis function selection procedure.

    PubMed

    Brown, James; Carrington, Tucker

    2016-06-28

    In this paper we show that it is possible to use an iterative eigensolver in conjunction with Halverson and Poirier's symmetrized Gaussian (SG) basis [T. Halverson and B. Poirier, J. Chem. Phys. 137, 224101 (2012)] to compute accurate vibrational energy levels of molecules with as many as five atoms. This is done, without storing and manipulating large matrices, by solving a regular eigenvalue problem that makes it possible to exploit direct-product structure. These ideas are combined with a new procedure for selecting which basis functions to use. The SG basis we work with is orders of magnitude smaller than the basis made by using a classical energy criterion. We find significant convergence errors in previous calculations with SG bases. For sum-of-product Hamiltonians, SG bases large enough to compute accurate levels are orders of magnitude larger than even simple pruned bases composed of products of harmonic oscillator functions.

  7. Reduced basis technique for calculating sensitivity coefficients of nonlinear structural response

    NASA Technical Reports Server (NTRS)

    Noor, Ahmed K.; Peters, Jeanne M.

    1992-01-01

    An efficient reduced basis technique is presented for calculating the sensitivity of nonlinear structural response to variations in the design variables. The structure is discretized by using two-field mixed finite element models. The vector of structural response and its sensitivity coefficients (derivatives with respect to design variables) are each expressed as a linear combination of a small number of basis (or global approximation) vectors. The Bubnov-Galerkin technique is then used to approximate each of the finite element equations governing the response and the sensitivity coefficients by a small number of algebraic equations in the amplitudes of these vectors. The path derivatives (derivatives of the response vector with respect to path parameters, e.g., load parameters) are used as basis vectors for approximating the response. A combination of the path derivatives and their derivatives with respect to the design variables is used for approximating the sensitivity coefficients. The potential of the proposed technique is discussed and its effectiveness is demonstrated by means of numerical examples of laminated composite plates subjected to mechanical and thermal loads.

  8. Clutter and target discrimination in forward-looking ground penetrating radar using sparse structured basis pursuits

    NASA Astrophysics Data System (ADS)

    Camilo, Joseph A.; Malof, Jordan M.; Torrione, Peter A.; Collins, Leslie M.; Morton, Kenneth D.

    2015-05-01

    Forward-looking ground penetrating radar (FLGPR) is a remote sensing modality that has recently been investigated for buried threat detection. FLGPR offers greater standoff than other downward-looking modalities such as electromagnetic induction and downward-looking GPR, but it suffers from high false alarm rates due to surface and ground clutter. A stepped frequency FLGPR system consists of multiple radars with varying polarizations and bands, each of which interacts differently with subsurface materials and therefore might potentially be able to discriminate clutter from true buried targets. However, it is unclear which combinations of bands and polarizations would be most useful for discrimination or how to fuse them. This work applies sparse structured basis pursuit, a supervised statistical model which searches for sets of bands that are collectively effective for discriminating clutter from targets. The algorithm works by trying to minimize the number of selected items in a dictionary of signals; in this case the separate bands and polarizations make up the dictionary elements. A structured basis pursuit algorithm is employed to gather groups of modes together in collections to eliminate whole polarizations or sensors. The approach is applied to a large collection of FLGPR data for data around emplaced target and non-target clutter. The results show that a sparse structure basis pursuits outperforms a conventional CFAR anomaly detector while also pruning out unnecessary bands of the FLGPR sensor.

  9. Structural Basis for the Catalytic Activity of Human SER/THR Protein Phosphatase-5

    NASA Technical Reports Server (NTRS)

    Swingle, M. R.; Honkanen, R.; Ciszak, E.

    2004-01-01

    Serinekhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth. Here we report the 1.6 Angstrom resolution crystal structure of PP5 catalytic domain with metal and phosphate ions in the active site. The structure reveals a mechanism for PPS-mediated catalysis that requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1),-M(sub 2)-His(sup 427)-W(sup 2)-His(sup 304)-Asp(sup 274) catalytic motif, and provides a structural basis for the exceptional catalytic proficiency of protein phosphatases placing them among the most powerful catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of PP5 should aid development of specific inhibitors.

  10. Structural basis of GDP release and gating in G protein coupled Fe[superscript 2+] transport

    SciTech Connect

    Guilfoyle, Amy; Maher, Megan J.; Rapp, Mikaela; Clarke, Ronald; Harrop, Stephen; Jormakka, Mika

    2009-09-29

    G proteins are key molecular switches in the regulation of membrane protein function and signal transduction. The prokaryotic membrane protein FeoB is involved in G protein coupled Fe{sup 2+} transport, and is unique in that the G protein is directly tethered to the membrane domain. Here, we report the structure of the soluble domain of FeoB, including the G protein domain, and its assembly into an unexpected trimer. Comparisons between nucleotide free and liganded structures reveal the closed and open state of a central cytoplasmic pore, respectively. In addition, these data provide the first observation of a conformational switch in the nucleotide-binding G5 motif, defining the structural basis for GDP release. From these results, structural parallels are drawn to eukaryotic G protein coupled membrane processes.

  11. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase [delta

    SciTech Connect

    Swan, Michael K.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2009-09-25

    DNA polymerase {delta} (Pol {delta}) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol {delta} in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-{angstrom} resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol {delta}.

  12. Comparison of advanced reduced-basis methods for transient structural analysis

    NASA Technical Reports Server (NTRS)

    Mcgowan, David M.; Bostic, Susan W.

    1991-01-01

    Two advanced reduced-basis methods for linear, transient structural analysis, the force-derivative method and the Lanczos method, are compared to two widely-used modal methods, the mode-displacement method and the mode-acceleration method. Comparisons are made for two linear example problems: a proportionally-damped cantilevered beam subject to a discrete tip load which varies linearly with time, and a discretely-damped multispan beam subject to a uniformly distributed load which varies as a quintic function of time. Results from the methods are compared in terms of the number of basis vectors required to obtain a desired level of accuracy and the associated computational times. The results are problem dependent, and it is shown that for the cantilevered beam problem, the mode-acceleration and force-derivative methods are the most efficient in terms of the number of basis vectors and computational time. The force-derivative method is shown to be the most effective method for solving the multispan beam problem with closely-spaced frequencies. In general, the force-derivative method is shown to produce an accurate solution using very few basis vectors and to require less computational time as compared to the other methods studied.

  13. Simulation of structural response using a recurrent radial basis function network

    SciTech Connect

    Paez, T.L.

    1994-08-01

    System behaviors can be accurately simulated using artificial neural networks (ANNs), and one that performs well in simulation of structural response is the radial basis function network. A specific implementation of this is the connectionist normalized linear spline (CNLS) network, investigated in this study. A useful framework for ANN simulation of structural response is the recurrent network. This framework simulates the response of a structure one step at a time. It requires as inputs some measures of the excitation, and the response at previous times. On output, the recurrent ANN yields the response at some time in the future. This framework is practical to implement because every ANN requires training, and this is executed by showing the ANN examples of correct input/output behavior (exemplars), and requiring the ANN to simulate this behavior. In practical applications, hundreds or, perhaps, thousands, of exemplars are required for ANN training. The usual laboratory and non-neural numerical applications to be simulated by ANNs produce these amounts of information. Once the recurrent ANN is trained, it can be provided with excitation information, and used to propagate structural response, simulating the response it was trained to approximate. The structural characteristics, parameters in the CNLS network, and degree of training influence the accuracy of approximation. This investigation studies the accuracy of structural response simulation for a single-degree-of-freedom (SDF), nonlinear system excited by random vibration loading. The ANN used to simulate structural response is a recurrent CNLS network. We investigate the error in structural system simulation.

  14. Structural concepts and techniques I. Basis concepts, folding, and structural techniques

    SciTech Connect

    Foster, N.H.; Beaumont, E.A.

    1988-01-01

    This publication is a reprint volume belowging to a series that is called the Treatise of Petroleum Geology. This particular volume contains papers on pore pressure effects; stress analysis; folding processes and geometries; and methods to solve structural problems encountered during exploration and development. These papers relate ways to visualize and predict the three-dimensional arrangement and location of strata in the subsurface.

  15. Structural Basis for Modulation of Quality Control Fate in a Marginally Stable Protein

    PubMed Central

    Brock, Kelly P.; Abraham, Ayelet-chen; Amen, Triana; Kaganovich, Daniel; England, Jeremy L.

    2015-01-01

    Summary The human von Hippel-Lindau (VHL) tumor suppressor is a marginally stable protein previously used as a model substrate of eukaryotic refolding and degradation pathways. When expressed in the absence of its cofactors, VHL cannot fold and is quickly degraded by the quality control machinery of the cell. We combined computational methods with in vivo experiments to examine the basis of the misfolding propensity of VHL. By expressing a set of randomly mutated VHL sequences in yeast, we discovered a more stable mutant form. Subsequent modeling suggested the mutation had caused a conformational change affecting cofactor and chaperone interaction, and this hypothesis was then confirmed by additional knockout and overexpression experiments targeting a yeast cofactor homolog. These findings offer a detailed structural basis for the modulation of quality control fate in a model misfolded protein and highlight burial mode modeling as a rapid means to detect functionally important conformational changes in marginally stable globular domains. PMID:26027734

  16. Structural basis of motility in the microtubular axostyle: implications for cytoplasmic microtubule structure and function

    PubMed Central

    1980-01-01

    The gross morphology of the protozoan microtubule axostyle of Saccinobaculus ambloaxostylus can now be described in macromolecular detail. The left-handed coil of the axostyle is seen to be dependent upon the asymmetry inherent in the constituent microtubules as expressed by the specific array of linkages between microtubules and by a possible tendency for microtubules to coil into left-handed helices. The laminated sheets of microtubules are not aligned parallel to the long axis of the organelle, but become increasingly tilted off-axis as one descends through the sheets of microtubules from the convex to the concave surface of the axostyle. Fine-structural analysis of the axostyle indicates similarities of the linkages to dynein. The potential loci of the force-generating protein(s) are discussed as well as implications of the axostyle's structure on general microtubule function. PMID:6448863

  17. Crystal structure of human interferon-γ receptor 2 reveals the structural basis for receptor specificity.

    PubMed

    Mikulecký, Pavel; Zahradník, Jirí; Kolenko, Petr; Černý, Jiří; Charnavets, Tatsiana; Kolářová, Lucie; Nečasová, Iva; Pham, Phuong Ngoc; Schneider, Bohdan

    2016-09-01

    Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π-cation motif of stacked residues KWRWRH, a NAG-W-NAG sandwich (where NAG stands for N-acetyl-D-glucosamine) and finally a helix formed by residues 78-85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2. PMID:27599734

  18. Crystal structure of human interferon-γ receptor 2 reveals the structural basis for receptor specificity

    PubMed Central

    Mikulecký, Pavel; Zahradník, Jirí; Kolenko, Petr; Černý, Jiří; Charnavets, Tatsiana; Kolářová, Lucie; Nečasová, Iva; Pham, Phuong Ngoc; Schneider, Bohdan

    2016-01-01

    Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π–cation motif of stacked residues KWRWRH, a NAG–W–NAG sandwich (where NAG stands for N-acetyl-d-glucosamine) and finally a helix formed by residues 78–85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2. PMID:27599734

  19. Structural basis for the mechanism of inhibition of uridine phosphorylase from Salmonella typhimurium

    SciTech Connect

    Lashkov, A. A.; Zhukhlistova, N. E.; Sotnichenko, S. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2010-01-15

    The three-dimensional structures of three complexes of Salmonella typhimurium uridine phosphorylase with the inhibitor 2,2'-anhydrouridine, the substrate PO{sub 4}, and with both the inhibitor 2,2'-anhydrouridine and the substrate PO{sub 4} (a binary complex) were studied in detail by X-ray diffraction. The structures of the complexes were refined at 2.38, 1.5, and 1.75 A resolution, respectively. Changes in the three-dimensional structure of the subunits in different crystal structures are considered depending on the presence or absence of the inhibitor molecule and (or) the phosphate ion in the active site of the enzyme. The presence of the phosphate ion in the phosphate-binding site was found to substantially change the orientations of the side chains of the amino-acid residues Arg30, Arg91, and Arg48 coordinated to this ion. A comparison showed that the highly flexible loop L9 is unstable. The atomic coordinates of the refined structures of the complexes and the corresponding structure factors were deposited in the Protein Data Bank (their PDB ID codes are 3DD0 and 3C74). The experimental data on the spatial reorganization of the active site caused by changes in its functional state from the unligated to the completely inhibited state suggest the structural basis for the mechanism of inhibition of Salmonella typhimurium uridine phosphorylase.

  20. Structural basis of CX-4945 binding to human protein kinase CK2

    SciTech Connect

    Ferguson, Andrew D.; Sheth, Payal R.; Basso, Andrea D.; Paliwal, Sunil; Gray, Kimberly; Fischmann, Thierry O.; Le, Hung V.

    2012-02-07

    Protein kinase CK2 (CK2), a constitutively active serine/threonine kinase, is involved in a variety of roles essential to the maintenance of cellular homeostasis. Elevated levels of CK2 expression results in the dysregulation of key signaling pathways that regulate transcription, and has been implicated in cancer. The adenosine-5'-triphosphate-competitive inhibitor CX-4945 has been reported to show broad spectrum anti-proliferative activity in multiple cancer cell lines. Although the enzymatic IC{sub 50} of CX-4945 has been reported, the thermodynamics and structural basis of binding to CK2{alpha} remained elusive. Presented here are the crystal structures of human CK2{alpha} in complex with CX-4945 and adenylyl phosphoramidate at 2.7 and 1.3 {angstrom}, respectively. Biophysical analysis of CX-4945 binding is also described. This data provides the structural rationale for the design of more potent inhibitors against this emerging cancer target.

  1. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    NASA Astrophysics Data System (ADS)

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-07-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or `invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

  2. The Structural Basis of Cooperative Regulation at an Alternate Genetic Switch

    SciTech Connect

    Pinkett,H.; Shearwin, K.; Stayrook, S.; Dodd, I.; Burr, T.; Hochschild, A.; Egan, J.; Lewis, M.

    2006-01-01

    Bacteriophage {gamma} is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of {gamma} and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 Angstroms resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 Angstroms resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and {gamma} repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.

  3. Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

    SciTech Connect

    Schlunzen, Frank; Zarivach, Raz; Harms, Jörg; Bashan, Anat; Tocilj, Ante; Albrecht, Renate; Yonath, Ada; Franceschi, Francois

    2009-10-07

    Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

  4. Photosynthesis. Structural basis for energy transfer pathways in the plant PSI-LHCI supercomplex.

    PubMed

    Qin, Xiaochun; Suga, Michihiro; Kuang, Tingyun; Shen, Jian-Ren

    2015-05-29

    Photosynthesis converts solar energy to chemical energy by means of two large pigment-protein complexes: photosystem I (PSI) and photosystem II (PSII). In higher plants, the PSI core is surrounded by a large light-harvesting complex I (LHCI) that captures sunlight and transfers the excitation energy to the core with extremely high efficiency. We report the structure of PSI-LHCI, a 600-kilodalton membrane protein supercomplex, from Pisum sativum (pea) at a resolution of 2.8 angstroms. The structure reveals the detailed arrangement of pigments and other cofactors—especially within LHCI—as well as numerous specific interactions between the PSI core and LHCI. These results provide a firm structural basis for our understanding on the energy transfer and photoprotection mechanisms within the PSI-LHCI supercomplex.

  5. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    PubMed Central

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-01-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions. PMID:26138143

  6. Structural basis for alcohol modulation of a pentameric ligand-gated ion channel.

    PubMed

    Howard, Rebecca J; Murail, Samuel; Ondricek, Kathryn E; Corringer, Pierre-Jean; Lindahl, Erik; Trudell, James R; Harris, R Adron

    2011-07-19

    Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.

  7. Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

    PubMed Central

    Malito, Enrico; Bursulaya, Badry; Chen, Connie; Surdo, Paola Lo; Picchianti, Monica; Balducci, Enrico; Biancucci, Marco; Brock, Ansgar; Berti, Francesco; Bottomley, Matthew James; Nissum, Mikkel; Costantino, Paolo; Rappuoli, Rino; Spraggon, Glen

    2012-01-01

    CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197. PMID:22431623

  8. Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197.

    PubMed

    Malito, Enrico; Bursulaya, Badry; Chen, Connie; Lo Surdo, Paola; Picchianti, Monica; Balducci, Enrico; Biancucci, Marco; Brock, Ansgar; Berti, Francesco; Bottomley, Matthew James; Nissum, Mikkel; Costantino, Paolo; Rappuoli, Rino; Spraggon, Glen

    2012-04-01

    CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.

  9. Somatic mutations in PI3K[alpha]: Structural basis for enzyme activation and drug design

    SciTech Connect

    Gabelli, Sandra B.; Mandelker, Diana; Schmidt-Kittler, Oleg; Vogelstein, Bert; Amzel, L. Mario

    2011-09-06

    The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3K{alpha} harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.

  10. Somatic Mutations in PI3Kalpha: Structural Basis for Enzyme Activation and Drug Design

    SciTech Connect

    S Gabelli; D Mandelker; O Schmidt-Kittler; B Vogelstein; L Amzel

    2011-12-31

    The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3K{alpha} harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.

  11. The structure of human GALNS reveals the molecular basis for mucopolysaccharidosis IV A

    PubMed Central

    Rivera-Colón, Yadilette; Schutsky, Emily K.; Kita, Adriana Z.; Garman, Scott C.

    2012-01-01

    Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here we report the three-dimensional structure of human GALNS, determined by x-ray crystallography at 2.2 Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active site geometries in the family, but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases. PMID:22940367

  12. Structural Basis of Mucopolysaccharidosis Type II and Construction of a Database of Mutant Iduronate 2-Sulfatases

    PubMed Central

    Saito, Seiji; Ohno, Kazuki; Okuyama, Torayuki; Sakuraba, Hitoshi

    2016-01-01

    Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked genetic disorder caused by a deficiency of iduronate 2-sulfatase (IDS), and missense mutations comprising about 30% of the mutations responsible for MPS II result in heterogeneous phenotypes ranging from the severe to the attenuated form. To elucidate the basis of MPS II from the structural viewpoint, we built structural models of the wild type and mutant IDS proteins resulting from 131 missense mutations (phenotypes: 67 severe and 64 attenuated), and analyzed the influence of each amino acid substitution on the IDS structure by calculating the accessible surface area, the number of atoms affected and the root-mean-square distance. The results revealed that the amino acid substitutions causing MPS II were widely spread over the enzyme molecule and that the structural changes of the enzyme protein were generally larger in the severe group than in the attenuated one. Coloring of the atoms influenced by different amino acid substitutions at the same residue showed that the structural changes influenced the disease progression. Based on these data, we constructed a database of IDS mutations as to the structures of mutant IDS proteins. PMID:27695081

  13. Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A.

    PubMed

    Gorelik, Alexei; Illes, Katalin; Superti-Furga, Giulio; Nagar, Bhushan

    2016-03-18

    Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins. PMID:26792860

  14. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    PubMed

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  15. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

    PubMed Central

    Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-01-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  16. Consistent structures and interactions by density functional theory with small atomic orbital basis sets.

    PubMed

    Grimme, Stefan; Brandenburg, Jan Gerit; Bannwarth, Christoph; Hansen, Andreas

    2015-08-01

    A density functional theory (DFT) based composite electronic structure approach is proposed to efficiently compute structures and interaction energies in large chemical systems. It is based on the well-known and numerically robust Perdew-Burke-Ernzerhoff (PBE) generalized-gradient-approximation in a modified global hybrid functional with a relatively large amount of non-local Fock-exchange. The orbitals are expanded in Ahlrichs-type valence-double zeta atomic orbital (AO) Gaussian basis sets, which are available for many elements. In order to correct for the basis set superposition error (BSSE) and to account for the important long-range London dispersion effects, our well-established atom-pairwise potentials are used. In the design of the new method, particular attention has been paid to an accurate description of structural parameters in various covalent and non-covalent bonding situations as well as in periodic systems. Together with the recently proposed three-fold corrected (3c) Hartree-Fock method, the new composite scheme (termed PBEh-3c) represents the next member in a hierarchy of "low-cost" electronic structure approaches. They are mainly free of BSSE and account for most interactions in a physically sound and asymptotically correct manner. PBEh-3c yields good results for thermochemical properties in the huge GMTKN30 energy database. Furthermore, the method shows excellent performance for non-covalent interaction energies in small and large complexes. For evaluating its performance on equilibrium structures, a new compilation of standard test sets is suggested. These consist of small (light) molecules, partially flexible, medium-sized organic molecules, molecules comprising heavy main group elements, larger systems with long bonds, 3d-transition metal systems, non-covalently bound complexes (S22 and S66×8 sets), and peptide conformations. For these sets, overall deviations from accurate reference data are smaller than for various other tested DFT methods

  17. Consistent structures and interactions by density functional theory with small atomic orbital basis sets

    NASA Astrophysics Data System (ADS)

    Grimme, Stefan; Brandenburg, Jan Gerit; Bannwarth, Christoph; Hansen, Andreas

    2015-08-01

    A density functional theory (DFT) based composite electronic structure approach is proposed to efficiently compute structures and interaction energies in large chemical systems. It is based on the well-known and numerically robust Perdew-Burke-Ernzerhoff (PBE) generalized-gradient-approximation in a modified global hybrid functional with a relatively large amount of non-local Fock-exchange. The orbitals are expanded in Ahlrichs-type valence-double zeta atomic orbital (AO) Gaussian basis sets, which are available for many elements. In order to correct for the basis set superposition error (BSSE) and to account for the important long-range London dispersion effects, our well-established atom-pairwise potentials are used. In the design of the new method, particular attention has been paid to an accurate description of structural parameters in various covalent and non-covalent bonding situations as well as in periodic systems. Together with the recently proposed three-fold corrected (3c) Hartree-Fock method, the new composite scheme (termed PBEh-3c) represents the next member in a hierarchy of "low-cost" electronic structure approaches. They are mainly free of BSSE and account for most interactions in a physically sound and asymptotically correct manner. PBEh-3c yields good results for thermochemical properties in the huge GMTKN30 energy database. Furthermore, the method shows excellent performance for non-covalent interaction energies in small and large complexes. For evaluating its performance on equilibrium structures, a new compilation of standard test sets is suggested. These consist of small (light) molecules, partially flexible, medium-sized organic molecules, molecules comprising heavy main group elements, larger systems with long bonds, 3d-transition metal systems, non-covalently bound complexes (S22 and S66×8 sets), and peptide conformations. For these sets, overall deviations from accurate reference data are smaller than for various other tested DFT methods

  18. Consistent structures and interactions by density functional theory with small atomic orbital basis sets

    SciTech Connect

    Grimme, Stefan Brandenburg, Jan Gerit; Bannwarth, Christoph; Hansen, Andreas

    2015-08-07

    A density functional theory (DFT) based composite electronic structure approach is proposed to efficiently compute structures and interaction energies in large chemical systems. It is based on the well-known and numerically robust Perdew-Burke-Ernzerhoff (PBE) generalized-gradient-approximation in a modified global hybrid functional with a relatively large amount of non-local Fock-exchange. The orbitals are expanded in Ahlrichs-type valence-double zeta atomic orbital (AO) Gaussian basis sets, which are available for many elements. In order to correct for the basis set superposition error (BSSE) and to account for the important long-range London dispersion effects, our well-established atom-pairwise potentials are used. In the design of the new method, particular attention has been paid to an accurate description of structural parameters in various covalent and non-covalent bonding situations as well as in periodic systems. Together with the recently proposed three-fold corrected (3c) Hartree-Fock method, the new composite scheme (termed PBEh-3c) represents the next member in a hierarchy of “low-cost” electronic structure approaches. They are mainly free of BSSE and account for most interactions in a physically sound and asymptotically correct manner. PBEh-3c yields good results for thermochemical properties in the huge GMTKN30 energy database. Furthermore, the method shows excellent performance for non-covalent interaction energies in small and large complexes. For evaluating its performance on equilibrium structures, a new compilation of standard test sets is suggested. These consist of small (light) molecules, partially flexible, medium-sized organic molecules, molecules comprising heavy main group elements, larger systems with long bonds, 3d-transition metal systems, non-covalently bound complexes (S22 and S66×8 sets), and peptide conformations. For these sets, overall deviations from accurate reference data are smaller than for various other tested DFT

  19. The structural basis for function in diamond-like carbon binding peptides.

    PubMed

    Gabryelczyk, Bartosz; Szilvay, Géza R; Linder, Markus B

    2014-07-29

    The molecular structural basis for the function of specific peptides that bind to diamond-like carbon (DLC) surfaces was investigated. For this, a competition assay that provided a robust way of comparing relative affinities of peptide variants was set up. Point mutations of specific residues resulted in significant effects, but it was shown that the chemical composition of the peptide was not sufficient to explain peptide affinity. More significantly, rearrangements in the sequence indicated that the binding is a complex recognition event that is dependent on the overall structure of the peptide. The work demonstrates the unique properties of peptides for creating functionality at interfaces via noncovalent binding for potential applications in, for example, nanomaterials, biomedical materials, and sensors. PMID:25007096

  20. Structural basis of RNA binding discrimination between bacteriophages Qbeta and MS2.

    PubMed

    Horn, Wilf T; Tars, Kaspars; Grahn, Elin; Helgstrand, Charlotte; Baron, Andrew J; Lago, Hugo; Adams, Chris J; Peabody, David S; Phillips, Simon E V; Stonehouse, Nicola J; Liljas, Lars; Stockley, Peter G

    2006-03-01

    Sequence-specific interactions between RNA stem-loops and coat protein (CP) subunits play vital roles in the life cycles of the RNA bacteriophages, e.g., by allowing translational repression of their replicase cistrons and tagging their own RNA genomes for encapsidation. The CPs of bacteriophages Qbeta and MS2 each discriminate in favor of their cognate translational operators, even in the presence of closely related operators from other phages in vivo. Discrete mutations within the MS2 CP have been shown to relax this discrimination in vitro. We have determined the structures of eight complexes between such mutants and both MS2 and Qbeta stem-loops with X-ray crystallography. In conjunction with previously determined in vivo repression data, the structures enable us to propose the molecular basis for the discrimination mechanism. PMID:16531233

  1. Low field domain wall dynamics in artificial spin-ice basis structure

    SciTech Connect

    Kwon, J.; Goolaup, S.; Lim, G. J.; Kerk, I. S.; Lew, W. S.; Chang, C. H.; Roy, K.

    2015-10-28

    Artificial magnetic spin-ice nanostructures provide an ideal platform for the observation of magnetic monopoles. The formation of a magnetic monopole is governed by the motion of a magnetic charge carrier via the propagation of domain walls (DWs) in a lattice. To date, most experiments have been on the static visualization of DW propagation in the lattice. In this paper, we report on the low field dynamics of DW in a unit spin-ice structure measured by magnetoresistance changes. Our results show that reversible DW propagation can be initiated within the spin-ice basis. The initial magnetization configuration of the unit structure strongly influences the direction of DW motion in the branches. Single or multiple domain wall nucleation can be induced in the respective branches of the unit spin ice by the direction of the applied field.

  2. Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

    SciTech Connect

    Westfall, Corey S.; Zubieta, Chloe; Herrmann, Jonathan; Kapp, Ulrike; Nanao, Max H.; Jez, Joseph M.

    2013-04-08

    Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

  3. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    NASA Astrophysics Data System (ADS)

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-10-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

  4. Structural basis for copper/silver binding by the Synechocystis metallochaperone CopM.

    PubMed

    Zhao, Shun; Wang, Xiao; Niu, Guoqi; Dong, Wei; Wang, Jia; Fang, Ying; Lin, Yajing; Liu, Lin

    2016-09-01

    Copper homeostasis integrates multiple processes from sensing to storage and efflux out of the cell. CopM is a cyanobacterial metallochaperone, the gene for which is located upstream of a two-component system for copper resistance, but the molecular basis for copper recognition by this four-helical bundle protein is unknown. Here, crystal structures of CopM in apo, copper-bound and silver-bound forms are reported. Monovalent copper/silver ions are buried within the bundle core; divalent copper ions are found on the surface of the bundle. The monovalent copper/silver-binding site is constituted by two consecutive histidines and is conserved in a previously functionally unknown protein family. The structural analyses show two conformational states and suggest that flexibility in the first α-helix is related to the metallochaperone function. These results also reveal functional diversity from a protein family with a simple four-helical fold. PMID:27599732

  5. Structural basis and selectivity of tankyrase inhibition by a Wnt signaling inhibitor WIKI4.

    PubMed

    Haikarainen, Teemu; Venkannagari, Harikanth; Narwal, Mohit; Obaji, Ezeogo; Lee, Hao-Wei; Nkizinkiko, Yves; Lehtiö, Lari

    2013-01-01

    Recently a novel inhibitor of Wnt signaling was discovered. The compound, WIKI4, was found to act through tankyrase inhibition and regulate β-catenin levels in many cancer cell lines and human embryonic stem cells. Here we confirm that WIKI4 is a high potency tankyrase inhibitor and that it selectively inhibits tankyrases over other ARTD enzymes tested. The binding mode of the compound to tankyrase 2 was determined by protein X-ray crystallography to 2.4 Å resolution. The structure revealed a novel binding mode to the adenosine subsite of the donor NAD(+) binding groove of the catalytic domain. Our results form a structural basis for further development of potent and selective tankyrase inhibitors based on the WIKI4 scaffold. PMID:23762361

  6. Structural Basis and Selectivity of Tankyrase Inhibition by a Wnt Signaling Inhibitor WIKI4

    PubMed Central

    Haikarainen, Teemu; Venkannagari, Harikanth; Narwal, Mohit; Obaji, Ezeogo; Lee, Hao-Wei; Nkizinkiko, Yves; Lehtiö, Lari

    2013-01-01

    Recently a novel inhibitor of Wnt signaling was discovered. The compound, WIKI4, was found to act through tankyrase inhibition and regulate β-catenin levels in many cancer cell lines and human embryonic stem cells. Here we confirm that WIKI4 is a high potency tankyrase inhibitor and that it selectively inhibits tankyrases over other ARTD enzymes tested. The binding mode of the compound to tankyrase 2 was determined by protein X-ray crystallography to 2.4 Å resolution. The structure revealed a novel binding mode to the adenosine subsite of the donor NAD+ binding groove of the catalytic domain. Our results form a structural basis for further development of potent and selective tankyrase inhibitors based on the WIKI4 scaffold. PMID:23762361

  7. Structural basis for the inhibition of firefly luciferase by a general anesthetic.

    PubMed

    Franks, N P; Jenkins, A; Conti, E; Lieb, W R; Brick, P

    1998-11-01

    The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

  8. Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

    SciTech Connect

    Singh, Harkewal; Reilly, Thomas J.; Tanner, John J.

    2012-01-20

    The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

  9. Structural Basis for a Reciprocating Mechanism of Negative Cooperativity in Dimeric Phosphagen Kinase Activity

    SciTech Connect

    Wu, X.; Ye, S; Guo, S; Yan, W; Bartlam, M; Rao, Z

    2010-01-01

    Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 {angstrom}, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widely than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase. - Wu, X., Ye, S., Guo, S., Yan, W., Bartlam, M., Rao, Z. Structural basis for a reciprocating mechanism of negative cooperativity in dimeric phosphagen kinase activity.

  10. The dynamics of zinc sites in proteins: electronic basis for coordination sphere expansion at structural sites.

    PubMed

    Daniel, A Gerard; Farrell, Nicholas P

    2014-12-01

    The functional role assumed by zinc in proteins is closely tied to the variable dynamics around its coordination sphere arising by virtue of its flexibility in bonding. Modern experimental and computational methods allow the detection and study of previously unknown features of bonding between zinc and its ligands in protein environment. These discoveries are occurring just in time as novel biological functions of zinc, which involve rather unconventional coordination trends, are emerging. In this sense coordination sphere expansion of structural zinc sites, as observed in our previous experiments, is a novel phenomenon. Here we explore the electronic and structural requirements by simulating this phenomenon in structural zinc sites using DFT computations. For this purpose, we have chosen MPW1PW91 and a mixed basis set combination as the DFT method through benchmarking, because it accurately reproduces structural parameters of experimentally characterized zinc compounds. Using appropriate models, we show that the greater ionic character of zinc coordination would allow for coordination sphere expansion if the steric and electrostatic repulsions of the ligands are attenuated properly. Importantly, through the study of electronic and structural aspects of the models used, we arrive at a comprehensive bonding model, explaining the factors that influence coordination of zinc in proteins. The proposed model along with the existing knowledge would enhance our ability to predict zinc binding sites in proteins, which is today of growing importance given the predicted enormity of the zinc proteome.

  11. Structural Basis for Glycyl Radical Formation By Pyruvate Formate-Lyase Activating Enzyme

    SciTech Connect

    Vey, J.L.; Yang, J.; Li, M.; Broderick, W.E.; Broderick, J.B.; Drennan, C.L.

    2009-05-26

    Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G{sup 734} of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G{sup 734}. Our structures provide fundamental insights into the interactions between the activase and the G{sup 734} loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G{sup 734}4 of pyruvate formate-lyase.

  12. Structural basis of Ac-SDKP hydrolysis by Angiotensin-I converting enzyme.

    PubMed

    Masuyer, Geoffrey; Douglas, Ross G; Sturrock, Edward D; Acharya, K Ravi

    2015-09-25

    Angiotensin-I converting enzyme (ACE) is a zinc dipeptidylcarboxypeptidase with two active domains and plays a key role in the regulation of blood pressure and electrolyte homeostasis, making it the principal target in the treatment of cardiovascular disease. More recently, the tetrapetide N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) has emerged as a potent antifibrotic agent and negative regulator of haematopoietic stem cell differentiation which is processed exclusively by ACE. Here we provide a detailed biochemical and structural basis for the domain preference of Ac-SDKP. The high resolution crystal structures of N-domain ACE in complex with the dipeptide products of Ac-SDKP cleavage were obtained and offered a template to model the mechanism of substrate recognition of the enzyme. A comprehensive kinetic study of Ac-SDKP and domain co-operation was performed and indicated domain interactions affecting processing of the tetrapeptide substrate. Our results further illustrate the molecular basis for N-domain selectivity and should help design novel ACE inhibitors and Ac-SDKP analogues that could be used in the treatment of fibrosis disorders.

  13. Structural basis of Sorcin-mediated calcium-dependent signal transduction

    PubMed Central

    Ilari, Andrea; Fiorillo, Annarita; Poser, Elena; Lalioti, Vasiliki S.; Sundell, Gustav N.; Ivarsson, Ylva; Genovese, Ilaria; Colotti, Gianni

    2015-01-01

    Sorcin is an essential penta-EF hand calcium binding protein, able to confer the multi-drug resistance phenotype to drug-sensitive cancer cells and to reduce Endoplasmic Reticulum stress and cell death. Sorcin silencing blocks cell cycle progression in mitosis and induces cell death by triggering apoptosis. Sorcin participates in the modulation of calcium homeostasis and in calcium-dependent cell signalling in normal and cancer cells. The molecular basis of Sorcin action is yet unknown. The X-ray structures of Sorcin in the apo (apoSor) and in calcium bound form (CaSor) reveal the structural basis of Sorcin action: calcium binding to the EF1-3 hands promotes a large conformational change, involving a movement of the long D-helix joining the EF1-EF2 sub-domain to EF3 and the opening of EF1. This movement promotes the exposure of a hydrophobic pocket, which can accommodate in CaSor the portion of its N-terminal domain displaying the consensus binding motif identified by phage display experiments. This domain inhibits the interaction of sorcin with PDCD6, a protein that carries the Sorcin consensus motif, co-localizes with Sorcin in the perinuclear region of the cell and in the midbody and is involved in the onset of apoptosis. PMID:26577048

  14. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  15. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  16. Structural basis for the glucan phosphatase activity of Starch Excess4

    SciTech Connect

    Vander Kooi, Craig W.; Taylor, Adam O.; Pace, Rachel M.; Meekins, David A.; Guo, Hou-Fu; Kim, Youngjun; Gentry, Matthew S.

    2010-11-12

    Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.

  17. Crystal Structure of CTP: Glycerol-3-Phosphate Cytidylyl Tranferase from Staphylococcus Aurues: Examination of Structural Basis for Kinetic Mechanism

    SciTech Connect

    Fong,D.; Yim, V.; D'elia, M.; Brown, E.; Berghuis, A.

    2006-01-01

    Integrity of the cell wall is essential for bacterial survival, and as a consequence components involved in its biosynthesis can potentially be exploited as targets for antibiotics. One such potential target is CTP:glycerol-3-phosphate cytidylyltransferase. This enzyme (TarD{sub Sa} in Staphylococcus aureus and TagD{sub Bs} in Bacillus subtilis) catalyzes the formation of CDP-glycerol, which is used for the assembly of linkages between peptidoglycan and teichoic acid polymer in Gram-positive bacteria. Intriguingly, despite the high sequence identity between TarD{sub Sa} and TagD{sub Bs} (69% identity), kinetic studies show that these two enzymes differ markedly in their kinetic mechanism and activity. To examine the basis for the disparate enzymological properties, we have determined the crystal structure of TarD{sub Sa} in the apo state to 3 Angstroms resolution, and performed equilibrium sedimentation analysis. Comparison of the structure with that of CTP- and CDP-glycerol-bound TagD{sub Bs} crystal structures reveals that the overall structure of TarD{sub Sa} is essentially the same as that of TagD{sub Bs}, except in the C-terminus, where it forms a helix in TagD{sub Bs} but is disordered in the apo TarDSa structure. In addition, TarD{sub Sa} can exist both as a tetramer and as a dimer, unlike TagD{sub Bs}, which is a dimer. These observations shed light on the structural basis for the differing kinetic characteristics between TarD{sub Sa} and TagD{sub Bs}.

  18. Structural Basis for Distinct Binding Properties of the Human Galectins to Thomsen-Friedenreich Antigen

    PubMed Central

    Bian, Cheng-Feng; Zhang, Ying; Sun, Hui; Li, De-Feng; Wang, Da-Cheng

    2011-01-01

    The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif (51AHGDA55) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins. PMID:21949831

  19. Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1

    PubMed Central

    Wu, Donghui; Kaan, Hung Yi Kristal; Zheng, Xiaoxia; Tang, Xuhua; He, Yang; Vanessa Tan, Qianmin; Zhang, Neng; Song, Haiwei

    2015-01-01

    Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation. PMID:26443277

  20. Structural Basis of Human PXR Activation by the Hops Constituent Colupulone

    PubMed Central

    Teotico, Denise G.; Bischof, Jason J.; Peng, Li; Kliewer, Steven A.; Redinbo, Matthew R.

    2008-01-01

    Hops extracts are used to alleviate menopausal symptoms and as an alternative to hormone replacement therapy, but can produce potentially harmful drug-drug interactions. The nuclear xenobiotic receptor PXR is promiscuously activated by a range of structurally distinct chemicals. It has a key role in the transcriptional regulation of genes that encode xenobiotic metabolism enzymes. In this study, hops extracts are shown to induce the expression of numerous drug metabolism and excretion proteins. The β-bitter acid colupulone is demonstrated to be a bioactive component and direct activator of human PXR. The 2.8 Å resolution crystal structure of the ligand binding domain of human PXR in complex with colupulone was elucidated, and colupulone was observed to bind in a single orientation stabilized by both Van der Waals and hydrogen bonding contacts. The crystal structure also indicates that related α- and β-bitter acids have the capacity to serve as PXR agonists, as well. Taken together, these results reveal the structural basis for drug-drug interactions mediated by colupulone and related constituents of hops extracts. PMID:18768384

  1. Structural basis for the Smad5 MH1 domain to recognize different DNA sequences

    PubMed Central

    Chai, Nan; Li, Wan-Xin; Wang, Jue; Wang, Zhi-Xin; Yang, Shi-Ming; Wu, Jia-Wei

    2015-01-01

    Smad proteins are important intracellular mediators of TGF-β signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 domain contacts the GC-rich site with the same β-hairpin, but the detailed interaction modes are different. Conserved β-hairpin residues make base specific contacts with the minimal GC-rich site, 5′-GGC-3′. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 domain with the DNA targets. PMID:26304548

  2. Structural Basis of Multifunctionality in a Vitamin B[subscript 12]-processing Enzyme

    SciTech Connect

    Koutmos, Markos; Gherasim, Carmen; Smith, Janet L.; Banerjee, Ruma

    2012-07-11

    An early step in the intracellular processing of vitamin B{sub 12} involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B{sub 12}), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.

  3. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

    PubMed

    Erbilgin, Onur; Sutter, Markus; Kerfeld, Cheryl A

    2016-03-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  4. The structure of an authentic spore photoproduct lesion in DNA suggests a basis for recognition.

    PubMed

    Singh, Isha; Jian, Yajun; Lian, Yajun; Li, Lei; Georgiadis, Millie M

    2014-03-01

    The spore photoproduct lesion (SP; 5-thymine-5,6-dihydrothymine) is the dominant photoproduct found in UV-irradiated spores of some bacteria such as Bacillus subtilis. Upon spore germination, this lesion is repaired in a light-independent manner by a specific repair enzyme: the spore photoproduct lyase (SP lyase). In this work, a host-guest approach in which the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (MMLV RT) serves as the host and DNA as the guest was used to determine the crystal structures of complexes including 16 bp oligonucleotides with and without the SP lesion at 2.14 and 1.72 Å resolution, respectively. In contrast to other types of thymine-thymine lesions, the SP lesion retains normal Watson-Crick hydrogen bonding to the adenine bases of the complementary strand, with shorter hydrogen bonds than found in the structure of the undamaged DNA. However, the lesion induces structural changes in the local conformation of what is otherwise B-form DNA. The region surrounding the lesion differs significantly in helical form from B-DNA, and the minor groove is widened by almost 3 Å compared with that of the undamaged DNA. Thus, these unusual structural features associated with SP lesions may provide a basis for recognition by the SP lyase. PMID:24598744

  5. Structural basis for the indispensable role of a unique zinc finger motif in LNX2 ubiquitination

    PubMed Central

    Nayak, Digant; Sivaraman, J.

    2015-01-01

    LNX (Ligand of Numb Protein-X) proteins, LNX1 and LNX2, are RING- and PDZ-based E3-ubiquitin ligases known to interact with Numb. Silencing of LNX2 has been reported to down-regulate WNT and NOTCH, two key signaling pathways in tumorigenesis. Here we report the identification of the domain boundary of LNX2 to confer its ubiquitination activity, its crystal structure along with functional studies. We show that the RING domain in LNX2 is flanked by two Zinc-binding motifs (Zn-RING-Zn), in which the N-terminal Zinc-binding motif adopts novel conformation. Although this motif follows the typical Cys2His2-type zinc finger configuration, it is devoid of any secondary structure and forms an open circle conformation, which has not been reported yet. This unique N-terminal Zn-finger motif is indispensable for the activity and stability of LNX2, as verified using mutational studies. The Zn-RING-Zn domain of LNX2 is a dimer and assumes a rigid elongated structure that undergoes autoubiquitination and undergoes N-terminal polyubiquitination. The ubiquitin chains consist of all seven possible isopeptide linkages. These results were validated using full-length LNX2. Moreover we have demonstrated the ubiquitination of cell fate determinant protein, Numb by LNX2. Our study provides a structural basis for the functional machinery of LNX2 and thus provides the opportunity to investigate suitable drug targets against LNX2. PMID:26451611

  6. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle

    PubMed Central

    Kerfeld, Cheryl A.

    2016-01-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  7. Structural basis for the modular recognition of single-stranded RNA by PPR proteins.

    PubMed

    Yin, Ping; Li, Quanxiu; Yan, Chuangye; Liu, Ying; Liu, Junjie; Yu, Feng; Wang, Zheng; Long, Jiafu; He, Jianhua; Wang, Hong-Wei; Wang, Jiawei; Zhu, Jian-Kang; Shi, Yigong; Yan, Nieng

    2013-12-01

    Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

  8. Structural basis for the modular recognition of single-stranded RNA by PPR proteins

    NASA Astrophysics Data System (ADS)

    Yin, Ping; Li, Quanxiu; Yan, Chuangye; Liu, Ying; Liu, Junjie; Yu, Feng; Wang, Zheng; Long, Jiafu; He, Jianhua; Wang, Hong-Wei; Wang, Jiawei; Zhu, Jian-Kang; Shi, Yigong; Yan, Nieng

    2013-12-01

    Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

  9. Functional assessment and structural basis of antibody binding to human papillomavirus capsid.

    PubMed

    Zhang, Xiao; Li, Shaowei; Modis, Yorgo; Li, Zhihai; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

    2016-03-01

    Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs. PMID:26676802

  10. Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.

    PubMed

    Mishima, Masaki; Maesaki, Ryoko; Kasa, Miyuki; Watanabe, Takashi; Fukata, Masaki; Kaibuchi, Kozo; Hakoshima, Toshio

    2007-06-19

    Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end. PMID:17563362

  11. Structural basis for antifreeze activity of ice-binding protein from arctic yeast.

    PubMed

    Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

    2012-03-30

    Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins. PMID:22303017

  12. Structural Basis for Error-free Replication of Oxidatively Damaged DNA by Yeast DNA Polymerase eta

    SciTech Connect

    T Silverstein; R Jain; R Johnson; L Prakash; S Prakash; A Aggarwal

    2011-12-31

    7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase {eta} (Pol{eta}). We show that the open active site cleft of Pol{eta} can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Pol to prevent the mutagenic replication of 8-oxoG lesions in cells.

  13. Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

    SciTech Connect

    Hu, Liya; Ramani, Sasirekha; Czako, Rita; Sankaran, Banumathi; Yu, Ying; Smith, David F.; Cummings, Richard D.; Estes, Mary K.; Venkataram Prasad, B. V.

    2015-09-30

    We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.

  14. Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

    DOE PAGES

    Hu, Liya; Ramani, Sasirekha; Czako, Rita; Sankaran, Banumathi; Yu, Ying; Smith, David F.; Cummings, Richard D.; Estes, Mary K.; Venkataram Prasad, B. V.

    2015-09-30

    We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

  15. Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

    PubMed Central

    Hu, Liya; Ramani, Sasirekha; Czako, Rita; Sankaran, Banumathi; Yu, Ying; Smith, David F.; Cummings, Richard D.; Estes, Mary K.; Venkataram Prasad, B. V.

    2015-01-01

    Strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. Such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses. PMID:26420502

  16. Structural basis for KCNE3 modulation of potassium recycling in epithelia.

    PubMed

    Kroncke, Brett M; Van Horn, Wade D; Smith, Jarrod; Kang, CongBao; Welch, Richard C; Song, Yuanli; Nannemann, David P; Taylor, Keenan C; Sisco, Nicholas J; George, Alfred L; Meiler, Jens; Vanoye, Carlos G; Sanders, Charles R

    2016-09-01

    The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K(+)) channel to enable K(+) recycling coupled to transepithelial chloride ion (Cl(-)) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K(+) recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated "up" state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the "CF gender gap." PMID:27626070

  17. Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds

    NASA Astrophysics Data System (ADS)

    Harada, Shusaku; Hiromori, Youhei; Nakamura, Shota; Kawahara, Kazuki; Fukakusa, Shunsuke; Maruno, Takahiro; Noda, Masanori; Uchiyama, Susumu; Fukui, Kiichi; Nishikawa, Jun-Ichi; Nagase, Hisamitsu; Kobayashi, Yuji; Yoshida, Takuya; Ohkubo, Tadayasu; Nakanishi, Tsuyoshi

    2015-02-01

    Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.

  18. Structural basis for stabilization of Z-DNA by cobalt hexaammine and magnesium cations

    NASA Technical Reports Server (NTRS)

    Gessner, R. V.; Quigley, G. J.; Wang, A. H.; van der Marel, G. A.; van Boom, J. H.; Rich, A.

    1985-01-01

    In the equilibrium between B-DNA and Z-DNA in poly(dC-dG), the [Co(NH3)6]3+ ion stabilizes the Z form 4 orders of magnitude more effectively than the Mg2+ ion. The structural basis of this difference is revealed in Z-DNA crystal structures of d(CpGpCpGpCpG) stabilized by either Na+/Mg2+ or Na+/Mg2+ plus [Co(NH3)6]3+. The crystals diffract X-rays to high resolution, and the structures were refined at 1.25 A. The [Co(NH3)6]3+ ion forms five hydrogen bonds onto the surface of Z-DNA, bonding to a guanine O6 and N7 as well as to a phosphate group in the ZII conformation. The Mg2+ ion binds through its hydration shell with up to three hydrogen bonds to guanine N7 and O6. Higher charge, specific fitting of more hydrogen bonds, and a more stable complex all contribute to the great effectiveness of [Co(NH3)6]3+ in stabilizing Z-DNA.

  19. Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products

    PubMed Central

    Kuzuyama, Tomohisa; Noel, Joseph P.; Richard, Stéphane B.

    2010-01-01

    The anti-oxidant naphterpin is a natural product containing a polyketide-based aromatic core with an attached 10-carbon geranyl group derived from isoprenoid (terpene) metabolism1–3. Hybrid natural products such as naphterpin that contain 5-carbon (dimethylallyl), 10-carbon (geranyl) or 15-carbon (farnesyl) isoprenoid chains possess biological activities distinct from their non-prenylated aromatic precursors4. These hybrid natural products represent new anti-microbial, anti-oxidant, anti-inflammatory, anti-viral and anti-cancer compounds. A small number of aromatic prenyltransferases (PTases) responsible for prenyl group attachment have only recently been isolated and characterized5,6. Here we report the gene identification, biochemical characterization and high-resolution X-ray crystal structures of an architecturally novel aromatic PTase, Orf2 from Streptomyces sp. strain CL190, with substrates and substrate analogues bound. In vivo, Orf2 attaches a geranyl group to a 1,3,6,8-tetra-hydroxynaphthalene-derived polyketide during naphterpin biosynthesis. In vitro, Orf2 catalyses carbon–carbon-based and carbon–oxygen-based prenylation of a diverse collection of hydroxyl-containing aromatic acceptors of synthetic, microbial and plant origin. These crystal structures, coupled with in vitro assays, provide a basis for understanding and potentially manipulating the regio-specific prenylation of aromatic small molecules using this structurally unique family of aromatic PTases. PMID:15959519

  20. Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils

    PubMed Central

    Paravastu, Anant K.; Leapman, Richard D.; Yau, Wai-Ming; Tycko, Robert

    2008-01-01

    We describe a full structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1–40), based on numerous constraints from solid state NMR and electron microscopy. This model applies specifically to fibrils with a periodically twisted morphology, with twist period equal to 120 ± 20 nm (defined as the distance between apparent minima in fibril width in negatively stained transmission electron microscope images). The structure has threefold symmetry about the fibril growth axis, implied by mass-per-length data and the observation of a single set of 13C NMR signals. Comparison with a previously reported model for Aβ1–40 fibrils with a qualitatively different, striated ribbon morphology reveals the molecular basis for polymorphism. At the molecular level, the 2 Aβ1–40 fibril morphologies differ in overall symmetry (twofold vs. threefold), the conformation of non-β-strand segments, and certain quaternary contacts. Both morphologies contain in-register parallel β-sheets, constructed from nearly the same β-strand segments. Because twisted and striated ribbon morphologies are also observed for amyloid fibrils formed by other polypeptides, such as the amylin peptide associated with type 2 diabetes, these structural variations may have general implications. PMID:19015532

  1. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching.

    PubMed

    Lee, Lawrence K; Ginsburg, Michael A; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

    2010-08-19

    The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation. PMID:20676082

  2. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching

    SciTech Connect

    Lee, Lawrence K.; Ginsburg, Michael A.; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

    2010-09-13

    The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.

  3. Structural basis for KCNE3 modulation of potassium recycling in epithelia

    PubMed Central

    Kroncke, Brett M.; Van Horn, Wade D.; Smith, Jarrod; Kang, CongBao; Welch, Richard C.; Song, Yuanli; Nannemann, David P.; Taylor, Keenan C.; Sisco, Nicholas J.; George, Alfred L.; Meiler, Jens; Vanoye, Carlos G.; Sanders, Charles R.

    2016-01-01

    The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.” PMID:27626070

  4. Structural basis of VDR–DNA interactions on direct repeat response elements

    PubMed Central

    Shaffer, Paul L.; Gewirth, Daniel T.

    2002-01-01

    The vitamin D receptor (VDR) forms homo- or heterodimers on response elements composed of two hexameric half-sites separated by 3 bp of spacer DNA. We describe here the crystal structures at 2.7–2.8 Å resolution of the VDR DNA-binding region (DBD) in complex with response elements from three different promoters: osteopontin (SPP), canonical DR3 and osteocalcin (OC). These structures reveal the chemical basis for the increased affinity of VDR for the SPP response element, and for the poor stability of the VDR–OC complex, relative to the canonical DR3 response element. The homodimeric protein–protein interface is stabilized by van der Waals interactions and is predominantly non-polar. An extensive α-helix at the C-terminal end of the VDR DBD resembles that found in the thyroid hormone receptor (TR), and suggests a mechanism by which VDR and TR discriminate among response elements. Selective structure-based mutations in the asymmetric homodimeric interface result in a VDR DBD protein that is defective in homodimerization but now forms heterodimers with the 9-cis retinoic acid receptor (RXR) DBD. PMID:11980721

  5. Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

    SciTech Connect

    Tomita, Takeo; Fushinobu, Shinya; Kuzuyama, Tomohisa; Nishiyama, Makoto . E-mail: umanis@mail.ecc.u-tokyo.ac.jp

    2006-08-25

    To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.

  6. Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products.

    PubMed

    Kuzuyama, Tomohisa; Noel, Joseph P; Richard, Stéphane B

    2005-06-16

    The anti-oxidant naphterpin is a natural product containing a polyketide-based aromatic core with an attached 10-carbon geranyl group derived from isoprenoid (terpene) metabolism. Hybrid natural products such as naphterpin that contain 5-carbon (dimethylallyl), 10-carbon (geranyl) or 15-carbon (farnesyl) isoprenoid chains possess biological activities distinct from their non-prenylated aromatic precursors. These hybrid natural products represent new anti-microbial, anti-oxidant, anti-inflammatory, anti-viral and anti-cancer compounds. A small number of aromatic prenyltransferases (PTases) responsible for prenyl group attachment have only recently been isolated and characterized. Here we report the gene identification, biochemical characterization and high-resolution X-ray crystal structures of an architecturally novel aromatic PTase, Orf2 from Streptomyces sp. strain CL190, with substrates and substrate analogues bound. In vivo, Orf2 attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during naphterpin biosynthesis. In vitro, Orf2 catalyses carbon-carbon-based and carbon-oxygen-based prenylation of a diverse collection of hydroxyl-containing aromatic acceptors of synthetic, microbial and plant origin. These crystal structures, coupled with in vitro assays, provide a basis for understanding and potentially manipulating the regio-specific prenylation of aromatic small molecules using this structurally unique family of aromatic PTases.

  7. Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement

    PubMed Central

    Esser, Lothar; Yu, Chang-An; Xia, Di

    2016-01-01

    The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc1 complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc1 inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc1 complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc1 by various quinol oxidation- and reduction-site inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives. PMID:23688079

  8. Structural basis of resistance to anti-cytochrome bc₁ complex inhibitors: implication for drug improvement.

    PubMed

    Esser, Lothar; Yu, Chang-An; Xia, Di

    2014-01-01

    The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc₁ complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc₁ inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc₁ complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc₁ by various quinol oxidation- and reductionsite inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives.

  9. Structural Basis for Promoter ;#8722;10 Element Recognition by the Bacterial RNA Polymerase [sigma] Subunit

    SciTech Connect

    Feklistov, Andrey; Darst, Seth A.

    2011-12-15

    The key step in bacterial promoter opening is recognition of the -10 promoter element (T-{sub 12}A-{sub 11}T-{sub 10}A-{sub 9}A-{sub 8}T{sub -7} consensus sequence) by the RNA polymerase {alpha} subunit. We determined crystal structures of {alpha} domain 2 bound to single-stranded DNA bearing -10 element sequences. Extensive interactions occur between the protein and the DNA backbone of every -10 element nucleotide. Base-specific interactions occur primarily with A{sub -11} and T{sub -7}, which are flipped out of the single-stranded DNA base stack and buried deep in protein pockets. The structures, along with biochemical data, support a model where the recognition of the -10 element sequence drives initial promoter opening as the bases of the nontemplate strand are extruded from the DNA double-helix and captured by {alpha}. These results provide a detailed structural basis for the critical roles of A{sub -11} and T{sub -7} in promoter melting and reveal important insights into the initiation of transcription bubble formation.

  10. Structural basis for the suppression of skin cancers by DNA polymerase [eta

    SciTech Connect

    Silverstein, Timothy D.; Johnson, Robert E.; Jain, Rinku; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2010-09-13

    DNA polymerase {eta} (Pol{eta}) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Pol{eta} (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Pol{eta} (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Pol{eta} to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln55, Arg73 and Met74. Together, these features define the basis for Pol{eta}'s action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.

  11. Structural basis for KCNE3 modulation of potassium recycling in epithelia

    PubMed Central

    Kroncke, Brett M.; Van Horn, Wade D.; Smith, Jarrod; Kang, CongBao; Welch, Richard C.; Song, Yuanli; Nannemann, David P.; Taylor, Keenan C.; Sisco, Nicholas J.; George, Alfred L.; Meiler, Jens; Vanoye, Carlos G.; Sanders, Charles R.

    2016-01-01

    The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”

  12. Structural basis for DNA cleavage by the potent antiproliferative agent (–)-lomaiviticin A

    PubMed Central

    Woo, Christina M.; Li, Zhenwu; Herzon, Seth B.

    2016-01-01

    (–)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar–picomolar concentrations. (–)-Lomaiviticin A (1) possesses a C2-symmetric structure that contains two unusual diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC)2 by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule–DNA association. PMID:26929332

  13. Structural Basis of Interaction Between Urokinase-type Plasminogen Activator and its Receptor

    SciTech Connect

    Barinka,C.; Parry, G.; Callahan, J.; Shaw, D.; Kuo, A.; Cines, B.; Mazar, A.; Lubkowski, J.

    2006-01-01

    Recent studies indicate that binding of the urokinase-type plasminogen activator (uPA) to its high-affinity receptor (uPAR) orchestrates uPAR interactions with other cellular components that play a pivotal role in diverse (patho-)physiological processes, including wound healing, angiogenesis, inflammation, and cancer metastasis. However, notwithstanding the wealth of biochemical data available describing the activities of uPAR, little is known about the exact mode of uPAR/uPA interactions or the presumed conformational changes that accompany uPA/uPAR engagement. Here, we report the crystal structure of soluble urokinase plasminogen activator receptor (suPAR), which contains the three domains of the wild-type receptor but lacks the cell-surface anchoring sequence, in complex with the amino-terminal fragment of urokinase-type plasminogen activator (ATF), at the resolution of 2.8 {angstrom}. We report the 1.9 {angstrom} crystal structure of free ATF. Our results provide a structural basis, represented by conformational changes induced in uPAR, for several published biochemical observations describing the nature of uPAR/uPA interactions and provide insight into mechanisms that may be responsible for the cellular responses induced by uPA binding.

  14. Efficient O(N) integration for all-electron electronic structure calculation using numeric basis functions

    SciTech Connect

    Havu, V. Blum, V.; Havu, P.; Scheffler, M.

    2009-12-01

    We consider the problem of developing O(N) scaling grid-based operations needed in many central operations when performing electronic structure calculations with numeric atom-centered orbitals as basis functions. We outline the overall formulation of localized algorithms, and specifically the creation of localized grid batches. The choice of the grid partitioning scheme plays an important role in the performance and memory consumption of the grid-based operations. Three different top-down partitioning methods are investigated, and compared with formally more rigorous yet much more expensive bottom-up algorithms. We show that a conceptually simple top-down grid partitioning scheme achieves essentially the same efficiency as the more rigorous bottom-up approaches.

  15. Electrical heart disease: Genetic and molecular basis of cardiac arrhythmias in normal structural hearts.

    PubMed

    Farwell, David; Gollob, Michael H

    2007-08-01

    Purely electrical heart diseases, defined by the absence of any structural cardiac defects, are responsible for a large number of sudden, unexpected deaths in otherwise healthy, young individuals. These conditions include the long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia and the short QT syndrome. Collectively, these conditions have been referred to as channelopathies. Ion channels provide the molecular basis for cardiac electrical activity. These channels have specific ion selectivity and are responsible for the precise and timely regulation of the passage of charged ions across the cell membrane in myocytes, and the summation of their activity in cardiac muscle defines the surface electrocardiogram. Impairment in the flow of these ions in heart cells may mean the difference between a normal, prosperous life and the tragedy of a sudden, unexpected death due to ventricular arrhythmia. The present paper reviews the current clinical and molecular understanding of the electrical diseases of the heart associated with sudden cardiac death.

  16. Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

    SciTech Connect

    Rickert, Keith W.; Patel, Sangita B.; Allison, Timothy J.; Byrne, Noel J.; Darke, Paul L.; Ford, Rachael E.; Guerin, David J.; Hall, Dawn L.; Kornienko, Maria; Lu, Jun; Munshi, Sanjeev K.; Reid, John C.; Shipman, Jennifer M.; Stanton, Elizabeth F.; Wilson, Kevin J.; Young, Jonathon R.; Soisson, Stephen M.; Lumb, Kevin J.

    2012-03-15

    The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix {alpha}C and the G loop to generate a viable active site. Helix {alpha}C adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

  17. Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa

    SciTech Connect

    Han, Seungil; Zaniewski, Richard P.; Marr, Eric S.; Lacey, Brian M.; Tomaras, Andrew P.; Evdokimov, Artem; Miller, J. Richard; Shanmugasundaram, Veerabahu

    2012-02-08

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by {beta}-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed {beta}-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.

  18. Structural basis for cell-cycle-dependent nuclear import mediated by the karyopherin Kap121p.

    PubMed

    Kobayashi, Junya; Matsuura, Yoshiyuki

    2013-06-12

    Kap121p (also known as Pse1p) is an essential karyopherin that mediates nuclear import of a plethora of cargoes including cell cycle regulators, transcription factors, and ribosomal proteins in Saccharomyces cerevisiae. It has been proposed that the spindle assembly checkpoint signaling triggers molecular rearrangements of nuclear pore complexes and thereby arrests Kap121p-mediated nuclear import at metaphase, while leaving import mediated by other karyopherins unaffected. The Kap121p-specific import inhibition is required for normal progression through mitosis. To understand the structural basis for Kap121p-mediated nuclear import and its unique regulatory mechanism during mitosis, we determined crystal structures of Kap121p in isolation and also in complex with either its import cargoes or nucleoporin Nup53p or RanGTP. Kap121p has a superhelical structure composed of 24 HEAT repeats. The structures of Kap121p-cargo complexes define a non-conventional nuclear localization signal (NLS) that has a consensus sequence of KV/IxKx1-2K/H/R. The structure of Kap121p-Nup53p complex shows that cargo and Nup53p compete for the same high-affinity binding site, explaining how Nup53p binding forces cargo release when the Kap121p-binding site of Nup53p is exposed during mitosis. Comparison of the NLS and RanGTP complexes reveals that RanGTP binding not only occludes the cargo-binding site but also forces Kap121p into a conformation that is incompatible with NLS recognition.

  19. Structural Basis for the Differential Regulation of DNA by the Methionine Repressor MetJ

    SciTech Connect

    Augustus, Anne; Reardon, Patrick; Heller, William T; Spicer, Leonard D.

    2006-01-01

    The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.

  20. Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa

    PubMed Central

    Han, Seungil; Zaniewski, Richard P.; Marr, Eric S.; Lacey, Brian M.; Tomaras, Andrew P.; Evdokimov, Artem; Miller, J. Richard; Shanmugasundaram, Veerabahu

    2010-01-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by β-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed β-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs. PMID:21135211

  1. hERG potassium channels and the structural basis of drug-induced arrhythmias.

    PubMed

    Mitcheson, John S

    2008-05-01

    hERG potassium channels have a critical role in the normal electrical activity of the heart. The block of hERG channels can cause the drug-induced form of long QT syndrome, a cardiac disorder that carries an increased risk of cardiac arrhythmias and sudden death. hERG channels are extraordinarily sensitive to block by large numbers of structurally diverse drugs. In previous years, the risk of compounds causing this cardiotoxic side effect has been a common reason for the failure of compounds in preclinical safety trials. Pharmaceutical companies have successfully utilized and developed higher throughput techniques for the early detection of compounds that block hERG, and this has helped reduce the number of compounds that fail in the late stages of development. Nevertheless, this screening-based approach is expensive, consumes chemistry resources, and bypasses the problem rather than shedding light on it. Crystal structures of potassium channels have facilitated studies into the structural basis for the gating and block of hERG channels. Most drugs bind within the inner cavity, and the individual amino acids that form the drug binding site have been identified by site-directed mutagenesis approaches. Gating processes have an important influence on the drug-binding site. Recent advances in our understanding of channel activation and inactivation are providing insight into why hERG channels are more susceptible to block than other K (+) channels. Knowledge of the structure of the drug-binding site and precise nature of interactions with drug molecules should assist efforts to develop drugs without the propensity to cause cardiac arrhythmias. PMID:18447395

  2. Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

    PubMed Central

    Hatayama, Minoru; Tomizawa, Tadashi; Sakai-Kato, Kumiko; Bouvagnet, Patrice; Kose, Shingo; Imamoto, Naoko; Yokoyama, Shigeyuki; Utsunomiya-Tate, Naoko; Mikoshiba, Katsuhiko; Kigawa, Takanori; Aruga, Jun

    2008-01-01

    Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS. PMID:18716025

  3. Structural basis for antagonism of human interleukin 18 by poxvirus interleukin 18-binding protein

    SciTech Connect

    Krumm, Brian; Meng, Xiangzhi; Li, Yongchao; Xiang, Yan; Deng, Junpeng

    2009-07-10

    Human interleukin-18 (hIL-18) is a cytokine that plays an important role in inflammation and host defense against microbes. Its activity is regulated in vivo by a naturally occurring antagonist, the human IL-18-binding protein (IL-18BP). Functional homologs of human IL-18BP are encoded by all orthopoxviruses, including variola virus, the causative agent of smallpox. They contribute to virulence by suppressing IL-18-mediated immune responses. Here, we describe the 2.0-{angstrom} resolution crystal structure of an orthopoxvirus IL-18BP, ectromelia virus IL-18BP (ectvIL-18BP), in complex with hIL-18. The hIL-18 structure in the complex shows significant conformational change at the binding interface compared with the structure of ligand-free hIL-18, indicating that the binding is mediated by an induced-fit mechanism. EctvIL-18BP adopts a canonical Ig fold and interacts via one edge of its {beta}-sandwich with 3 cavities on the hIL-18 surface through extensive hydrophobic and hydrogen bonding interactions. Most of the ectvIL-18BP residues that participate in these interactions are conserved in both human and viral homologs, explaining their functional equivalence despite limited sequence homology. EctvIL-18BP blocks a putative receptor-binding site on IL-18, thus preventing IL-18 from engaging its receptor. Our structure provides insights into how IL-18BPs modulate hIL-18 activity. The revealed binding interface provides the basis for rational design of inhibitors against orthopoxvirus IL-18BP (for treating orthopoxvirus infection) or hIL-18 (for treating certain inflammatory and autoimmune diseases).

  4. Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.

    PubMed

    Feng, Chong; Liu, Yang; Wang, Guoqiang; Deng, Zengqin; Zhang, Qi; Wu, Wei; Tong, Yufeng; Cheng, Changmei; Chen, Zhongzhou

    2014-04-25

    N(6)-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct "lid" region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a "flipping" motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members. PMID:24616105

  5. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

    PubMed

    Hayes, Robert P; Xiao, Yibei; Ding, Fran; van Erp, Paul B G; Rajashankar, Kanagalaghatta; Bailey, Scott; Wiedenheft, Blake; Ke, Ailong

    2016-02-25

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process. PMID:26863189

  6. Structural basis of nuclear import of flap endonuclease 1 (FEN1).

    PubMed

    de Barros, Andrea C; Takeda, Agnes A S; Chang, Chiung Wen; Kobe, Boštjan; Fontes, Marcos R M

    2012-07-01

    Flap endonuclease 1 (FEN1) is a member of the nuclease family and is structurally conserved from bacteriophages to humans. This protein is involved in multiple DNA-processing pathways, including Okazaki fragment maturation, stalled replication-fork rescue, telomere maintenance, long-patch base-excision repair and apoptotic DNA fragmentation. FEN1 has three functional motifs that are responsible for its nuclease, PCNA-interaction and nuclear localization activities, respectively. It has been shown that the C-terminal nuclear localization sequence (NLS) facilitates nuclear localization of the enzyme during the S phase of the cell cycle and in response to DNA damage. To determine the structural basis of the recognition of FEN1 by the nuclear import receptor importin α, the crystal structure of the complex of importin α with a peptide corresponding to the FEN1 NLS was solved. Structural studies confirmed the binding of the FEN1 NLS as a classical bipartite NLS; however, in contrast to the previously proposed (354)KRKX(8)KKK(367) sequence, it is the (354)KRX(10)KKAK(369) sequence that binds to importin α. This result explains the incomplete inhibition of localization that was observed on mutating residues (365)KKK(367). Acidic and polar residues in the X(10) linker region close to the basic clusters play an important role in binding to importin α. These results suggest that the basic residues in the N-terminal basic cluster of bipartite NLSs may play roles that are more critical than those of the many basic residues in the C-terminal basic cluster.

  7. Structural Basis for the Interconversion of Maltodextrins by MalQ, the Amylomaltase of Escherichia coli*

    PubMed Central

    Weiss, Simon C.; Skerra, Arne; Schiefner, André

    2015-01-01

    Amylomaltase MalQ is essential for the metabolism of maltose and maltodextrins in Escherichia coli. It catalyzes transglycosylation/disproportionation reactions in which glycosyl or dextrinyl units are transferred among linear maltodextrins of various lengths. To elucidate the molecular basis of transglycosylation by MalQ, we have determined three crystal structures of this enzyme, i.e. the apo-form, its complex with maltose, and an inhibitor complex with the transition state analog acarviosine-glucose-acarbose, at resolutions down to 2.1 Å. MalQ represents the first example of a mesophilic bacterial amylomaltase with known structure and exhibits an N-terminal extension of about 140 residues, in contrast with previously described thermophilic enzymes. This moiety seems unique to amylomaltases from Enterobacteriaceae and folds into two distinct subdomains that associate with different parts of the catalytic core. Intriguingly, the three MalQ crystal structures appear to correspond to distinct states of this enzyme, revealing considerable conformational changes during the catalytic cycle. In particular, the inhibitor complex highlights the requirement of both a 3-OH group and a 4-OH group (or α1–4-glycosidic bond) at the acceptor subsite +1 for the catalytically competent orientation of the acid/base catalyst Glu-496. Using an HPLC-based MalQ enzyme assay, we could demonstrate that the equilibrium concentration of maltodextrin products depends on the length of the initial substrate; with increasing numbers of glycosidic bonds, less glucose is formed. Thus, both structural and enzymatic data are consistent with the extremely low hydrolysis rates observed for amylomaltases and underline the importance of MalQ for the metabolism of maltodextrins in E. coli. PMID:26139606

  8. Structural basis of Lewisb antigen binding by the Helicobacter pylori adhesin BabA

    PubMed Central

    Hage, Naim; Howard, Tina; Phillips, Chris; Brassington, Claire; Overman, Ross; Debreczeni, Judit; Gellert, Paul; Stolnik, Snow; Winkler, G. Sebastiaan; Falcone, Franco H.

    2015-01-01

    Helicobacter pylori is a leading cause of peptic ulceration and gastric cancer worldwide. To achieve colonization of the stomach, this Gram-negative bacterium adheres to Lewisb (Leb) antigens in the gastric mucosa using its outer membrane protein BabA. Structural information for BabA has been elusive, and thus, its molecular mechanism for recognizing Leb antigens remains unknown. We present the crystal structure of the extracellular domain of BabA, from H. pylori strain J99, in the absence and presence of Leb at 2.0- and 2.1-Å resolutions, respectively. BabA is a predominantly α-helical molecule with a markedly kinked tertiary structure containing a single, shallow Leb binding site at its tip within a β-strand motif. No conformational change occurs in BabA upon binding of Leb, which is characterized by low affinity under acidic [KD (dissociation constant) of ~227 μM] and neutral (KD of ~252 μM) conditions. Binding is mediated by a network of hydrogen bonds between Leb Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions. The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Leb, respectively. Knowledge of the molecular basis of Leb recognition by BabA provides a platform for the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa. PMID:26601230

  9. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

    PubMed

    Hayes, Robert P; Xiao, Yibei; Ding, Fran; van Erp, Paul B G; Rajashankar, Kanagalaghatta; Bailey, Scott; Wiedenheft, Blake; Ke, Ailong

    2016-02-25

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

  10. Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)

    SciTech Connect

    Wang, Hui; Hota, Prasanta K.; Tong, Yufeng; Li, Buren; Shen, Limin; Nedyalkova, Lyudmila; Borthakur, Susmita; Kim, SoonJeung; Tempel, Wolfram; Buck, Matthias; Park, Hee-Won

    2011-09-20

    Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD {center_dot} Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD {beta}3-{beta}4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

  11. Crystal Structure of Human Dihydrolipoamide Dehydrogenase: NAD[superscript +]/NADH Binding and the Structural Basis of Disease-causing Mutations

    SciTech Connect

    Brautigam, Chad A.; Chuang, Jacinta L.; Tomchick, Diana R.; Machius, Mischa; Chuang, David T.

    2010-07-13

    Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial {alpha}-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: noncovalently bound FAD and a transiently bound substrate, NAD{sup +}. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD{sup +} or NADH have been determined at resolutions of 2.5 {angstrom} and 2.1 {angstrom}, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD{sup +} bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD{sup +} or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD{sup +}-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.

  12. Structural Basis for Substrate Selectivity of the E3 Ligase COP1.

    PubMed

    Uljon, Sacha; Xu, Xiang; Durzynska, Izabela; Stein, Sarah; Adelmant, Guillaume; Marto, Jarrod A; Pear, Warren S; Blacklow, Stephen C

    2016-05-01

    COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation. PMID:27041596

  13. Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

    PubMed Central

    Maqbool, A; Saitoh, H; Franceschetti, M; Stevenson, CEM; Uemura, A; Kanzaki, H; Kamoun, S; Terauchi, R; Banfield, MJ

    2015-01-01

    Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001 PMID:26304198

  14. Structural basis of nucleotide exchange and client binding by the novel Hsp70-cochaperone Bag2

    PubMed Central

    Xu, Zhen; Page, Richard C; Gomes, Michelle M; Kohli, Ekta; Nix, Jay C; Herr, Andrew B; Patterson, Cam; Misra, Saurav

    2009-01-01

    Cochaperones are essential for Hsp70/Hsc70-mediated folding of proteins and include nucleotide exchange factors (NEF) that assist protein folding by accelerating ADP/ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as a NEF, but the molecular basis of its functions is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the “Brand New Bag” (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client-binding sites and Hsc70 interaction sites of Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp-70-mediated protein folding by Bag2. PMID:19029896

  15. Molecular And Structural Basis of Cytokine Receptor Pleiotropy in the Interleukin-4/13 System

    SciTech Connect

    LaPorte, S.L.; Juo, Z.S.; Vaclavikova, J.; Colf, L.A.; Qi, X.; Heller, N.M.; Keegan, A.D.; Garcia, K.C.

    2009-05-20

    Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R{alpha}/{gamma}{sub c}/IL-4) and type II (IL-4R/IL-13R{alpha}1/IL-4, IL-4R{alpha}/IL-13R{alpha}1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for {gamma}{sub c}'s ability to recognize six different {gamma}{sub c}-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R{alpha}1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

  16. Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

    SciTech Connect

    McLaughlin, K.J.; Soares, A.; Strain-Damerell, C. M.; Xie, K.; Brekasis, D.; Pagent, M. S. B.; Kielkopf, C. L.

    2010-05-28

    Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD{sup +} is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD{sup +} ratio. Here we investigate the molecular mechanism for NADH/NAD{sup +} sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD{sup +}, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40{sup o} closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.

  17. Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling

    PubMed Central

    Hospenthal, Manuela K.; Redzej, Adam; Dodson, Karen; Ukleja, Marta; Frenz, Brandon; Rodrigues, Catarina; Hultgren, Scott J.; DiMaio, Frank; Egelman, Edward H.; Waksman, Gabriel

    2016-01-01

    Summary Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod’s remarkable mechanical properties and illuminates its role in pilus secretion. PMID:26724865

  18. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    NASA Astrophysics Data System (ADS)

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

    2015-08-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  19. Structural and thermodynamic basis of proline-induced transmembrane complex stabilization

    PubMed Central

    Schmidt, Thomas; Situ, Alan J.; Ulmer, Tobias S.

    2016-01-01

    In membrane proteins, proline-mediated helix kinks are indispensable for the tight packing of transmembrane (TM) helices. However, kinks invariably affect numerous interhelical interactions, questioning the acceptance of proline substitutions and evolutionary origin of kinks. Here, we present the structural and thermodynamic basis of proline-induced integrin αIIbβ3 TM complex stabilization to understand the introduction of proline kinks in membrane proteins. In phospholipid bicelles, the A711P substitution in the center of the β3 TM helix changes the direction of adjacent helix segments to form a 35 ± 2° angle and predominantly repacks the segment in the inner membrane leaflet due to a swivel movement. This swivel repacks hydrophobic and electrostatic interhelical contacts within intracellular lipids, resulting in an overall TM complex stabilization of −0.82 ± 0.01 kcal/mol. Thus, proline substitutions can directly stabilize membrane proteins and such substitutions are proposed to follow the structural template of integrin αIIbβ3(A711P). PMID:27436065

  20. Structural basis of GM1 ganglioside recognition by simian virus 40.

    PubMed

    Neu, Ursula; Woellner, Karin; Gauglitz, Guenter; Stehle, Thilo

    2008-04-01

    Simian virus 40 (SV40) has been a paradigm for understanding attachment and entry of nonenveloped viruses, viral DNA replication, and virus assembly, as well as for endocytosis pathways associated with caveolin and cholesterol. We find by glycan array screening that SV40 recognizes its ganglioside receptor GM1 with a quite narrow specificity, but isothermal titration calorimetry shows that individual binding sites have a relatively low affinity, with a millimolar dissociation constant. The high-resolution crystal structure of recombinantly produced SV40 capsid protein, VP1, in complex with the carbohydrate portion of GM1, reveals that the receptor is bound in a shallow solvent-exposed groove at the outer surface of the capsid. Through a complex network of interactions, VP1 recognizes a conformation of GM1 that is the dominant one in solution. Analysis of contacts provides a structural basis for the observed specificity and suggests binding mechanisms for additional physiologically relevant GM1 variants. Comparison with murine Polyomavirus (Polyoma) receptor complexes reveals that SV40 uses a different mechanism of sialic acid binding, which has implications for receptor binding of human polyomaviruses. The SV40-GM1 complex reveals a parallel to cholera toxin, which uses a similar cell entry pathway and binds GM1 in the same conformation.

  1. Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA

    SciTech Connect

    Babayeva, Nigar D.; Wilder, Phillip J.; Shiina, Masaaki; Mino, Koshiki; Desler, Michelle; Ogata, Kazuhiro; Rizzino, Angie; Tahirov, Tahir H.

    2010-09-03

    Ets1 is a member of the Ets family of transcription factors. Ets1 is autoinhibited and its activation requires heterodimerization with a partner protein or DNA-mediated homodimerization for cooperative DNA binding. In the latter case, Ets1 molecules bind to palindromic sequences in which two Ets-binding sites (EBS) are separated by four base pairs, for example in the promoters of stromelysin-1 and p53. Interestingly, counteraction of autoinhibition requires the autoinhibitory region encoded by exon VII of the gene. The structural basis for the requirement of autoinhibitory sequences for Ets1 binding to palindromic EBS still remains unresolved. Here we report the crystal structure of two Ets1 molecules bound to an EBS palindrome of the stromelysin-1 promoter DNA, providing a plausible explanation for the requirement of exon VII-encoded sequences for Ets1 cooperative DNA binding. The proposed mechanism was verified both in vitro by surface plasmon resonance and in vivo by transcription-based assays.

  2. Structural basis for haem piracy from host haemopexin by Haemophilus influenzae.

    PubMed

    Zambolin, Silvia; Clantin, Bernard; Chami, Mohamed; Hoos, Sylviane; Haouz, Ahmed; Villeret, Vincent; Delepelaire, Philippe

    2016-05-18

    Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin.

  3. Structural Basis of Arc Binding to Synaptic Proteins: Implications for Cognitive Disease

    PubMed Central

    Zhang, Wenchi; Wu, Jing; Ward, Matthew D.; Yang, Sunggu; Chuang, Yang-An; Xiao, Meifang; Li, Ruojing; Leahy, Daniel J.; Worley, Paul F.

    2015-01-01

    SUMMARY Arc is a cellular immediate early gene (IEG) that functions at excitatory synapses and is required for learning and memory. We report crystal structures of Arc subdomains that form a bi-lobar architecture remarkably similar to the capsid domain of human immunodeficiency virus (HIV) gag protein. Analysis indicates Arc originated from the Ty3/Gypsy retrotransposon family and was “domesticated” in higher vertebrates for synaptic functions. The Arc N-terminal lobe evolved a unique hydrophobic pocket that mediates intermolecular binding with synaptic proteins as resolved in complexes with TARPγ2 (Stargazin) and CaMKII peptides, and is essential for Arc’s synaptic function. A consensus sequence for Arc binding identifies several additional partners that include genes implicated in schizophrenia. Arc N-lobe binding is inhibited by small chemicals suggesting Arc’s synaptic action may be druggable. These studies reveal the remarkable evolutionary origin of Arc and provide a structural basis for understanding Arc’s contribution to neural plasticity and disease. PMID:25864631

  4. Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling

    NASA Astrophysics Data System (ADS)

    Sohmen, Daniel; Chiba, Shinobu; Shimokawa-Chiba, Naomi; Innis, C. Axel; Berninghausen, Otto; Beckmann, Roland; Ito, Koreaki; Wilson, Daniel N.

    2015-04-01

    Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream membrane protein biogenesis factor YidC2 (YqjG) in Bacillus subtilis, but not in Escherichia coli. In the absence of structures of Gram-positive bacterial ribosomes, a molecular basis for species-specific stalling has remained unclear. Here we present the structure of a Gram-positive B. subtilis MifM-stalled 70S ribosome at 3.5-3.9 Å, revealing a network of interactions between MifM and the ribosomal tunnel, which stabilize a non-productive conformation of the PTC that prevents aminoacyl-tRNA accommodation and thereby induces translational arrest. Complementary genetic analyses identify a single amino acid within ribosomal protein L22 that dictates the species specificity of the stalling event. Such insights expand our understanding of how the synergism between the ribosome and the nascent chain is utilized to modulate the translatome in a species-specific manner.

  5. Structural Basis for Platelet Collagen Responses by the Immune-type Receptor Glycoprotein VI

    SciTech Connect

    Horii,K.; Kahn, M.; Herr, A.

    2006-01-01

    Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immunetype receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.

  6. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

    PubMed

    Joachimiak, Lukasz A; Walzthoeni, Thomas; Liu, Corey W; Aebersold, Ruedi; Frydman, Judith

    2014-11-20

    The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates. PMID:25416944

  7. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT

    PubMed Central

    Joachimiak, Lukasz A.; Walzthoeni, Thomas; Liu, Corey; Aebersold, Ruedi; Frydman, Judith

    2014-01-01

    Summary The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved, pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to its unique ability to fold obligate substrates. PMID:25416944

  8. Structural basis for haem piracy from host haemopexin by Haemophilus influenzae

    PubMed Central

    Zambolin, Silvia; Clantin, Bernard; Chami, Mohamed; Hoos, Sylviane; Haouz, Ahmed; Villeret, Vincent; Delepelaire, Philippe

    2016-01-01

    Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin. PMID:27188378

  9. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

    PubMed

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-12-12

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

  10. Structural basis for LIN54 recognition of CHR elements in cell cycle-regulated promoters.

    PubMed

    Marceau, Aimee H; Felthousen, Jessica G; Goetsch, Paul D; Iness, Audra N; Lee, Hsiau-Wei; Tripathi, Sarvind M; Strome, Susan; Litovchick, Larisa; Rubin, Seth M

    2016-01-01

    The MuvB complex recruits transcription factors to activate or repress genes with cell cycle-dependent expression patterns. MuvB contains the DNA-binding protein LIN54, which directs the complex to promoter cell cycle genes homology region (CHR) elements. Here we characterize the DNA-binding properties of LIN54 and describe the structural basis for recognition of a CHR sequence. We biochemically define the CHR consensus as TTYRAA and determine that two tandem cysteine rich regions are required for high-affinity DNA association. A crystal structure of the LIN54 DNA-binding domain in complex with a CHR sequence reveals that sequence specificity is conferred by two tyrosine residues, which insert into the minor groove of the DNA duplex. We demonstrate that this unique tyrosine-mediated DNA binding is necessary for MuvB recruitment to target promoters. Our results suggest a model in which MuvB binds near transcription start sites and plays a role in positioning downstream nucleosomes.

  11. Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1.

    PubMed

    Eustermann, Sebastian; Wu, Wing-Fung; Langelier, Marie-France; Yang, Ji-Chun; Easton, Laura E; Riccio, Amanda A; Pascal, John M; Neuhaus, David

    2015-12-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1's function remained obscure; inherent dynamics of SSBs and PARP-1's multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1's signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins. PMID:26626479

  12. Structural basis of redox-dependent modulation of galectin-1 dynamics and function

    PubMed Central

    Guardia, Carlos M; Caramelo, Julio J; Trujillo, Madia; Méndez-Huergo, Santiago P; Radi, Rafael; Estrin, Darío A; Rabinovich, Gabriel A

    2014-01-01

    Galectin-1 (Gal-1), a member of a family of multifunctional lectins, plays key roles in diverse biological processes including cell signaling, immunomodulation, neuroprotection and angiogenesis. The presence of an unusual number of six cysteine residues within Gal-1 sequence prompted a detailed analysis of the impact of the redox environment on the functional activity of this lectin. We examined the role of each cysteine residue in the structure and function of Gal-1 using both experimental and computational approaches. Our results show that: (i) only three cysteine residues present in each carbohydrate recognition domain (CRD) (Cys2, Cys16 and Cys88) were important in protein oxidation, (ii) oxidation promoted the formation of the Cys16–Cys88 disulfide bond, as well as multimers through Cys2, (iii) the oxidized protein did not bind to lactose, probably due to poor interactions with Arg48 and Glu71, (iv) in vitro oxidation by air was completely reversible and (v) oxidation by hydrogen peroxide was relatively slow (1.7 ± 0.2 M−1 s−1 at pH 7.4 and 25°C). Finally, an analysis of key cysteines in other human galectins is also provided in order to predict their behaviour in response to redox variations. Collectively, our data provide new insights into the structural basis of Gal-1 redox regulation with critical implications in physiology and pathology. PMID:24451991

  13. Structural basis for LIN54 recognition of CHR elements in cell cycle-regulated promoters

    PubMed Central

    Marceau, Aimee H.; Felthousen, Jessica G.; Goetsch, Paul D.; Iness, Audra N.; Lee, Hsiau-Wei; Tripathi, Sarvind M.; Strome, Susan; Litovchick, Larisa; Rubin, Seth M.

    2016-01-01

    The MuvB complex recruits transcription factors to activate or repress genes with cell cycle-dependent expression patterns. MuvB contains the DNA-binding protein LIN54, which directs the complex to promoter cell cycle genes homology region (CHR) elements. Here we characterize the DNA-binding properties of LIN54 and describe the structural basis for recognition of a CHR sequence. We biochemically define the CHR consensus as TTYRAA and determine that two tandem cysteine rich regions are required for high-affinity DNA association. A crystal structure of the LIN54 DNA-binding domain in complex with a CHR sequence reveals that sequence specificity is conferred by two tyrosine residues, which insert into the minor groove of the DNA duplex. We demonstrate that this unique tyrosine-mediated DNA binding is necessary for MuvB recruitment to target promoters. Our results suggest a model in which MuvB binds near transcription start sites and plays a role in positioning downstream nucleosomes. PMID:27465258

  14. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    PubMed Central

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-01-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design. PMID:27708429

  15. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity.

    PubMed

    Winkelmann, Donald A; Forgacs, Eva; Miller, Matthew T; Stock, Ann M

    2015-08-06

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  16. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

    PubMed Central

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-01-01

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. DOI: http://dx.doi.org/10.7554/eLife.05553.001 PMID:25497229

  17. Structural basis for CDK6 activation by a virus-encoded cyclin

    SciTech Connect

    Schulze-Gahmen, Ursula; Kim, Sung-Hou

    2002-01-17

    Cyclin from herpesvirus saimiri (Vcyclin) preferentially forms complexes with cyclin-dependent kinase 6 (CDK6) from primate host cells. These complexes show higher kinase activity than host cell CDK complexes with cellular cyclins and are resistant to cyclin-dependent inhibitory proteins (CDKIs). The crystal structure of human CDK6-Vcyclin in an active state was determined to 3.1 Angstrom resolution to get a better understanding of the structural basis of CDK6 activation by viral cyclins. The unphosphorylated CDK6 complexed to Vcyclin has many features characteristic of cyclinA-activated, phosphorylated CDK2. There are, however, differences in the conformation at the tip of the T-loop and its interactions with Vcyclin. Residues in the N-terminal extension of Vcyclin wrap around the tip of the CDK6 T-loop and form a short b-sheet with the T-loop backbone. These interactions lead to a 20 percent larger buried surface in the CDK6-Vcyclin interface than in the CDK2-cyclinA complex and are probably largely responsible for Vcyclin specificity for CDK6 and resistance of the complex to inhibition by INK-typeCDKIs.

  18. Molecular and Structural Basis of Inner Core Lipopolysaccharide Alterations in Escherichia coli

    PubMed Central

    Klein, Gracjana; Müller-Loennies, Sven; Lindner, Buko; Kobylak, Natalia; Brade, Helmut; Raina, Satish

    2013-01-01

    It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn2+. PMID:23372159

  19. Energetic and structural basis for activation of the epithelial sodium channel by matriptase.

    PubMed

    Kota, Pradeep; García-Caballero, Agustin; Dang, Hong; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2012-04-24

    Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors, and other proteins synthesized as inactive precursors. Serine proteases make up one of the largest and most conserved families of endopeptidases involved in diverse cellular activities, including wound healing, blood coagulation, and immune responses. Heteromeric α,β,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle's syndrome are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for the enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that has previously not been identified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. On the basis of our results, we propose a dynamics-driven "anglerfish" mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase than by furin. PMID:22471557

  20. Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC.

    PubMed

    Lee, Chul-Jin; Liang, Xiaofei; Gopalaswamy, Ramesh; Najeeb, Javaria; Ark, Eugene D; Toone, Eric J; Zhou, Pei

    2014-01-17

    The LpxC enzyme in the lipid A biosynthetic pathway is one of the most promising and clinically unexploited antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Progress in medicinal chemistry has led to the discovery of potent LpxC inhibitors with a variety of chemical scaffolds and distinct antibiotic profiles. The vast majority of these compounds, including the nanomolar inhibitors L-161,240 and BB-78485, are highly effective in suppressing the activity of Escherichia coli LpxC (EcLpxC) but not divergent orthologs such as Pseudomonas aeruginosa LpxC (PaLpxC) in vitro. The molecular basis for such promiscuous inhibition of EcLpxC has remained poorly understood. Here, we report the crystal structure of EcLpxC bound to L-161,240, providing the first molecular insight into L-161,240 inhibition. Additionally, structural analysis of the EcLpxC/L-161,240 complex together with the EcLpxC/BB-78485 complex reveals an unexpected backbone flipping of the Insert I βa-βb loop in EcLpxC in comparison with previously reported crystal structures of EcLpxC complexes with l-threonyl-hydroxamate-based broad-spectrum inhibitors. Such a conformational switch, which has only been observed in EcLpxC but not in divergent orthologs such as PaLpxC, results in expansion of the active site of EcLpxC, enabling it to accommodate LpxC inhibitors with a variety of head groups, including compounds containing single (R- or S-enantiomers) or double substitutions at the neighboring Cα atom of the hydroxamate warhead group. These results highlight the importance of understanding inherent conformational plasticity of target proteins in lead optimization. PMID:24117400

  1. Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

    PubMed

    Modak, Joyanta K; Rut, Wioletta; Wijeyewickrema, Lakshmi C; Pike, Robert N; Drag, Marcin; Roujeinikova, Anna

    2016-02-01

    The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

  2. Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram*

    PubMed Central

    Galkin, Andrey; Kulakova, Liudmila; Lim, Kap; Chen, Catherine Z.; Zheng, Wei; Turko, Illarion V.; Herzberg, Osnat

    2014-01-01

    Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing. PMID:24558036

  3. Structural basis for the coupling between activation and inactivation gates in K+ channels

    PubMed Central

    Cuello, Luis G.; Jogini, Vishwanath; Cortes, D. Marien.; Pan, Albert C; Gagnon, Dominique G.; Dalmas, Olivier; Cordero-Morales, Julio F.; Chakrapani, Sudha; Roux, Benoit; Perozo, Eduardo

    2011-01-01

    The coupled interplay between activation and inactivation gating is a functional hallmark of K+ channels1,2. This coupling has been experimentally demonstrated from ion interaction effects3,4, cysteine accessibility1 and is associated with a well-defined boundary of energetically coupled residues2. The structure of KcsA in its fully open conformation, as well as four other partial openings, richly illustrates the structural basis of activation-inactivation gating5. Here, we have identified the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that as a consequence of the hinge bending and rotation of TM2, the aromatic ring of Phe103 tilts towards residues Thr74 and Thr75 in the pore helix as well as Ile100 in the neighboring subunit. This allows the network of hydrogen bonds among residues W67, E71, and D80 to destabilize the selectivity filter6,7, facilitating entry to its non-conductive conformation. Mutations at position 103, affect gating kinetics in a size-dependent way: small side chain substitutions F103A and F103C severely impair inactivation kinetics, while larger side chains (F103W) have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open state interaction-energies between Phe103 and surrounding residues. Similar interactions were probed in the Shaker K-channel where inactivation was impaired in the mutant I470A. We propose that side chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K channels. PMID:20613845

  4. Structural basis of importin-α-mediated nuclear transport for Ku70 and Ku80.

    PubMed

    Takeda, Agnes A S; de Barros, Andrea C; Chang, Chiung-Wen; Kobe, Boštjan; Fontes, Marcos R M

    2011-09-16

    Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.

  5. Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

    PubMed

    Modak, Joyanta K; Rut, Wioletta; Wijeyewickrema, Lakshmi C; Pike, Robert N; Drag, Marcin; Roujeinikova, Anna

    2016-02-01

    The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases. PMID:26616008

  6. Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

    SciTech Connect

    Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric; Pioszak, Augen A.

    2012-05-09

    The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides. The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.

  7. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  8. Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment.

    PubMed Central

    Coulombe, R; Grochulski, P; Sivaraman, J; Ménard, R; Mort, J S; Cygler, M

    1996-01-01

    Cathepsin L is a member of the papain superfamily of cysteine proteases and, like many other proteases, it is synthesized as an inactive proenzyme. Its prosegment shows little homology to that of procathepsin B, whose structure, the first for a cysteine protease proenzyme, has been determined recently. We report here the 3-D structure of a mutant of human procathepsin L determined at 2.2 A resolution, describe the mode of binding employed by the prosegment and discuss the molecular basis for other possible roles of the prosegment. The N-terminal part of the prosegment is globular and contains three alpha-helices with a small hydrophobic core built around aromatic side chains. This domain packs against a loop on the enzyme's surface, with the aromatic side chain from the prosegment being located in the center of this loop and providing a large contact area. The C-terminal portion of the prosegment assumes an extended conformation and follows along the substrate binding cleft toward the N-terminus of the mature enzyme. The direction of the prosegment in the substrate binding cleft is opposite to that of substrates. The previously described role of the prosegment in the interactions with membranes is supported by the structure of its N-terminal domain. The fold of the prosegment and the mechanism by which it inhibits the enzymatic activity of procathepsin L is similar to that observed in procathepsin B despite differences in length and sequence, suggesting that this mode of inhibition is common to all enzymes from the papain superfamily. Images PMID:8896443

  9. Structural Basis of Substrate Recognition in Human Nicotinamide N-Methyltransferase

    SciTech Connect

    Peng, Yi; Sartini, Davide; Pozzi, Valentina; Wilk, Dennis; Emanuelli, Monica; Yee, Vivien C.

    2012-05-02

    Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7 {angstrom} resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k{sub cat}/K{sub m} decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K{sub m} for both NCA and AdoMet and modestly decreased k{sub cat}. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K{sub m}(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

  10. Structural Basis for the Rescue of Stalled Ribosomes: Structure of YaeJ Bound to the Ribosome

    SciTech Connect

    Gagnon, Matthieu G.; Seetharaman, Sai V.; Bulkley, David; Steitz, Thomas A.

    2012-06-19

    In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA{sub i}{sup fMet} and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.

  11. The higher-order structure in the cells nucleus as the structural basis of the post-mitotic state.

    PubMed

    Aranda-Anzaldo, Armando; Dent, Myrna A R; Martínez-Gómez, Alejandro

    2014-05-01

    In metazoan cells during the interphase nuclear DNA is organized in supercoiled, topologically constrained loops anchored to a proteinaceous compartment or substructure commonly known as the nuclear matrix (NM). The DNA-NM interactions result from a thermodynamically-driven process leading to the necessary dissipation of structural stress along chromosomal DNA, otherwise the chromosomes would break into pieces. Such DNA-NM interactions define a nuclear higher-order structure that is independent of chromatin proteins. On the other hand, a metazoan cell no longer able to undergo mitosis is defined as post-mitotic and this condition indicates a terminally differentiated cell that may survive in such a state for indefinite time. The non-reversible nature of the post-mitotic state suggests a non-genetic basis for it since no spontaneous or induced mutations can revert it. Yet in individual cells the loss of proliferative potential has both a developmental and a stochastic component. Here we discuss evidence suggesting that the stability of the nuclear higher-order structure is the factor that links the stochastic and developmental components leading to the post-mitotic state. PMID:24556025

  12. Optical-structural machine analysis of heterogeneous materials as a basis for forming its physical mechanical properties

    NASA Astrophysics Data System (ADS)

    Ulianov, Eduard I.; Ivanov, Konstantin M.

    1994-01-01

    The microstructural measurements method was realized on the basis of the information-gage complex `the scanning microscope -- the desk-top computer.' The microstructural measurements method is based on analysis of the structure's morphology with the help of the optical-structural machine analysis method and enables us to determine the information entropy of the structure and quantitative estimation of the morphology structures' well regulated degree.

  13. On the structural and mechanistic basis of function, classification, and ligand design for 5-HT receptors.

    PubMed

    Weinstein, H; Osman, R

    1990-01-01

    . The recognition site is considered to consist of two aromatic residues separated by a hydrophilic residue. In contrast to the model for 5-HT1A, the recognition is based on the interaction of neutral molecules and the stabilization is provided by dispersion forces. The resulting activation mechanism is based on a structural rearrangement. These detailed descriptions of elements in the ligand-receptor interactions at the two receptor subtypes lead to a new basis for rational design of receptor-selective compounds with predetermined efficacy.

  14. Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain

    PubMed Central

    Olsen, Shaun K.; Li, James Y.H.; Bromleigh, Carrie; Eliseenkova, Anna V.; Ibrahimi, Omar A.; Lao, Zhimin; Zhang, Fuming; Linhardt, Robert J.; Joyner, Alexandra L.; Mohammadi, Moosa

    2006-01-01

    Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the “c” splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the “b” isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8bF32A mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution. PMID:16384934

  15. Structural Basis by Which Alternative Splicing Modulates the Organizer Activity of FGF8 in the Brain

    SciTech Connect

    Olsen,S.; Li, J.; Eliseenkova, A.; Ibrahimi, O.; Lao, Z.; Zhang, F.; Linhardt, R.; Joyner, A.; Mohammadi, M.

    2006-01-01

    Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the 'c' splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the 'b' isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b{sup F32A} mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution.

  16. Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin.

    PubMed

    Lee, Kwangwoon; Alphonse, Sébastien; Piserchio, Andrea; Tavares, Clint D J; Giles, David H; Wellmann, Rebecca M; Dalby, Kevin N; Ghose, Ranajeet

    2016-09-01

    Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells. PMID:27499441

  17. The structural basis of germline-encoded VH3 immunoglobulin binding to staphylococcal protein A

    PubMed Central

    1993-01-01

    The ability of human VH3 immunoglobulins (Ig) to bind to staphylococcal protein A (SPA) via their Fab region is analogous to the binding of bacterial superantigens to T cell receptors. The present report establishes the structural basis for the interaction of SPA and VH3 Ig. We have studied a panel of 27 human monoclonal IgM that were derived from fetal B lymphocytes. As such, these IgM were expected to be encoded by unmutated germline genes. Binding to SPA in ELISA occurred with 15 of 15 VH3 IgM, but none of 12 IgM from the VH1, VH4, VH5, or VH6 families. The VH sequences of the 27 IgM were derived from 20 distinct VH elements, including 11 from the VH3 family. Use of D, JH, and CL genes was similar among VH3 and non-VH3 IgM. A comparison of the corresponding VH protein sequences, and those of previously studied IgM, identified a probable site for SPA binding that includes VH3 residues in framework region 3 (FR3), and perhaps FR1 and 3' complementary determining region 2. The results thus demonstrate that among human IgM, specificity for SPA is encoded by at least 11 different VH3 germline genes. Furthermore, like the T cell superantigens, SPA likely binds to residues in the VH framework region, outside the classical antigen-binding site of the hypervariable loops. PMID:8315388

  18. The structural basis of tail-anchored membrane protein recognition by Get3

    SciTech Connect

    Mateja, Agnieszka; Szlachcic, Anna; Downing, Maureen E.; Dobosz, Malgorzata; Mariappan, Malaiyalam; Hegde, Ramanujan S.; Keenan, Robert J.

    2009-10-05

    Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are 'tail-anchored' by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP {center_dot} AlF{sub 4}{sup -}-bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.

  19. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase*

    PubMed Central

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L.

    2015-01-01

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  20. “Smooth Muscle Cell Stiffness Syndrome”—Revisiting the Structural Basis of Arterial Stiffness

    PubMed Central

    Sehgel, Nancy L.; Vatner, Stephen F.; Meininger, Gerald A.

    2015-01-01

    In recent decades, the pervasiveness of increased arterial stiffness in patients with cardiovascular disease has become increasingly apparent. Though, this phenomenon has been well documented in humans and animal models of disease for well over a century, there has been surprisingly limited development in a deeper mechanistic understanding of arterial stiffness. Much of the historical literature has focused on changes in extracellular matrix proteins—collagen and elastin. However, extracellular matrix changes alone appear insufficient to consistently account for observed changes in vascular stiffness, which we observed in our studies of aortic stiffness in aging monkeys. This led us to examine novel mechanisms operating at the level of the vascular smooth muscle cell (VSMC)—that include increased cell stiffness and adhesion to extracellular matrix—which that may be interrelated with other mechanisms contributing to arterial stiffness. We introduce these observations as a new concept—the Smooth Muscle Cell Stiffness Syndrome (SMCSS)—within the field of arterial stiffness and posit that stiffening of vascular cells impairs vascular function and may contribute stiffening to the vasculature with aging and cardiovascular disease. Importantly, this review article revisits the structural basis of arterial stiffness in light of these novel findings. Such classification of SMCSS and its contextualization into our current understanding of vascular mechanics may be useful in the development of strategic therapeutics to directly target arterial stiffness. PMID:26635621

  1. Structural basis of synaptic vesicle assembly promoted by α-synuclein

    PubMed Central

    Fusco, Giuliana; Pape, Tillmann; Stephens, Amberley D.; Mahou, Pierre; Costa, Ana Rita; Kaminski, Clemens F.; Kaminski Schierle, Gabriele S.; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M.; De Simone, Alfonso

    2016-01-01

    α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this ‘double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts. PMID:27640673

  2. Structural design and analysis of a mixer pump for beyond-design- basis load

    SciTech Connect

    Rezvani, M.A.; Strehlow, J.P.; Baliga, R.; Kok, S.B.

    1994-03-01

    This paper presents the results of the structural evaluation of a mixer pump for a postulated drop accident. The mixer pump will be installed in a double-shell tank at the Hanford Site, near Richland, Washington. This tank has a 1,000,000-gallon (3,785,000 liter) capacity and is used to store radioactive waste before final disposal. The beyond-design-basis load case presented here is a postmulated drop of the pump during installation or removal. It is assumed that the pump assembly might be dropped approximateely 140 ft (15 m) from a height at which the bottom of the pump assembly is slightly above the top of the access riser to the bottom of the tank. The acceptance criterion for this load case is that the pump assembly shall not penetrate the primary tank liner. To ensure the integrity of the liner, the kinetic energy (developed in the pump drop) must be absorbed by some means to limit the impact force on the tank dome and thereby keep the pump from contacting the bottom of the tank. The limited clearance near the mounting assembly warranted an innovative two-step design of the energy absorbing system to limit the impact force on the tank dome to an acceptable value. This innovative design incorporates two energy absorbers in a unique series arrangement, one with the pump assembly and tile other in the pump pit.

  3. Structural Basis for Gating and Activation of RyR1.

    PubMed

    des Georges, Amédée; Clarke, Oliver B; Zalk, Ran; Yuan, Qi; Condon, Kendall J; Grassucci, Robert A; Hendrickson, Wayne A; Marks, Andrew R; Frank, Joachim

    2016-09-22

    The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.

  4. Structural Basis for Gating and Activation of RyR1.

    PubMed

    des Georges, Amédée; Clarke, Oliver B; Zalk, Ran; Yuan, Qi; Condon, Kendall J; Grassucci, Robert A; Hendrickson, Wayne A; Marks, Andrew R; Frank, Joachim

    2016-09-22

    The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain. PMID:27662087

  5. Structural basis of synaptic vesicle assembly promoted by α-synuclein.

    PubMed

    Fusco, Giuliana; Pape, Tillmann; Stephens, Amberley D; Mahou, Pierre; Costa, Ana Rita; Kaminski, Clemens F; Kaminski Schierle, Gabriele S; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M; De Simone, Alfonso

    2016-01-01

    α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this 'double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts. PMID:27640673

  6. Structural basis for cpSRP43 chromodomain selectivity and dynamics in Alb3 insertase interaction

    PubMed Central

    Horn, Annemarie; Hennig, Janosch; Ahmed, Yasar L.; Stier, Gunter; Wild, Klemens; Sattler, Michael; Sinning, Irmgard

    2015-01-01

    Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR). In contrast, high-throughput delivery of abundant light-harvesting chlorophyll a,b-binding proteins (LHCPs) in chloroplasts to the Alb3 insertase occurs post-translationally via a soluble transit complex including the cpSRP43/cpSRP54 heterodimer (cpSRP). Here we describe the molecular mechanisms of tethering cpSRP to the Alb3 insertase by specific interaction of cpSRP43 chromodomain 3 with a linear motif in the Alb3 C-terminal tail. Combining NMR spectroscopy, X-ray crystallography and biochemical analyses, we dissect the structural basis for selectivity of chromodomains 2 and 3 for their respective ligands cpSRP54 and Alb3, respectively. Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface. Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains. PMID:26568381

  7. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase.

    PubMed

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L

    2015-07-10

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  8. Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4[alpha

    SciTech Connect

    Lu, Peng; Rha, Geun Bae; Melikishvili, Manana; Wu, Guangteng; Adkins, Brandon C.; Fried, Michael G.; Chi, Young-In

    2010-11-09

    HNF4{alpha} (hepatocyte nuclear factor 4{alpha}) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic {beta}-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4{alpha} is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4{alpha} recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 {angstrom} crystal structure of human HNF4{alpha} DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1{alpha}, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4{alpha} molecular function can cause significant effects in afflicted MODY patients.

  9. Structural basis for the binding of tryptophan-based motifs by δ-COP

    PubMed Central

    Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

    2015-01-01

    Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

  10. Molecular Basis of Clay Mineral Structure and Dynamics in Subsurface Engineering Applications

    NASA Astrophysics Data System (ADS)

    Cygan, R. T.

    2015-12-01

    Clay minerals and their interfaces play an essential role in many geochemical, environmental, and subsurface engineering applications. Adsorption, dissolution, precipitation, nucleation, and growth mechanisms, in particular, are controlled by the interplay of structure, thermodynamics, kinetics, and transport at clay mineral-water interfaces. Molecular details of these processes are typically beyond the sensitivity of experimental and analytical methods, and therefore require accurate models and simulations. Also, basal surfaces and interlayers of clay minerals provide constrained interfacial environments to facilitate the evaluation of these complex processes. We have developed and used classical molecular and quantum methods to examine the complex behavior of clay mineral-water interfaces and dynamics of interlayer species. Bulk structures, swelling behavior, diffusion, and adsorption processes are evaluated and compared to experimental and spectroscopic findings. Analysis of adsorption mechanisms of radionuclides on clay minerals provides a scientific basis for predicting the suitability of engineered barriers associated with nuclear waste repositories and the fate of contaminants in the environment. Similarly, the injection of supercritical carbon dioxide into geological reservoirs—to mitigate the impact of climate change—is evaluated by molecular models of multi-fluid interactions with clay minerals. Molecular dynamics simulations provide insights into the wettability of different fluids—water, electrolyte solutions, and supercritical carbon dioxide—on clay surfaces, and which ultimately affects capillary fluid flow and the integrity of shale caprocks. This work is supported as part of Center for Frontiers of Subsurface Energy Security, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science and by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Geosciences Research Program

  11. Structural basis for the binding of tryptophan-based motifs by δ-COP.

    PubMed

    Suckling, Richard J; Poon, Pak Phi; Travis, Sophie M; Majoul, Irina V; Hughson, Frederick M; Evans, Philip R; Duden, Rainer; Owen, David J

    2015-11-17

    Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ'ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

  12. Structural Basis for WDR5 Interaction (Win) Motif Recognition in Human SET1 Family Histone Methyltransferases*

    PubMed Central

    Dharmarajan, Venkatasubramanian; Lee, Jeong-Heon; Patel, Anamika; Skalnik, David G.; Cosgrove, Michael S.

    2012-01-01

    Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies. PMID:22665483

  13. Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases.

    PubMed

    Dharmarajan, Venkatasubramanian; Lee, Jeong-Heon; Patel, Anamika; Skalnik, David G; Cosgrove, Michael S

    2012-08-10

    Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies. PMID:22665483

  14. Structural basis for androgen specificity and oestrogen synthesis in human aromatase

    SciTech Connect

    Ghosh, Debashis; Griswold, Jennifer; Erman, Mary; Pangborn, Walter

    2009-03-06

    Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer. In a three-step process, each step requiring 1 mol of O{sub 2}, 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16{alpha}-hydroxytestosterone to oestrone, 17{beta}-oestradiol and 17{beta},16{alpha}-oestriol, respectively. The first two steps are C19-methyl hydroxylation steps, and the third involves the aromatization of the steroid A-ring, unique to aromatase. Whereas most P450s are not highly substrate selective, it is the hallmark androgenic specificity that sets aromatase apart. The structure of this enzyme of the endoplasmic reticulum membrane has remained unknown for decades, hindering elucidation of the biochemical mechanism. Here we present the crystal structure of human placental aromatase, the only natural mammalian, full-length P450 and P450 in hormone biosynthetic pathways to be crystallized so far. Unlike the active sites of many microsomal P450s that metabolize drugs and xenobiotics, aromatase has an androgen-specific cleft that binds the androstenedione molecule snugly. Hydrophobic and polar residues exquisitely complement the steroid backbone. The locations of catalytically important residues shed light on the reaction mechanism. The relative juxtaposition of the hydrophobic amino-terminal region and the opening to the catalytic cleft shows why membrane anchoring is necessary for the lipophilic substrates to gain access to the active site. The molecular basis for the enzyme's androgenic specificity and unique catalytic mechanism can be used for developing next-generation aromatase inhibitors.

  15. Molecule-optimized basis sets and Hamiltonians for accelerated electronic structure calculations of atoms and molecules.

    PubMed

    Gidofalvi, Gergely; Mazziotti, David A

    2014-01-16

    Molecule-optimized basis sets, based on approximate natural orbitals, are developed for accelerating the convergence of quantum calculations with strongly correlated (multireferenced) electrons. We use a low-cost approximate solution of the anti-Hermitian contracted Schrödinger equation (ACSE) for the one- and two-electron reduced density matrices (RDMs) to generate an approximate set of natural orbitals for strongly correlated quantum systems. The natural-orbital basis set is truncated to generate a molecule-optimized basis set whose rank matches that of a standard correlation-consistent basis set optimized for the atoms. We show that basis-set truncation by approximate natural orbitals can be viewed as a one-electron unitary transformation of the Hamiltonian operator and suggest an extension of approximate natural-orbital truncations through two-electron unitary transformations of the Hamiltonian operator, such as those employed in the solution of the ACSE. The molecule-optimized basis set from the ACSE improves the accuracy of the equivalent standard atom-optimized basis set at little additional computational cost. We illustrate the method with the potential energy curves of hydrogen fluoride and diatomic nitrogen. Relative to the hydrogen fluoride potential energy curve from the ACSE in a polarized triple-ζ basis set, the ACSE curve in a molecule-optimized basis set, equivalent in size to a polarized double-ζ basis, has a nonparallelity error of 0.0154 au, which is significantly better than the nonparallelity error of 0.0252 au from the polarized double-ζ basis set.

  16. The structural basis of modified nucleosome recognition by 53BP1.

    PubMed

    Wilson, Marcus D; Benlekbir, Samir; Fradet-Turcotte, Amélie; Sherker, Alana; Julien, Jean-Philippe; McEwan, Andrea; Noordermeer, Sylvie M; Sicheri, Frank; Rubinstein, John L; Durocher, Daniel

    2016-08-01

    DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

  17. The study of basis sets for the calculation of the structure and dynamics of the benzene-Kr complex

    SciTech Connect

    Shirkov, Leonid; Makarewicz, Jan

    2015-05-28

    An ab initio intermolecular potential energy surface (PES) has been constructed for the benzene-krypton (BKr) van der Waals (vdW) complex. The interaction energy has been calculated at the coupled cluster level of theory with single, double, and perturbatively included triple excitations using different basis sets. As a result, a few analytical PESs of the complex have been determined. They allowed a prediction of the complex structure and its vibrational vdW states. The vibrational energy level pattern exhibits a distinct polyad structure. Comparison of the equilibrium structure, the dipole moment, and vibrational levels of BKr with their experimental counterparts has allowed us to design an optimal basis set composed of a small Dunning’s basis set for the benzene monomer, a larger effective core potential adapted basis set for Kr and additional midbond functions. Such a basis set yields vibrational energy levels that agree very well with the experimental ones as well as with those calculated from the available empirical PES derived from the microwave spectra of the BKr complex. The basis proposed can be applied to larger complexes including Kr because of a reasonable computational cost and accurate results.

  18. The study of basis sets for the calculation of the structure and dynamics of the benzene-Kr complex.

    PubMed

    Shirkov, Leonid; Makarewicz, Jan

    2015-05-28

    An ab initio intermolecular potential energy surface (PES) has been constructed for the benzene-krypton (BKr) van der Waals (vdW) complex. The interaction energy has been calculated at the coupled cluster level of theory with single, double, and perturbatively included triple excitations using different basis sets. As a result, a few analytical PESs of the complex have been determined. They allowed a prediction of the complex structure and its vibrational vdW states. The vibrational energy level pattern exhibits a distinct polyad structure. Comparison of the equilibrium structure, the dipole moment, and vibrational levels of BKr with their experimental counterparts has allowed us to design an optimal basis set composed of a small Dunning's basis set for the benzene monomer, a larger effective core potential adapted basis set for Kr and additional midbond functions. Such a basis set yields vibrational energy levels that agree very well with the experimental ones as well as with those calculated from the available empirical PES derived from the microwave spectra of the BKr complex. The basis proposed can be applied to larger complexes including Kr because of a reasonable computational cost and accurate results.

  19. The study of basis sets for the calculation of the structure and dynamics of the benzene-Kr complex.

    PubMed

    Shirkov, Leonid; Makarewicz, Jan

    2015-05-28

    An ab initio intermolecular potential energy surface (PES) has been constructed for the benzene-krypton (BKr) van der Waals (vdW) complex. The interaction energy has been calculated at the coupled cluster level of theory with single, double, and perturbatively included triple excitations using different basis sets. As a result, a few analytical PESs of the complex have been determined. They allowed a prediction of the complex structure and its vibrational vdW states. The vibrational energy level pattern exhibits a distinct polyad structure. Comparison of the equilibrium structure, the dipole moment, and vibrational levels of BKr with their experimental counterparts has allowed us to design an optimal basis set composed of a small Dunning's basis set for the benzene monomer, a larger effective core potential adapted basis set for Kr and additional midbond functions. Such a basis set yields vibrational energy levels that agree very well with the experimental ones as well as with those calculated from the available empirical PES derived from the microwave spectra of the BKr complex. The basis proposed can be applied to larger complexes including Kr because of a reasonable computational cost and accurate results. PMID:26026434

  20. 1Energetic and structural basis for activation of the epithelial sodium channel by matriptase

    PubMed Central

    Kota, Pradeep; García-Caballero, Agustin; Dang, Hong; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2012-01-01

    Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors and other proteins synthesized as inactive precursors. Serine proteases are one of the largest and conserved families of endopeptidases involved in diverse cellular activities including wound healing, blood coagulation and immune responses. Heteromeric α,β,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle’s syndrome, are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/Channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that is previously unidentified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC, and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. Based on our results, we propose a dynamics-driven “anglerfish” mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase compared to furin. PMID:22471557

  1. Structural Basis for Multiple Sugar Recognition of Jacalin-related Human ZG16p Lectin*

    PubMed Central

    Kanagawa, Mayumi; Liu, Yan; Hanashima, Shinya; Ikeda, Akemi; Chai, Wengang; Nakano, Yukiko; Kojima-Aikawa, Kyoko; Feizi, Ten; Yamaguchi, Yoshiki

    2014-01-01

    ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1–3Man and Glcβ1–3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using 15N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition. PMID:24790092

  2. Structural Basis for Degenerate Recognition of Natural HIV Peptide Variants by Cytotoxic Lymphocytes

    SciTech Connect

    Martinez-Hackert,E.; Anikeeva, N.; Kalams, S.; Walker, B.; Hendrickson, W.; Sykulev, Y.

    2006-01-01

    It is well established that even small changes in amino acid side chains of antigenic peptide bound to MHC protein may completely abrogate recognition of the peptide-MHC (pMHC) complex by the T-cell receptor (TCR). Often, however, several non-conservative substitutions in the peptide antigen are accommodated and do not impair its recognition by TCR. For example, a preponderance of natural sequence variants of the HIV p17 Gag-derived peptide SLYNTVATL (SL9) are recognized by cytotoxic T lymphocytes (CTL), which implies that interactions with SL9 variants are degenerate both with respect to the class I MHC molecule and with respect to TCR. Here we study the molecular basis for this degenerate recognition of SL9 variants. We show that several SL9 variants bind comparably well to soluble HLA-A2 and to a particular soluble TCR and that these variants are active in the cognate cytotoxicity assay. Natural SL9 variation is restricted by its context in the HIV p17 matrix protein, and we have used synthetic variants to explore the wider spectrum of recognition. High-resolution crystal structures of seven selected SL9 variants bound to HLA-A2 all have remarkably similar peptide conformations and side-chain dispositions outside sites of substitution. This preservation of the peptide conformation despite epitope variations suggests a mechanism for the observed degeneracy in pMHC recognition by TCR, and may contribute to the persistence of SL9-mediated immune responses in chronically infected individuals.

  3. "LPO Lite" : Representing Lattice Preferred Orientation and its Evolution Using Structured Basis Functions

    NASA Astrophysics Data System (ADS)

    Ribe, N. M.; Castelnau, O.

    2008-12-01

    Current methods for calculating the evolution of flow-induced seismic anisotropy in the upper mantle describe Lattice Preferred Orientation (LPO) using ensembles of 103-104 individual grains, and are nowaday too computationally expensive to be incorporated into three-dimensional time-dependent convection models. We propose a much faster (by a factor ~ 103) method wherein LPO is described by a small number of 'structured basis functions' (SBFs.) The number of SBFs required is equal to the number of active slip systems (= 3 for olivine), and each SBF represents the 'virtual' LPO that would be produced by the action of just one of those systems. Analytical expressions for the SBFs are obtained using a simple 'single-slip' (SS) model, and are then tested against the predictions of the second-order (SO) self-consistent model of Ponte-Castaneda (J. Mech. Phys. Solids 50, 737-757, 2002) in which several slip systems act simultaneously. Remarkably, the SS model reproduces exactly (99.9% variance reduction) the orientation- dependence of the slip rate ·γ predicted by the SO model for each active slip system, once the overall amplitude of the SS expression for ·γ has been determined by least-squares fitting to the SO prediction. Having thus demonstrated that the analytical SBFs are physically realistic, we develop a scheme for representing an arbitrary LPO as a superposition of the SBFs and for determining the evolution equations satisfied by the expansion coefficients. We illustrate the method both for simple uniform deformations (uniaxial compression, simple shear) and for more geophysically realistic nonuniform deformation histories.

  4. Structural and energetic basis of protein kinetic destabilization in human phosphoglycerate kinase 1 deficiency.

    PubMed

    Pey, Angel L; Mesa-Torres, Noel; Chiarelli, Laurent R; Valentini, Giovanna

    2013-02-19

    Protein kinetic destabilization is a common feature of many human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency is a rare genetic disease caused by mutations in the PGK1 protein, which often shows reduced kinetic stability. In this work, we have performed an in-depth characterization of the thermal stability of the wild type and four disease-causing mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1 thermal denaturation is a process under kinetic control, and it is described well by a two-state irreversible denaturation model. Kinetic analysis of differential scanning calorimetry profiles shows that the disease-causing mutations decrease PGK1 kinetic stability from ~5-fold (E252A) to ~100000-fold (L89P) compared to that of wild-type PGK1, and in some cases, mutant enzymes are denatured on a time scale of a few minutes at physiological temperature. We show that changes in protein kinetic stability are associated with large differences in enthalpic and entropic contributions to denaturation free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may have important implications for the development of native state kinetic stabilizers for the treatment of this disease. PMID:23336698

  5. Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

    SciTech Connect

    Critton, D.; Tortajada, A; Stetson, G; Peti, W; Page, R

    2008-01-01

    Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPs as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.

  6. The construction of graph models for calculations of the properties of substitution isomers of basis structures on the basis of additivity of energy contributions

    NASA Astrophysics Data System (ADS)

    Nilov, D. Yu.; Smolyakov, V. M.

    2012-05-01

    A method for the construction of additive models for calculations of the properties of substitution isomers of basis structures is described for the example of a series of X-substituted methylsilanes CH3 - k X k -SiH3 - l X l (where X = CH3, F, Cl, …, k, l = 0, 1, 2, 3). The method is based on similarity of subgraphs in graphs of several molecules and the arrangement of polygonal numbers (triangular, tetrahedral) of the Pascal triangle. Parameters taking into account multiple nonvalence interactions (-C-Si<, >C-Si<, …) through two atoms along the molecular chain of an X-substituted methylsilane (X = CH3) were for the first time explicitly included in the calculation scheme. Taking these interactions into account allows us to completely differentiate all the structural isomers of certain molecules and obtain numerical parameter values for predicting properties P under consideration in various approximations. Numerical calculations of Δf H {g,298/K o} were performed for 16 alkylsilanes (as X-substituted methylsilanes), including 7 compounds not studied experimentally.

  7. Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2.

    PubMed

    Wang, Yuhong; Zhang, Mei; Xu, Yu; Jiang, Min; Zankov, Dimitar P; Cui, Meng; Tseng, Gea-Ny

    2012-12-01

    KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a "repolarization reserve" in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics. PMID:23183700

  8. Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2.

    PubMed

    Wang, Yuhong; Zhang, Mei; Xu, Yu; Jiang, Min; Zankov, Dimitar P; Cui, Meng; Tseng, Gea-Ny

    2012-12-01

    KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a "repolarization reserve" in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.

  9. On the feasibility of ab initio electronic structure calculations for Cu using a single s orbital basis

    SciTech Connect

    Hegde, Ganesh Bowen, R. Chris

    2015-10-15

    The accuracy of a single s-orbital representation of Cu towards enabling multi-thousand atom ab initio calculations of electronic structure is evaluated in this work. If an electrostatic compensation charge of 0.3 electron per atom is used in this basis representation, the electronic transmission in bulk and nanocrystalline Cu can be made to compare accurately to that obtained with a Double Zeta Polarized basis set. The use of this representation is analogous to the use of single band effective mass representation for semiconductor electronic structure. With a basis of just one s-orbital per Cu atom, the representation is extremely computationally efficient and can be used to provide much needed ab initio insight into electronic transport in nanocrystalline Cu interconnects at realistic dimensions of several thousand atoms.

  10. The Structural Basis of Action of Vanadyl (VO(2+)) Chelates in Cells.

    PubMed

    Makinen, Marvin W; Salehitazangi, Marzieh

    2014-11-01

    Much emphasis has been given to vanadium compounds as potential therapeutic reagents for the treatment of diabetes mellitus. Thus far, no vanadium compound has proven efficacious for long-term treatment of this disease in humans. Therefore, in review of the research literature, our goal has been to identify properties of vanadium compounds that are likely to favor physiological and biochemical compatibility for further development as therapeutic reagents. We have, therefore, limited our review to those vanadium compounds that have been used in both in vivo experiments with small, laboratory animals and in in vitro studies with primary or cultured cell systems and for which pharmacokinetic and pharmacodynamics results have been reported, including vanadium tissue content, vanadium and ligand lifetime in the bloodstream, structure in solution, and interaction with serum transport proteins. Only vanadyl (VO(2+)) chelates fulfill these requirements despite the large variety of vanadium compounds of different oxidation states, ligand structure, and coordination geometry synthesized as potential therapeutic agents. Extensive review of research results obtained with use of organic VO(2+)-chelates shows that the vanadyl chelate bis(acetylacetonato)oxidovanadium(IV) [hereafter abbreviated as VO(acac)2], exhibits the greatest capacity to enhance insulin receptor kinase activity in cells compared to other organic VO(2+)-chelates, is associated with a dose-dependent capacity to lower plasma glucose in diabetic laboratory animals, and exhibits a sufficiently long lifetime in the blood stream to allow correlation of its dose-dependent action with blood vanadium content. The properties underlying this behavior appear to be its high stability and capacity to remain intact upon binding to serum albumin. We relate the capacity to remain intact upon binding to serum albumin to the requirement to undergo transcytosis through the vascular endothelium to gain access to target tissues in

  11. The Structural Basis of Action of Vanadyl (VO2+) Chelates in Cells

    PubMed Central

    Makinen, Marvin W.; Salehitazangi, Marzieh

    2014-01-01

    Much emphasis has been given to vanadium compounds as potential therapeutic reagents for the treatment of diabetes mellitus. Thus far, no vanadium compound has proven efficacious for long-term treatment of this disease in humans. Therefore, in review of the research literature, our goal has been to identify properties of vanadium compounds that are likely to favor physiological and biochemical compatibility for further development as therapeutic reagents. We have, therefore, limited our review to those vanadium compounds that have been used in both in vivo experiments with small, laboratory animals and in in vitro studies with primary or cultured cell systems and for which pharmacokinetic and pharmacodynamics results have been reported, including vanadium tissue content, vanadium and ligand lifetime in the bloodstream, structure in solution, and interaction with serum transport proteins. Only vanadyl (VO2+) chelates fulfill these requirements despite the large variety of vanadium compounds of different oxidation states, ligand structure, and coordination geometry synthesized as potential therapeutic agents. Extensive review of research results obtained with use of organic VO2+-chelates shows that the vanadyl chelate bis(acetylacetonato)oxidovanadium(IV) [hereafter abbreviated as VO(acac)2], exhibits the greatest capacity to enhance insulin receptor kinase activity in cells compared to other organic VO2+-chelates, is associated with a dose-dependent capacity to lower plasma glucose in diabetic laboratory animals, and exhibits a sufficiently long lifetime in the blood stream to allow correlation of its dose-dependent action with blood vanadium content. The properties underlying this behavior appear to be its high stability and capacity to remain intact upon binding to serum albumin. We relate the capacity to remain intact upon binding to serum albumin to the requirement to undergo transcytosis through the vascular endothelium to gain access to target tissues in the

  12. Invertebrate muscles: thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

    PubMed Central

    Hooper, Scott L.; Hobbs, Kevin H.; Thuma, Jeffrey B.

    2008-01-01

    This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5 nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vetebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca++ binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved. PMID:18616971

  13. Homology modeling studies of yeast Mitogen-Activated Protein Kinases (MAPKS): structural motifs as a basis for specificity.

    PubMed

    Smith, D L; Nilar, S H

    2010-06-01

    Mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction. It is the objective of this communication to demonstrate that insight into protein-protein interactions in the Common Docking motif of yeast mitogen-activated protein kinases can be obtained based on homology models. Homology models for four yeast MAPKs, FUS3, KSS1, HOG1 and MPK1 were built based on the X-ray structures of active and inactive rat ERK2. The structural motifs required for the basis of specificity were rationalized based on these structures. PMID:19995338

  14. Conformational Variations of Both Phosphodiesterase-5 and Inhibitors Provide the Structural Basis for the Physiological Effects of Verdenafil and Sildenafil

    SciTech Connect

    Wang,H.; Ye, M.; Robinson, H.; Francis, S.; Ke, H.

    2008-01-01

    Vardenafil has higher affinity to phosphodiesterase-5 (PDE5) than sildenafil and lower administered dosage for the treatment of erectile dysfunction. However, the molecular basis for these differences is puzzling because two drugs have similar chemical structures. Reported here is a crystal structure of the fully active and nonmutated PDE5A1 catalytic domain in complex with vardenafil. The structure shows that the conformation of the H-loop in the PDE5A1-vardenafil complex is different from those of any known structures of the unliganded PDE5 and its complexes with the inhibitors. In addition, the molecular configuration of vardenafil differs from that of sildenafil when bound to PDE5. It is noteworthy that the binding of vardenafil causes loss of the divalent metal ions that have been observed in all the previously published PDE structures. The conformational variation of both PDE5 and the inhibitors provides structural insight into the different potencies of the drugs.

  15. Conformational Variations of Both Phosphodiesterase-5 and Inhibitors Provide the Structural Basis for the Physiological Effects of Vardenafil and Sildenafil

    SciTech Connect

    Wang, H.; Ye, M; Robinson, H; Fransis, S; Ke, H

    2007-01-01

    Vardenafil has higher affinity to phosphodiesterase-5 (PDE5) than sildenafil and lower administered dosage for the treatment of erectile dysfunction. However, the molecular basis for these differences is puzzling because two drugs have similar chemical structures. Reported here is a crystal structure of the fully active and nonmutated PDE5A1 catalytic domain in complex with vardenafil. The structure shows that the conformation of the H-loop in the PDE5A1-vardenafil complex is different from those of any known structures of the unliganded PDE5 and its complexes with the inhibitors. In addition, the molecular configuration of vardenafil differs from that of sildenafil when bound to PDE5. It is noteworthy that the binding of vardenafil causes loss of the divalent metal ions that have been observed in all the previously published PDE structures. The conformational variation of both PDE5 and the inhibitors provides structural insight into the different potencies of the drugs.

  16. Sequence–structure relationships in RNA loops: establishing the basis for loop homology modeling

    PubMed Central

    Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

    2010-01-01

    The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence–structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts. PMID:19923230

  17. Basis set convergence of CCSD(T) equilibrium geometries using a large and diverse set of molecular structures.

    PubMed

    Spackman, Peter R; Jayatilaka, Dylan; Karton, Amir

    2016-09-14

    We examine the basis set convergence of the CCSD(T) method for obtaining the structures of the 108 neutral first- and second-row species in the W4-11 database (with up to five non-hydrogen atoms). This set includes a total of 181 unique bonds: 75 H-X, 49 X-Y, 43 X=Y, and 14 X≡Y bonds (where X and Y are first- and second-row atoms). As reference values, geometries optimized at the CCSD(T)/aug'-cc-pV(6+d)Z level of theory are used. We consider the basis set convergence of the CCSD(T) method with the correlation consistent basis sets cc-pV(n+d)Z and aug'-cc-pV(n+d)Z (n = D, T, Q, 5) and the Weigend-Ahlrichs def2-n ZVPP basis sets (n = T, Q). For each increase in the highest angular momentum present in the basis set, the root-mean-square deviation (RMSD) over the bond distances is decreased by a factor of ∼4. For example, the following RMSDs are obtained for the cc-pV(n+d)Z basis sets 0.0196 (D), 0.0050 (T), 0.0015 (Q), and 0.0004 (5) Å. Similar results are obtained for the aug'-cc-pV(n+d)Z and def2-n ZVPP basis sets. The double-zeta and triple-zeta quality basis sets systematically and significantly overestimate the bond distances. A simple and cost-effective way to improve the performance of these basis sets is to scale the bond distances by an empirical scaling factor of 0.9865 (cc-pV(D+d)Z) and 0.9969 (cc-pV(T+d)Z). This results in RMSDs of 0.0080 (scaled cc-pV(D+d)Z) and 0.0029 (scaled cc-pV(T+d)Z) Å. The basis set convergence of larger basis sets can be accelerated via standard basis-set extrapolations. In addition, the basis set convergence of explicitly correlated CCSD(T)-F12 calculations is investigated in conjunction with the cc-pVnZ-F12 basis sets (n = D, T). Typically, one "gains" two angular momenta in the explicitly correlated calculations. That is, the CCSD(T)-F12/cc-pVnZ-F12 level of theory shows similar performance to the CCSD(T)/cc-pV(n+2)Z level of theory. In particular, the following RMSDs are obtained for the cc-pVnZ-F12 basis sets 0.0019 (D

  18. Basis set convergence of CCSD(T) equilibrium geometries using a large and diverse set of molecular structures.

    PubMed

    Spackman, Peter R; Jayatilaka, Dylan; Karton, Amir

    2016-09-14

    We examine the basis set convergence of the CCSD(T) method for obtaining the structures of the 108 neutral first- and second-row species in the W4-11 database (with up to five non-hydrogen atoms). This set includes a total of 181 unique bonds: 75 H-X, 49 X-Y, 43 X=Y, and 14 X≡Y bonds (where X and Y are first- and second-row atoms). As reference values, geometries optimized at the CCSD(T)/aug'-cc-pV(6+d)Z level of theory are used. We consider the basis set convergence of the CCSD(T) method with the correlation consistent basis sets cc-pV(n+d)Z and aug'-cc-pV(n+d)Z (n = D, T, Q, 5) and the Weigend-Ahlrichs def2-n ZVPP basis sets (n = T, Q). For each increase in the highest angular momentum present in the basis set, the root-mean-square deviation (RMSD) over the bond distances is decreased by a factor of ∼4. For example, the following RMSDs are obtained for the cc-pV(n+d)Z basis sets 0.0196 (D), 0.0050 (T), 0.0015 (Q), and 0.0004 (5) Å. Similar results are obtained for the aug'-cc-pV(n+d)Z and def2-n ZVPP basis sets. The double-zeta and triple-zeta quality basis sets systematically and significantly overestimate the bond distances. A simple and cost-effective way to improve the performance of these basis sets is to scale the bond distances by an empirical scaling factor of 0.9865 (cc-pV(D+d)Z) and 0.9969 (cc-pV(T+d)Z). This results in RMSDs of 0.0080 (scaled cc-pV(D+d)Z) and 0.0029 (scaled cc-pV(T+d)Z) Å. The basis set convergence of larger basis sets can be accelerated via standard basis-set extrapolations. In addition, the basis set convergence of explicitly correlated CCSD(T)-F12 calculations is investigated in conjunction with the cc-pVnZ-F12 basis sets (n = D, T). Typically, one "gains" two angular momenta in the explicitly correlated calculations. That is, the CCSD(T)-F12/cc-pVnZ-F12 level of theory shows similar performance to the CCSD(T)/cc-pV(n+2)Z level of theory. In particular, the following RMSDs are obtained for the cc-pVnZ-F12 basis sets 0.0019 (D

  19. Basis set convergence of CCSD(T) equilibrium geometries using a large and diverse set of molecular structures

    NASA Astrophysics Data System (ADS)

    Spackman, Peter R.; Jayatilaka, Dylan; Karton, Amir

    2016-09-01

    We examine the basis set convergence of the CCSD(T) method for obtaining the structures of the 108 neutral first- and second-row species in the W4-11 database (with up to five non-hydrogen atoms). This set includes a total of 181 unique bonds: 75 H—X, 49 X—Y, 43 X=Y, and 14 X≡Y bonds (where X and Y are first- and second-row atoms). As reference values, geometries optimized at the CCSD(T)/aug'-cc-pV(6+d)Z level of theory are used. We consider the basis set convergence of the CCSD(T) method with the correlation consistent basis sets cc-pV(n+d)Z and aug'-cc-pV(n+d)Z (n = D, T, Q, 5) and the Weigend-Ahlrichs def2-n ZVPP basis sets (n = T, Q). For each increase in the highest angular momentum present in the basis set, the root-mean-square deviation (RMSD) over the bond distances is decreased by a factor of ˜4. For example, the following RMSDs are obtained for the cc-pV(n+d)Z basis sets 0.0196 (D), 0.0050 (T), 0.0015 (Q), and 0.0004 (5) Å. Similar results are obtained for the aug'-cc-pV(n+d)Z and def2-n ZVPP basis sets. The double-zeta and triple-zeta quality basis sets systematically and significantly overestimate the bond distances. A simple and cost-effective way to improve the performance of these basis sets is to scale the bond distances by an empirical scaling factor of 0.9865 (cc-pV(D+d)Z) and 0.9969 (cc-pV(T+d)Z). This results in RMSDs of 0.0080 (scaled cc-pV(D+d)Z) and 0.0029 (scaled cc-pV(T+d)Z) Å. The basis set convergence of larger basis sets can be accelerated via standard basis-set extrapolations. In addition, the basis set convergence of explicitly correlated CCSD(T)-F12 calculations is investigated in conjunction with the cc-pVnZ-F12 basis sets (n = D, T). Typically, one "gains" two angular momenta in the explicitly correlated calculations. That is, the CCSD(T)-F12/cc-pVnZ-F12 level of theory shows similar performance to the CCSD(T)/cc-pV(n+2)Z level of theory. In particular, the following RMSDs are obtained for the cc-pVnZ-F12 basis sets 0

  20. Use of Structure as a Basis for Abstraction in Air Traffic Control

    NASA Technical Reports Server (NTRS)

    Davison, Hayley J.; Hansman, R. John

    2004-01-01

    The safety and efficiency of the air traffic control domain is highly dependent on the capabilities and limitations of its human controllers. Past research has indicated that structure provided by the airspace and procedures could aid in simplifying the controllers cognitive tasks. In this paper, observations, interviews, voice command data analyses, and radar analyses were conducted at the Boston Terminal Route Control (TRACON) facility to determine if there was evidence of controllers using structure to simplify their cognitive processes. The data suggest that controllers do use structure-based abstractions to simplify their cognitive processes, particularly the projection task. How structure simplifies the projection task and the implications of understanding the benefits structure provides to the projection task was discussed.

  1. Structural Basis for Antagonism by Suramin of Heparin Binding to Vaccinia Complement Protein

    SciTech Connect

    Ganesh, Vannakambadi K.; Muthuvel, Suresh Kumar; Smith, Scott A.; Kotwal, Girish J.; Murthy, Krishna H.M.

    2010-07-19

    Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.

  2. On the use of a hierarchical multi-level building block basis function scheme in periodic plasmonic structures

    NASA Astrophysics Data System (ADS)

    Zheng, X.; Valev, V. K.; Volskiy, V.; Vandenbosch, Guy A. E.; Moshchalkov, V. V.

    2014-05-01

    A Volumetric Method of Moments algorithm is applied to predict the plasmonic responses of chiral metamaterials. This algorithm is based on the use of a multi-level building block basis function scheme, in combination with a dedicated Kummer transformation in the calculation of periodic Green's functions. The validity of the algorithm is demonstrated by analyzing a Ninja Star periodic structure. A good agreement can be found between simulation and experiment.

  3. Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin

    PubMed Central

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Yokoyama, Shigeyuki; Shirouzu, Mikako

    2016-01-01

    The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs. PMID:27647526

  4. Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin.

    PubMed

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Yokoyama, Shigeyuki; Shirouzu, Mikako

    2016-01-01

    The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs. PMID:27647526

  5. Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

    PubMed

    Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-04-01

    Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

  6. Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters.

    PubMed

    Coincon, Mathieu; Uzdavinys, Povilas; Nji, Emmanuel; Dotson, David L; Winkelmann, Iven; Abdul-Hussein, Saba; Cameron, Alexander D; Beckstein, Oliver; Drew, David

    2016-03-01

    To fully understand the transport mechanism of Na(+)/H(+) exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog GltPh. Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na(+)/H(+) antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na(+)/H(+) antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions. PMID:26828964

  7. Structural basis of substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

    PubMed Central

    Yates, Susan P.; Edwards, Thomas E.; Bryan, Cassie M.; Stein, Adam J.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.; Zheng, Jimin; Jia, Zongchao

    2012-01-01

    Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). Based on the recent complex structure of AceK-ICDH from E. coli, we have classified the structures of homodimeric NADP+-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH which exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria. PMID:21870819

  8. Structural basis for cooperative oxygen binding and bracelet-assisted assembly of Lumbricus terrestris hemoglobin.

    PubMed

    Chen, Wei-Ting; Chen, Yu-Chuen; Liou, Horng-Huei; Chao, Chih-Yu

    2015-01-01

    The iron-containing hemoglobins (Hbs) are essential proteins to serve as oxygen transporters in the blood. Among various kinds of Hbs, the earthworm Hbs are the champions in carrying oxygen due to not only their large size but also the unusually high cooperativity of ligand binding. However, the cooperative oxygen binding mechanisms are still mostly unknown. Here we report the cryo-electron microscopy structure of Lumbricus terrestris Hb in its native, oxygenated state at 9.1 Å resolution, showing remarkable differences from the carbon monoxide-binding X-ray structure. Our structural analysis first indicates that the cooperative ligand binding of L. terrestris Hb requires tertiary and quaternary transitions in the heme pocket and a global subunit movement facilitated by intra-ring and inter-ring contacts. Moreover, the additional sinusoidal bracelet provides the confirmation for the long-standing debate about the additional electron densities absent in the X-ray crystal structure. PMID:25897633

  9. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.

    PubMed

    Li, Xin; Beeson, William T; Phillips, Christopher M; Marletta, Michael A; Cate, Jamie H D

    2012-06-01

    The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear. Here, we present structures of Neurospora crassa PMO-2 and PMO-3 at 1.10 and 1.37 Å resolution, respectively. In the structures, dioxygen species are found in the active sites, consistent with the proposed cleavage mechanism. Structural and sequence comparisons between PMOs also reveal that the enzyme substrate-binding surfaces contain highly varied aromatic amino acid and glycosylation positions. The structures reported here provide evidence for a wide range of PMO substrate recognition patterns in the plant cell wall, including binding modes that traverse multiple glucan chains. PMID:22578542

  10. Structural Basis of Allele Variation of Human Thiopurine-S-Methyltransferase

    PubMed Central

    Wu, Hong; Horton, John R.; Battaile, Kevin; Allali-Hassani, Abdellah; Martin, Fernando; Zeng, Hong; Loppnau, Peter; Vedadi, Masoud; Bochkarev, Alexey; Plotnikov, Alexander N.; Cheng, Xiaodong

    2009-01-01

    Human thiopurine S-methyltransferase (TPMT) exhibits considerable person-to-person variation in activity to thiopurine drugs. We have produced an N-terminal truncation of human TPMT protein, crystallized the protein in complex with the methyl donor product S-adenosyl-L-homocysteine, and determined the atomic structure to the resolution of 1.58 and 1.89 Å, respectively, for the seleno-methionine incorporated and wild type proteins. The structure of TPMT indicates that the naturally occurring amino acid polymorphisms scatter throughout the structure, and that the amino acids whose alteration have the most influence on function are those that form intra-molecular stabilizing interactions (mainly van der Waals contacts). Furthermore, we have produced four TPMT mutant proteins containing variant alleles of TPMT*2, *3A, *3B, and *3C and examined the structure-function relationship of the mutant proteins based on their expression and solubility in bacteria and their thermostability profile. PMID:17243178

  11. Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

    PubMed

    Petrou, Vasileios I; Herrera, Carmen M; Schultz, Kathryn M; Clarke, Oliver B; Vendome, Jérémie; Tomasek, David; Banerjee, Surajit; Rajashankar, Kanagalaghatta R; Belcher Dufrisne, Meagan; Kloss, Brian; Kloppmann, Edda; Rost, Burkhard; Klug, Candice S; Trent, M Stephen; Shapiro, Lawrence; Mancia, Filippo

    2016-02-01

    Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.

  12. Structural basis for cooperative oxygen binding and bracelet-assisted assembly of Lumbricus terrestris hemoglobin.

    PubMed

    Chen, Wei-Ting; Chen, Yu-Chuen; Liou, Horng-Huei; Chao, Chih-Yu

    2015-04-21

    The iron-containing hemoglobins (Hbs) are essential proteins to serve as oxygen transporters in the blood. Among various kinds of Hbs, the earthworm Hbs are the champions in carrying oxygen due to not only their large size but also the unusually high cooperativity of ligand binding. However, the cooperative oxygen binding mechanisms are still mostly unknown. Here we report the cryo-electron microscopy structure of Lumbricus terrestris Hb in its native, oxygenated state at 9.1 Å resolution, showing remarkable differences from the carbon monoxide-binding X-ray structure. Our structural analysis first indicates that the cooperative ligand binding of L. terrestris Hb requires tertiary and quaternary transitions in the heme pocket and a global subunit movement facilitated by intra-ring and inter-ring contacts. Moreover, the additional sinusoidal bracelet provides the confirmation for the long-standing debate about the additional electron densities absent in the X-ray crystal structure.

  13. Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose.

    PubMed

    Bourne, Yves; Astoul, Corinne Houlès; Zamboni, Véronique; Peumans, Willy J; Menu-Bouaouiche, Laurence; Van Damme, Els J M; Barre, Annick; Rougé, Pierre

    2002-05-15

    Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galbeta1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin-alpha-methyl-mannose complex at 2 A resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the beta-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities to a single lectin.

  14. Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose.

    PubMed Central

    Bourne, Yves; Astoul, Corinne Houlès; Zamboni, Véronique; Peumans, Willy J; Menu-Bouaouiche, Laurence; Van Damme, Els J M; Barre, Annick; Rougé, Pierre

    2002-01-01

    Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galbeta1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin-alpha-methyl-mannose complex at 2 A resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the beta-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities to a single lectin. PMID:11988090

  15. Structural Basis of Inhibition of the Human NAD+ -Dependent Deacetylase SIRT5 by Suramin

    SciTech Connect

    Schuetz,A.; Min, J.; Antoshenko, T.; Wang, C.; Allali-Hassani, A.; Dong, A.; Loppnau, P.; vedadi, M.; Bochkarev, A.; et al.

    2007-01-01

    Sirtuins are NAD+-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD+-dependent deacetylase activity with an IC50 value of 22 {mu}M. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD+, the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.

  16. Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein.

    PubMed

    Ohkubo, Takayuki; Tamiya, Minoru; Abe, Keiko; Ishiguro, Masaji

    2015-01-01

    Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness. PMID:26010443

  17. Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein

    PubMed Central

    Ohkubo, Takayuki; Tamiya, Minoru; Abe, Keiko; Ishiguro, Masaji

    2015-01-01

    Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness. PMID:26010443

  18. The structural basis of transferrin sequestration by transferrin-binding protein B

    SciTech Connect

    Calmettes, Charles; Alcantara, Joenel; Yu, Rong-Hua; Schryvers, Anthony B.; Moraes, Trevor F.

    2012-03-28

    Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin during infection through a surface transferrin receptor system composed of proteins TbpA and TbpB. Here we present the crystal structures of TbpB from N. meningitidis in its apo form and in complex with human transferrin. The structure reveals how TbpB sequesters and initiates iron release from human transferrin.

  19. Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease

    PubMed Central

    DuBois, Rebecca M.; Slavish, P. Jake; Baughman, Brandi M.; Yun, Mi-Kyung; Bao, Ju; Webby, Richard J.; Webb, Thomas R.; White, Stephen W.

    2012-01-01

    Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors. PMID:22876176

  20. Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.

    PubMed

    Welin, Martin; Wang, Liya; Eriksson, Staffan; Eklund, Hans

    2007-03-01

    Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

  1. The Structural Basis of Protein Acetylation by the p300/CBP Transcriptional Coactivator

    SciTech Connect

    Liu,X.; Wang, L.; Zhao, K.; Thompson, P.; Hwang, Y.; Marmorstein, R.; Cole, P.

    2008-01-01

    The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs. Here we describe a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with a bi-substrate inhibitor, Lys-CoA. This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals several novel features that explain the broad substrate specificity and preference for nearby basic residues. Based on this structure and accompanying biochemical data, we propose that p300/CBP uses an unusual 'hit-and-run' (Theorell-Chance) catalytic mechanism that is distinct from other characterized HATs. Several disease-associated mutations can also be readily accounted for by the p300 HAT structure. These studies pave the way for new epigenetic therapies involving modulation of p300/CBP HAT activity.

  2. Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein.

    PubMed

    Zhang, Lei; Yan, Feng; Zhang, Shengli; Lei, Dongsheng; Charles, M Arthur; Cavigiolio, Giorgio; Oda, Michael; Krauss, Ronald M; Weisgraber, Karl H; Rye, Kerry-Anne; Pownall, Henry J; Qiu, Xiayang; Ren, Gang

    2012-02-19

    Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

  3. Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

    PubMed Central

    Zhang, Lei; Yan, Feng; Zhang, Shengli; Lei, Dongsheng; Charles, M. Arthur; Cavigiolio, Giorgio; Oda, Michael; Krauss, Ronald M.; Weisgraber, Karl H.; Rye, Kerry-Anne; Pownall, Henry J.; Qiu, Xiayang; Ren, Gang

    2013-01-01

    Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition. PMID:22344176

  4. Structural and molecular basis for Ebola virus neutralization by protective human antibodies.

    PubMed

    Misasi, John; Gilman, Morgan S A; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S; Xiang, Ye; Sullivan, Nancy J; McLellan, Jason S

    2016-03-18

    Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.

  5. The Molecular and Structural Basis of HBV-resistance to Nucleos(t)ide Analogs

    PubMed Central

    Gupta, Nidhi; Goyal, Milky; Wu, Catherine H.; Wu, George Y.

    2014-01-01

    Infection with hepatitis B virus (HBV) is a worldwide health problem. Chronic hepatitis B can lead to fibrosis, liver cirrhosis, and hepatocellular carcinoma (HCC). Management of the latter two conditions often requires liver transplantation. Treatment with conventional interferon or pegylated interferon alpha can clear the virus, but the rates are very low. The likelihood, however, of viral resistance to interferon is minimal. The main problems with this therapy are the frequency and severity of side effects. In contrast, nucleos(t)ide analogs (NAs) have significantly lower side effects, but require long term treatment as sustained virological response rates are extremely low. However, long term treatment with NAs increases the risk for the development of anti-viral drug resistance. Only by understanding the molecular basis of resistance and using agents with multiple sites of action can drugs be designed to optimally prevent the occurrence of HBV antiviral resistance. PMID:26357626

  6. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    PubMed

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

  7. Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin

    PubMed Central

    Prakash, Ajit; Shin, Joon; Rajan, Sreekanth; Yoon, Ho Sup

    2016-01-01

    The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25–DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition. PMID:26762975

  8. Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.

    PubMed

    Prakash, Ajit; Shin, Joon; Rajan, Sreekanth; Yoon, Ho Sup

    2016-04-01

    The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition. PMID:26762975

  9. Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.

    PubMed

    Prakash, Ajit; Shin, Joon; Rajan, Sreekanth; Yoon, Ho Sup

    2016-04-01

    The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition.

  10. Structural Basis for Host Membrane Remodeling Induced by Protein 2B of Hepatitis A Virus

    PubMed Central

    Vives-Adrián, Laia; Garriga, Damià; Buxaderas, Mònica; Fraga, Joana; Pereira, Pedro José Barbosa

    2015-01-01

    ABSTRACT The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCE No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. PMID:25589659

  11. Structural Basis for the Aldolase and Epimerase Activities of Staphylococcus aureus Dihydroneopterin Aldolase

    SciTech Connect

    Blaszczyk,J.; Li, Y.; Gan, J.; Yan, H.; Ji, X.

    2007-01-01

    Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and also the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.

  12. Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization

    PubMed Central

    Xue, Bo; Leyrat, Cedric; Grimes, Jonathan M.; Robinson, Robert C.

    2014-01-01

    Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation. PMID:25313062

  13. Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase.

    PubMed

    Li, Zhi; Nair, Satish K

    2015-11-01

    Plant 4-coumarate:CoA ligase (4CL) serves as a central catalyst in the phenylpropanoid pathway that provides precursors for numerous metabolites and regulates carbon flow. Here, we present several high-resolution crystal structures of Nicotiana tabacum 4CL isoform 2 (Nt4CL2) in complex with Mg(2+) and ATP, with AMP and coenzyme A (CoA), and with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP. The Nt4CL2-Mg(2+)-ATP structure is captured in the adenylate-forming conformation, whereas the other structures are in the thioester-forming conformation. These structures represent a rare example of an ANL enzyme visualized in both conformations, and also reveal the binding determinants for both CoA and the hydroxycinnamate substrate. Kinetic studies of structure-based variants were used to identify residues crucial to catalysis, ATP binding, and hydroxycinnamate specificity. Lastly, we characterize a deletion mutant of Nt4CL2 that possesses the unusual sinapinate-utilizing activity. These studies establish a molecular framework for the engineering of this versatile biocatalyst. PMID:26412334

  14. Structural basis of empathy and the domain general region in the anterior insular cortex

    PubMed Central

    Mutschler, Isabella; Reinbold, Céline; Wankerl, Johanna; Seifritz, Erich; Ball, Tonio

    2013-01-01

    Empathy is key for healthy social functioning and individual differences in empathy have strong implications for manifold domains of social behavior. Empathy comprises of emotional and cognitive components and may also be closely linked to sensorimotor processes, which go along with the motivation and behavior to respond compassionately to another person's feelings. There is growing evidence for local plastic change in the structure of the healthy adult human brain in response to environmental demands or intrinsic factors. Here we have investigated changes in brain structure resulting from or predisposing to empathy. Structural MRI data of 101 healthy adult females was analyzed. Empathy in fictitious as well as real-life situations was assessed using a validated self-evaluation measure. Furthermore, empathy-related structural effects were also put into the context of a functional map of the anterior insular cortex (AIC) determined by activation likelihood estimate (ALE) meta-analysis of previous functional imaging studies. We found that gray matter (GM) density in the left dorsal AIC correlates with empathy and that this area overlaps with the domain general region (DGR) of the anterior insula that is situated in-between functional systems involved in emotion–cognition, pain, and motor tasks as determined by our meta-analysis. Thus, we propose that this insular region where we find structural differences depending on individual empathy may play a crucial role in modulating the efficiency of neural integration underlying emotional, cognitive, and sensorimotor information which is essential for global empathy. PMID:23675334

  15. Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2010-04-30

    Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A; R = 19.9%, R(free) = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg(2+) coordination in the ATP-binding site. l-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A; R = 18.1%, R(free) = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.

  16. Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody.

    PubMed

    Horwacik, Irena; Golik, Przemyslaw; Grudnik, Przemyslaw; Kolinski, Michal; Zdzalik, Michal; Rokita, Hanna; Dubin, Grzegorz

    2015-10-01

    Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a "key and lock" interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner. PMID:26179345

  17. Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

  18. Structural basis for the nonlinear mechanics of fibrin networks under compression.

    PubMed

    Kim, Oleg V; Litvinov, Rustem I; Weisel, John W; Alber, Mark S

    2014-08-01

    Fibrin is a protein polymer that forms a 3D filamentous network, a major structural component of protective physiological blood clots as well as life threatening pathological thrombi. It plays an important role in wound healing, tissue regeneration and is widely employed in surgery as a sealant and in tissue engineering as a scaffold. The goal of this study was to establish correlations between structural changes and mechanical responses of fibrin networks exposed to compressive loads. Rheological measurements revealed nonlinear changes of fibrin network viscoelastic properties under dynamic compression, resulting in network softening followed by its dramatic hardening. Repeated compression/decompression enhanced fibrin clot stiffening. Combining fibrin network rheology with simultaneous confocal microscopy provided direct evidence of structural modulations underlying nonlinear viscoelasticity of compressed fibrin networks. Fibrin clot softening in response to compression strongly correlated with fiber buckling and bending, while hardening was associated with fibrin network densification. Our results suggest a complex interplay of entropic and enthalpic mechanisms accompanying structural changes and accounting for the nonlinear mechanical response in fibrin networks undergoing compressive deformations. These findings provide new insight into the fibrin clot structural mechanics and can be useful for designing fibrin-based biomaterials with modulated viscoelastic properties.

  19. Structural basis for the nonlinear mechanics of fibrin networks under compression.

    PubMed

    Kim, Oleg V; Litvinov, Rustem I; Weisel, John W; Alber, Mark S

    2014-08-01

    Fibrin is a protein polymer that forms a 3D filamentous network, a major structural component of protective physiological blood clots as well as life threatening pathological thrombi. It plays an important role in wound healing, tissue regeneration and is widely employed in surgery as a sealant and in tissue engineering as a scaffold. The goal of this study was to establish correlations between structural changes and mechanical responses of fibrin networks exposed to compressive loads. Rheological measurements revealed nonlinear changes of fibrin network viscoelastic properties under dynamic compression, resulting in network softening followed by its dramatic hardening. Repeated compression/decompression enhanced fibrin clot stiffening. Combining fibrin network rheology with simultaneous confocal microscopy provided direct evidence of structural modulations underlying nonlinear viscoelasticity of compressed fibrin networks. Fibrin clot softening in response to compression strongly correlated with fiber buckling and bending, while hardening was associated with fibrin network densification. Our results suggest a complex interplay of entropic and enthalpic mechanisms accompanying structural changes and accounting for the nonlinear mechanical response in fibrin networks undergoing compressive deformations. These findings provide new insight into the fibrin clot structural mechanics and can be useful for designing fibrin-based biomaterials with modulated viscoelastic properties. PMID:24840618

  20. Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody*

    PubMed Central

    Horwacik, Irena; Golik, Przemyslaw; Grudnik, Przemyslaw; Kolinski, Michal; Zdzalik, Michal; Rokita, Hanna; Dubin, Grzegorz

    2015-01-01

    Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a “key and lock” interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner. PMID:26179345

  1. Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase

    PubMed Central

    McCorvie, Thomas J.; Kopec, Jolanta; Pey, Angel L.; Fitzpatrick, Fiona; Patel, Dipali; Chalk, Rod; Shrestha, Leela; Yue, Wyatt W.

    2016-01-01

    Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants. PMID:27005423

  2. Structural basis for the nonlinear mechanics of fibrin networks under compression

    PubMed Central

    Kim, Oleg V.; Litvinov, Rustem I.; Weisel, John W.; Alber, Mark S.

    2014-01-01

    Fibrin is a protein polymer that forms a 3D filamentous network, a major structural component of protective physiological blood clots as well as life threatening pathological thrombi. It plays an important role in wound healing, tissue regeneration and is widely employed in surgery as a sealant and in tissue engineering as a scaffold. The goal of this study was to establish correlations between structural changes and mechanical responses of fibrin networks exposed to compressive loads. Rheological measurements revealed nonlinear changes of fibrin network viscoelastic properties under dynamic compression, resulting in network softening followed by its dramatic hardening. Repeated compression/decompression enhanced fibrin clot stiffening. Combining fibrin network rheology with simultaneous confocal microscopy provided direct evidence of structural modulations underlying nonlinear viscoelasticity of compressed fibrin networks. Fibrin clot softening in response to compression strongly correlated with fiber buckling and bending, while hardening was associated with fibrin network densification. Our results suggest a complex interplay of entropic and enthalpic mechanisms accompanying structural changes and accounting for the nonlinear mechanical response in fibrin networks undergoing compressive deformations. These findings provide new insight into the fibrin clot structural mechanics and can be useful for designing fibrin-based biomaterials with modulated viscoelastic properties. PMID:24840618

  3. Structural Basis for the Assembly and Gate Closure Mechanisms of the Mycobacterium tuberculosis 20S Proteasome

    SciTech Connect

    Lin, D.; Li, H; Wang, T; Pan, H; Lin, G; Li, H

    2010-01-01

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  4. Structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors

    PubMed Central

    Lee, Youngjin; Ryu, Young Bae; Youn, Hyung-Seop; Cho, Jung Keun; Kim, Young Min; Park, Ji-Young; Lee, Woo Song; Park, Ki Hun; Eom, Soo Hyun

    2014-01-01

    Sialidase catalyzes the removal of a terminal sialic acid from glycoconjugates and plays a pivotal role in nutrition, cellular interactions and pathogenesis mediating various infectious diseases including cholera, influenza and sepsis. An array of antiviral sialidase agents have been developed and are commercially available, such as zanamivir and oseltamivir for treating influenza. However, the development of bacterial sialidase inhibitors has been much less successful. Here, natural polyphenolic geranylated flavonoids which show significant inhibitory effects against Cp-NanI, a sialidase from Clostridium perfringens, are reported. This bacterium causes various gastrointestinal diseases. The crystal structure of the Cp-NanI catalytic domain in complex with the best inhibitor, diplacone, is also presented. This structure explains how diplacone generates a stable enzyme–inhibitor complex. These results provide a structural framework for understanding the interaction between sialidase and natural flavonoids, which are promising scaffolds on which to discover new anti-sialidase agents. PMID:24816104

  5. Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

    SciTech Connect

    Tu, Chao; Tropea, Joseph E.; Austin, Brian P.; Court, Donald L.; Waugh, David S.; Ji, Xinhua

    2009-03-27

    Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs IVIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

  6. Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway

    PubMed Central

    Saxton, Robert A.; Knockenhauer, Kevin E.; Wolfson, Rachel L.; Chantranupong, Lynne; Pacold, Michael E.; Wang, Tim; Schwartz, Thomas U.; Sabatini, David M.

    2015-01-01

    Eukaryotic cells coordinate growth with the availability of nutrients through mTOR complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag GTPases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. We present the 2.7-Å crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway. PMID:26586190

  7. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

    SciTech Connect

    Li, D.; Li, H.; Li, H.; Wang, T.; Pan, H.; Lin, G.

    2010-06-16

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  8. Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation.

    PubMed

    Shu, Bo; Gong, Peng

    2016-07-12

    Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTP-induced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the template-product duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases. PMID:27339134

  9. Structural basis for the facilitative diffusion mechanism by SemiSWEET transporter

    NASA Astrophysics Data System (ADS)

    Lee, Yongchan; Nishizawa, Tomohiro; Yamashita, Keitaro; Ishitani, Ryuichiro; Nureki, Osamu

    2015-01-01

    SWEET family proteins mediate sugar transport across biological membranes and play crucial roles in plants and animals. The SWEETs and their bacterial homologues, the SemiSWEETs, are related to the PQ-loop family, which is characterized by highly conserved proline and glutamine residues (PQ-loop motif). Although the structures of the bacterial SemiSWEETs were recently reported, the conformational transition and the significance of the conserved motif in the transport cycle have remained elusive. Here we report crystal structures of SemiSWEET from Escherichia coli, in the both inward-open and outward-open states. A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer. The conserved PQ-loop motif serves as a molecular hinge that enables the ‘binder clip-like’ motion of SemiSWEET. The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins.

  10. Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

    PubMed Central

    Gaik, Monika; Flemming, Dirk; von Appen, Alexander; Kastritis, Panagiotis; Mücke, Norbert; Fischer, Jessica; Stelter, Philipp; Ori, Alessandro; Bui, Khanh Huy; Baßler, Jochen; Barbar, Elisar

    2015-01-01

    Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery. PMID:25646085

  11. Structural basis for Zn2+-dependent intercellular adhesion in staphylococcal biofilms

    PubMed Central

    Conrady, Deborah G.; Wilson, Jeffrey J.; Herr, Andrew B.

    2013-01-01

    Staphylococcal bacteria, including Staphylococcus epidermidis and Staphylococcus aureus, cause chronic biofilm-related infections. The homologous proteins Aap and SasG mediate biofilm formation in S. epidermidis and S. aureus, respectively. The self-association of these proteins in the presence of Zn2+ leads to the formation of extensive adhesive contacts between cells. This study reports the crystal structure of a Zn2+-bound construct from the self-associating region of Aap. Several unusual structural features include elongated β-sheets that are solvent-exposed on both faces and the lack of a canonical hydrophobic core. Zn2+-dependent dimers are observed in three distinct crystal forms, formed via pleomorphic coordination of Zn2+ in trans across the dimer interface. These structures illustrate how a long, flexible surface protein is able to form tight intercellular adhesion sites under adverse environmental conditions. PMID:23277549

  12. 1,2-Propanediol Dehydration in Roseburia inulinivorans: STRUCTURAL BASIS FOR SUBSTRATE AND ENANTIOMER SELECTIVITY.

    PubMed

    LaMattina, Joseph W; Keul, Nicholas D; Reitzer, Pierre; Kapoor, Suraj; Galzerani, Felipe; Koch, Daniel J; Gouvea, Iuri E; Lanzilotta, William N

    2016-07-22

    Glycyl radical enzymes (GREs) represent a diverse superfamily of enzymes that utilize a radical mechanism to catalyze difficult, but often essential, chemical reactions. In this work we present the first biochemical and structural data for a GRE-type diol dehydratase from the organism Roseburia inulinivorans (RiDD). Despite high sequence (48% identity) and structural similarity to the GRE-type glycerol dehydratase from Clostridium butyricum, we demonstrate that the RiDD is in fact a diol dehydratase. In addition, the RiDD will utilize both (S)-1,2-propanediol and (R)-1,2-propanediol as a substrate, with an observed preference for the S enantiomer. Based on the new structural information we developed and successfully tested a hypothesis that explains the functional differences we observe. PMID:27252380

  13. Structural Basis of Preexisting Immunity to the 2009 H1N1 Pandemic Influenza Virus

    SciTech Connect

    Xu, Rui; Ekiert, Damian C.; Krause, Jens C.; Hai, Rong; Crowe, Jr., James E.; Wilson, Ian A.

    2010-05-25

    The 2009 H1N1 swine flu is the first influenza pandemic in decades. The crystal structure of the hemagglutinin from the A/California/04/2009 H1N1 virus shows that its antigenic structure, particularly within the Sa antigenic site, is extremely similar to those of human H1N1 viruses circulating early in the 20th century. The cocrystal structure of the 1918 hemagglutinin with 2D1, an antibody from a survivor of the 1918 Spanish flu that neutralizes both 1918 and 2009 H1N1 viruses, reveals an epitope that is conserved in both pandemic viruses. Thus, antigenic similarity between the 2009 and 1918-like viruses provides an explanation for the age-related immunity to the current influenza pandemic.

  14. Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

    PubMed

    Yamaguchi, Hiroto; Miwa, Yukiko; Kasa, Miyuki; Kitano, Ken; Amano, Mutsuki; Kaibuchi, Kozo; Hakoshima, Toshio

    2006-09-01

    Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability. PMID:16891330

  15. Structural basis of the alternating-access mechanism in a bile acid transporter

    NASA Astrophysics Data System (ADS)

    Zhou, Xiaoming; Levin, Elena J.; Pan, Yaping; McCoy, Jason G.; Sharma, Ruchika; Kloss, Brian; Bruni, Renato; Quick, Matthias; Zhou, Ming

    2014-01-01

    Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBTNM) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na+ and a taurocholic acid. However, the structural changes that bring bile acid and Na+ across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na+ and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved `crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications

  16. Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

    SciTech Connect

    X Xu; Z Vysotskaya; Q Liu; L Zhou

    2011-12-31

    Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 {angstrom}. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a {approx}3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between {beta}4 and {beta}5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.

  17. Structural Basis of Substrate Recognition by Aldehyde Dehydrogenase 7A1

    PubMed Central

    2016-01-01

    Aldehyde dehydrogenase 7A1 (ALDH7A1) is part of lysine catabolism and catalyzes the NAD+-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Herein, we describe a structural study of human ALDH7A1 focused on substrate recognition. Five crystal structures and small-angle X-ray scattering data are reported, including the first crystal structure of any ALDH7 family member complexed with α-aminoadipate. The product binds with the ε-carboxylate in the oxyanion hole, the aliphatic chain packed into an aromatic box, and the distal end of the product anchored by electrostatic interactions with five conserved residues. This binding mode resembles that of glutamate bound to the proline catabolic enzyme ALDH4A1. Analysis of ALDH7A1 and ALDH4A1 structures suggests key interactions that underlie substrate discrimination. Structures of apo ALDH7A1 reveal dramatic conformational differences from the product complex. Product binding is associated with a 16 Å movement of the C-terminus into the active site, which stabilizes the active conformation of the aldehyde substrate anchor loop. The fact that the C-terminus is part of the active site was hitherto unknown. Interestingly, the C-terminus and aldehyde anchor loop are disordered in a new tetragonal crystal form of the apoenzyme, implying that these parts of the enzyme are highly flexible. Our results suggest that the active site of ALDH7A1 is disassembled when the aldehyde site is vacant, and the C-terminus is a mobile element that forms quaternary structural interactions that aid aldehyde binding. These results are relevant to the c.1512delG genetic deletion associated with pyridoxine-dependent epilepsy, which alters the C-terminus of ALDH7A1. PMID:26260980

  18. Structural basis of stable bending in DNA containing An tracts. Different types of bending.

    PubMed

    Chuprina, V P; Abagyan, R A

    1988-08-01

    Structural determinants of DNA bending of different types have been studied by theoretical conformational analysis of duplexes. Their terminal parts were fixed either in an ordinary low-energy B-like conformation or in "anomalous" conformations with a narrowed minor groove typical of An tracts. The anomalous conformations had different negative tilt angles (up to about zero), different propeller twists and minor groove widths. Calculations have been performed for DNA fragments AnTm, TnAm, AnGCTm, AnCGTm, TmGCAn, TmCGAn which are the models of the junction of two anomalous structures on An and Tm tracts. On the AT step of the AnTm fragment the minor groove can be easily narrowed so that a whole unbent fragment of anomalous structure is formed on AnTm. According to our energy estimates, there should not be any reliable bending on AnTm. In contrast, in all other cases there was a pronounced roll-like bending into the major groove in the chemical symmetry region. Calculations of the junction between the anomalous and ordinary B-like structure for GnTm and CnAm have shown that there is an equilibrium bending with a tilt component towards the chain having the anomalous structure at the 5'-end. From our calculations it is impossible to determine precisely the direction of bending, though it can be suggested that the roll component of bending might be directed towards the major groove. The anomalous structure is the main reason of bending; alternations of pyrimidines and purines can modulate the value and the direction of equilibrium bending (only the value in the case of self-complementary fragments).(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Nobel lecture. A structural basis of light energy and electron transfer in biology.

    PubMed Central

    Huber, R

    1989-01-01

    Aspects of intramolecular light energy and electron transfer will be discussed for three protein--cofactor complexes, whose three-dimensional structures have been elucidated by X-ray crystallography: components of light-harvesting cyanobacterial phycobilisomes; the purple bacterial reaction centre; and the blue multi-copper oxidases. A wealth of functional data is available for these systems which allows specific correlations between structure and function and general conclusions about light energy and electron transfer in biological materials to be made. Images PMID:2676513

  20. Structural basis of the mutagenicity of 1-amino-2-naphthol-based azo dyes.

    PubMed

    Rosenkranz, H S; Klopman, G

    1990-03-01

    A structure-activity study of 1-amino-2-naphthol derived azo dyes using CASE, the Computer Automated Structure Evaluation system, revealed that for optimal mutagenicity, reduction of the azo bond was required, thus suggesting that activity could be related to the liberated aromatic amines. Although it has long been known that sulfonation of azo dyes resulted in decreased carcinogenicity and mutagenicity, the present study elucidates the sites of sulfonation which will decrease mutagenicity maximally. Comparison of CASE predictions with available mutagenicity data indicates a concordance. Unexpectedly, CASE indicates that one of the aromatic amines obtained upon azo reduction of FD and C Red no. 40 is predicted to be mutagenic.

  1. Structural basis of unique ligand specificity of KAI2-like protein from parasitic weed Striga hermonthica.

    PubMed

    Xu, Yuqun; Miyakawa, Takuya; Nakamura, Hidemitsu; Nakamura, Akira; Imamura, Yusaku; Asami, Tadao; Tanokura, Masaru

    2016-01-01

    The perception of two plant germination inducers, karrikins and strigolactones, are mediated by the proteins KAI2 and D14. Recently, KAI2-type proteins from parasitic weeds, which are possibly related to seed germination induced by strigolactone, have been classified into three clades characterized by different responses to karrikin/strigolactone. Here we characterized a karrikin-binding protein in Striga (ShKAI2iB) that belongs to intermediate-evolving KAI2 and provided the structural bases for its karrikin-binding specificity. Binding assays showed that ShKAI2iB bound karrikins but not strigolactone, differing from other KAI2 and D14. The crystal structures of ShKAI2iB and ShKAI2iB-karrikin complex revealed obvious structural differences in a helix located at the entry of its ligand-binding cavity. This results in a smaller closed pocket, which is also the major cause of ShKAI2iB's specificity of binding karrikin. Our structural study also revealed that a few non-conserved amino acids led to the distinct ligand-binding profile of ShKAI2iB, suggesting that the evolution of KAI2 resulted in its diverse functions. PMID:27507097

  2. Structural basis of unique ligand specificity of KAI2-like protein from parasitic weed Striga hermonthica

    PubMed Central

    Xu, Yuqun; Miyakawa, Takuya; Nakamura, Hidemitsu; Nakamura, Akira; Imamura, Yusaku; Asami, Tadao; Tanokura, Masaru

    2016-01-01

    The perception of two plant germination inducers, karrikins and strigolactones, are mediated by the proteins KAI2 and D14. Recently, KAI2-type proteins from parasitic weeds, which are possibly related to seed germination induced by strigolactone, have been classified into three clades characterized by different responses to karrikin/strigolactone. Here we characterized a karrikin-binding protein in Striga (ShKAI2iB) that belongs to intermediate-evolving KAI2 and provided the structural bases for its karrikin-binding specificity. Binding assays showed that ShKAI2iB bound karrikins but not strigolactone, differing from other KAI2 and D14. The crystal structures of ShKAI2iB and ShKAI2iB-karrikin complex revealed obvious structural differences in a helix located at the entry of its ligand-binding cavity. This results in a smaller closed pocket, which is also the major cause of ShKAI2iB’s specificity of binding karrikin. Our structural study also revealed that a few non-conserved amino acids led to the distinct ligand-binding profile of ShKAI2iB, suggesting that the evolution of KAI2 resulted in its diverse functions. PMID:27507097

  3. Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

    SciTech Connect

    Pletnev, Sergei; Gurskaya, Nadya G.; Pletneva, Nadya V.; Lukyanov, Konstantin A.; Chudakov, Dmitri M.; Martynov, Vladimir I.; Popov, Vladimir O.; Kovalchuk, Mikhail V.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Vladimir

    2009-11-23

    KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the {beta}-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu68 and Ser119, located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

  4. Structural basis for suppression of hypernegative DNA supercoiling by E. coli topoisomerase I

    DOE PAGES

    Tan, Kemin; Zhou, Qingxuan; Cheng, Bokun; Zhang, Zhongtao; Joachimiak, Andrzej; Tse-Dinh, Yuk -Ching

    2015-10-20

    Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain andmore » zinc ribbon-like domain bind ssDNA with primarily π -stacking interactions. Finally, this novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3β from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.« less

  5. Structural Basis for Microcin C7 Inactivation by the MccE Acetyltransferase

    SciTech Connect

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K.

    2015-10-15

    The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE{sup AcTase}). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through p-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE{sup AcTase} can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.

  6. Structural basis for the prion-like MAVS filaments in antiviral innate immunity

    PubMed Central

    Xu, Hui; He, Xiaojing; Zheng, Hui; Huang, Lily J; Hou, Fajian; Yu, Zhiheng; de la Cruz, Michael Jason; Borkowski, Brian; Zhang, Xuewu; Chen, Zhijian J; Jiang, Qiu-Xing

    2014-01-01

    Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001 PMID:24569476

  7. Structural basis underlying the metallic-like conductivity of microbial nanowires

    NASA Astrophysics Data System (ADS)

    Malvankar, Nikhil; Vargas, Madeline; Tuominen, Mark; Lovley, Derek

    2014-03-01

    Microbial nanowires are electrically conductive proteinaceous pili nanofilaments secreted by Geobacter sulfurreducens. In contrast to current biochemical understanding that proteins are insulators, G. sulfurreducens pili show organic metallic-like conductivity. Pili also enable direct exchange of electrons among Geobacter co-cultures. Site-directed mutagenesis studies revealed that aromatic amino acids confer conductivity to pili. In order to develop a structural understanding of the pili to probe the conduction mechanism at a molecular level, we employed three complementary structural methods - X-ray microdiffraction using synchrotron radiation, rocking curve X-ray diffraction, and electron diffraction. Studies performed with all these three methods revealed a 3.2 Å periodic spacing in wild-type G. sulfurreducens pili, expected for metal-like conductivity and a lack of such spacing in genetically modified non-conductive pili. Notably, both the peak intensity and the conductivity increased 100-fold with lowering the pH from pH 10.5 to pH 2, demonstrating a structure-function correlation in pili. We also reconstructed the three dimensional tertiary structure of pili with homology modeling, which further suggested the 3.2 Å spacing among aromatics associated with metal-like conductivity. Funded by Office of Naval Research, DOE Genomic Sciences and NSF-NSEC Center for Hierarchical Manufacturing grant no. CMMI-1025020.

  8. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins

    PubMed Central

    Sun, Tianjun; Davies, Peter L.; Walker, Virginia K.

    2015-01-01

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein’s outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow. PMID:26488661

  9. Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

    PubMed Central

    Eiler, Daniel; Wang, Jimin; Steitz, Thomas A.

    2014-01-01

    Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket. PMID:25157168

  10. The Structural Basis for Allosteric Inhibition of a Threonine-sensitive Aspartokinase*

    PubMed Central

    Liu, Xuying; Pavlovsky, Alexander G.; Viola, Ronald E.

    2008-01-01

    The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK (Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry31 ,799 -8051731937). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation. PMID:18334478

  11. Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

    SciTech Connect

    Aller, Stephen G.; Yu, Jodie; Ward, Andrew; Weng, Yue; Chittaboina, Srinivas; Zhuo, Rupeng; Harrell, Patina M.; Trinh, Yenphuong T.; Zhang, Qinghai; Urbatsch, Ina L.; Chang, Geoffrey

    2009-04-22

    P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of -6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

  12. Structural Basis of APH(3)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

    SciTech Connect

    Fong, D.; Berghuis, A

    2009-01-01

    Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S)-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed. Unfortunately, butirosin, amikacin, and isepamicin are not resistant to inactivation by 3'-aminoglycoside O-phosphotransferase type IIIa [APH(3')-IIIa]. We report here the crystal structure of APH(3')-IIIa in complex with an ATP analog, AMPPNP [adenosine 5'-(?,{gamma}-imido)triphosphate], and butirosin A to 2.4-A resolution. The structure shows that butirosin A binds to the enzyme in a manner analogous to other 4,5-disubstituted aminoglycosides, and the flexible antibiotic-binding loop is key to the accommodation of structurally diverse substrates. Based on the crystal structure, we have also constructed a model of APH(3')-IIIa in complex with amikacin, a commonly used semisynthetic N1-substituted 4,6-disubstituted aminoglycoside. Together, these results suggest a strategy to further derivatize the AHB group in order to generate new aminoglycoside derivatives that can elude inactivation by resistance enzymes while maintaining their ability to bind to the ribosomal A site.

  13. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins.

    PubMed

    Sun, Tianjun; Davies, Peter L; Walker, Virginia K

    2015-10-20

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein's outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow. PMID:26488661

  14. Structural basis for autoinhibition and its relief of MOB1 in the Hippo pathway

    PubMed Central

    Kim, Sun-Yong; Tachioka, Yuka; Mori, Tomoyuki; Hakoshima, Toshio

    2016-01-01

    MOB1 protein is a key regulator of large tumor suppressor 1/2 (LATS1/2) kinases in the Hippo pathway. MOB1 is present in an autoinhibited form and is activated by MST1/2-mediated phosphorylation, although the precise mechanisms responsible for autoinhibition and activation are unknown due to lack of an autoinhibited MOB1 structure. Here, we report on the crystal structure of full-length MOB1B in the autoinhibited form and a complex between the MOB1B core domain and the N-terminal regulation (NTR) domain of LATS1. The structure of full-length MOB1B shows that the N-terminal extension forms a short β-strand, the SN strand, followed by a long conformationally flexible positively-charged linker and α-helix, the Switch helix, which blocks the LATS1 binding surface of MOB1B. The Switch helix is stabilized by β-sheet formation of the SN strand with the S2 strand of the MOB1 core domain. Phosphorylation of Thr12 and Thr35 residues structurally accelerates dissociation of the Switch helix from the LATS1-binding surface by the “pull-the-string” mechanism, thereby enabling LATS1 binding. PMID:27335147

  15. Histone deacetylase 6 structure and molecular basis of catalysis and inhibition.

    PubMed

    Hai, Yang; Christianson, David W

    2016-09-01

    Histone deacetylase 6 (HDAC6) is a critical target for drug design because of its role in oncogenic transformation and cancer metastasis, and is unique among all histone deacetylases in that it contains tandem catalytic domains designated CD1 and CD2. We now report the crystal structures of CD2 from Homo sapiens HDAC6 and of CD1 and CD2 from Danio rerio HDAC6. We correlated these structures with activity measurements using 13 different substrates. The catalytic activity of CD2 from both species exhibited broad substrate specificity, whereas that of CD1 was highly specific for substrates bearing C-terminal acetyllysine residues. Crystal structures of substrate complexes yielded unprecedented snapshots of the catalytic mechanism. Additionally, crystal structures of complexes with eight different inhibitors, including belinostat and panobinostat (currently used in cancer chemotherapy), the macrocyclic tetrapeptide HC toxin, and the HDAC6-specific inhibitor N-hydroxy-4-(2-((2-hydroxyethyl)(phenyl)amino)-2-oxoethyl)benzamide, revealed surprising new insight regarding changes in Zn(2+) coordination and isozyme-specific inhibition. PMID:27454933

  16. Unravelling the Structural and Molecular Basis Responsible for the Anti-Biofilm Activity of Zosteric Acid.

    PubMed

    Cattò, Cristina; Dell'Orto, Silvia; Villa, Federica; Villa, Stefania; Gelain, Arianna; Vitali, Alberto; Marzano, Valeria; Baroni, Sara; Forlani, Fabio; Cappitelli, Francesca

    2015-01-01

    The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to generate more potent anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to understand the structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Considerations concerning the relationship between structure and anti-biofilm activity revealed that i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the molecule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and, consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instrumental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down approach, combined with mass spectrometry, the herein-defined active structure of ZA was matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase, WrbA, suggesting a possible role of this protein in the biofilm formation process. PMID:26132116

  17. Structural Basis of High-Fidelity DNA Synthesis by Yeast DNA Polymerase δ

    SciTech Connect

    Swan, M.; Johnson, R; Prakash, L; Prakash, S; Aggarwal, A

    2009-01-01

    DNA polymerase ? (Pol ?) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol ? catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-A resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

  18. Structural Basis for β-Carboline Alkaloid Production by the Microbial Homodimeric Enzyme McbB.

    PubMed

    Mori, Takahiro; Hoshino, Shotaro; Sahashi, Shusaku; Wakimoto, Toshiyuki; Matsui, Takashi; Morita, Hiroyuki; Abe, Ikuro

    2015-07-23

    The β-carboline (βC) alkaloids occur throughout nature and exhibit diverse biological activities. In contrast to βC alkaloid synthesis in plants, the biosynthesis in microorganisms remains poorly understood. The recently reported McbB from Marinactinospora thermotolerans is a novel enzyme proposed to catalyze the Pictet-Spengler (PS) reaction of L-tryptophan and oxaloacetaldehyde to produce the βC scaffold of marinacarbolines. In this study, we solved the crystal structure of McbB complexed with L-tryptophan at 2.48 Å resolution, which revealed the novel protein folding of McbB and the totally different structure from those of other PS condensation catalyzing enzymes, such as strictosidine synthase and norcoclaurine synthase from plants. Structural analysis and site-directed mutagenesis confirmed that the previously proposed catalytic Glu97 at the active-site center functions as an acid and base catalyst. Remarkably, the structure-based mutants R72A and H87A, with expanded active-site cavities, newly accepted bulky phenylglyoxal as the aldehyde substrate, to produce 1-benzoyl-3-carboxy-β-carboline. PMID:26120001

  19. Catalytic mechanism of bleomycin N-acetyltransferase proposed on the basis of its crystal structure.

    PubMed

    Oda, Kosuke; Matoba, Yasuyuki; Noda, Masafumi; Kumagai, Takanori; Sugiyama, Masanori

    2010-01-01

    Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA(2), and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism. PMID:19889644

  20. Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange

    PubMed Central

    Pennington, Luke F.; Tarchevskaya, Svetlana; Brigger, Daniel; Sathiyamoorthy, Karthik; Graham, Michelle T.; Nadeau, Kari Christine; Eggel, Alexander; Jardetzky, Theodore S.

    2016-01-01

    Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions. PMID:27194387

  1. Determination and proof of structure of monosubstituted dihydrosilaazanthrones on the basis of PMR spectra

    SciTech Connect

    Prostakov, N.S.; Fomichev, A.A.; Golovtsov, N.I.; Varlamov, A.V.; Nazrul, I.; Polosin, V.M.

    1986-05-01

    PMR spectra have been used in establishing the structure of ten bromo-, nitro-, and amino-substituted 10,10-dimethyl-10-sila-2-azaanthrones. Anomalous effects of NO/sub 2/ and NH/sub 2/ groups adjacent to the carbonyl carbon have been observed and explained; these effects are related to the specific geometry of these molecules.

  2. Structural basis of the carbohydrate specificities of jacalin: an X-ray and modeling study.

    PubMed

    Jeyaprakash, A Arockia; Katiyar, S; Swaminathan, C P; Sekar, K; Surolia, A; Vijayan, M

    2003-09-01

    The structures of the complexes of tetrameric jacalin with Gal, Me-alpha-GalNAc, Me-alpha-T-antigen, GalNAcbeta1-3Gal-alpha-O-Me and Galalpha1-6Glc (mellibiose) show that the sugar-binding site of jacalin has three components: the primary site, secondary site A, and secondary site B. In these structures and in the two structures reported earlier, Gal or GalNAc occupy the primary site with the anomeric carbon pointing towards secondary site A. The alpha-substituents, when present, interact, primarily hydrophobically, with secondary site A which has variable geometry. O-H..., centered pi and C-H...pi hydrogen bonds involving this site also exist. On the other hand, beta-substitution leads to severe steric clashes. Therefore, in complexes involving beta-linked disaccharides, the reducing sugar binds at the primary site with the non-reducing end located at secondary site B. The interactions at secondary site B are primarily through water bridges. Thus, the nature of the linkage determines the mode of the association of the sugar with jacalin. The interactions observed in the crystal structures and modeling based on them provide a satisfactory qualitative explanation of the available thermodynamic data on jacalin-carbohydrate interactions. They also lead to fresh insights into the nature of the binding of glycoproteins by jacalin.

  3. Unravelling the Structural and Molecular Basis Responsible for the Anti-Biofilm Activity of Zosteric Acid

    PubMed Central

    Cattò, Cristina; Dell’Orto, Silvia; Villa, Federica; Villa, Stefania; Gelain, Arianna; Vitali, Alberto; Marzano, Valeria; Baroni, Sara; Forlani, Fabio; Cappitelli, Francesca

    2015-01-01

    The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to generate more potent anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to understand the structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Considerations concerning the relationship between structure and anti-biofilm activity revealed that i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the molecule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and, consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instrumental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down approach, combined with mass spectrometry, the herein-defined active structure of ZA was matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase, WrbA, suggesting a possible role of this protein in the biofilm formation process. PMID:26132116

  4. Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange

    DOE PAGES

    Pennington, Luke F.; Tarchevskaya, Svetlana; Brigger, Daniel; Sathiyamoorthy, Karthik; Graham, Michelle T.; Nadeau, Kari Christine; Eggel, Alexander; Jardetzky, Theodore S.

    2016-05-19

    Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, inmore » combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. Furthermore, this combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.« less

  5. Structural Basis for the Acyltransferase Activity of Lecithin: Retinol Acyltransferase-like Proteins

    SciTech Connect

    Golczak, Marcin; Kiser, Philip D.; Sears, Avery E.; Lodowski, David T.; Blaner, William S.; Palczewski, Krzysztof

    2012-10-10

    Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.

  6. The Structural Basis of Exopolygalacturonase Activity in a Family 28 Glycoside Hydrolase

    SciTech Connect

    Abbott,D.; Boraston, A.

    2007-01-01

    Family 28 glycoside hydrolases (polygalacturonases) are found in organisms across the plant, fungal and bacterial kingdoms, where they are central to diverse biological functions such as fruit ripening, biomass recycling and plant pathogenesis. The structures of several polygalacturonases have been reported; however, all of these enzymes utilize an endo-mode of digestion, which generates a spectrum of oligosaccharide products with varying degrees of polymerization. The structure of a complementary exo-acting polygalacturonase and an accompanying explanation of the molecular determinants for its specialized activity have been noticeably lacking. We present the structure of an exopolygalacturonase from Yersinia enterocolitica, YeGH28 in a native form (solved to 2.19 {angstrom} resolution) and a digalacturonic acid product complex (solved to 2.10 {angstrom} resolution). The activity of YeGH28 is due to inserted stretches of amino acid residues that transform the active site from the open-ended channel observed in the endopolygalacturonases to a closed pocket that restricts the enzyme to the exclusive attack of the non-reducing end of oligogalacturonide substrates. In addition, YeGH28 possesses a fused FN3 domain with unknown function, the first such structure described in pectin active enzymes.

  7. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    SciTech Connect

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  8. Structural Basis for Substrate Binding and the Catalytic Mechanism of Type III Pantothenate Kinase

    SciTech Connect

    Yang, Kun; Strauss, Erick; Huerta, Carlos; Zhang, Hong

    2008-07-15

    Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high K{sub m} for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the 'acetate and sugar kinase/heat shock protein 70/actin' (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.

  9. Eukaryotic class II cyclobutane pyrimidine dimer photolyase structure reveals basis for improved ultraviolet tolerance in plants.

    PubMed

    Hitomi, Kenichi; Arvai, Andrew S; Yamamoto, Junpei; Hitomi, Chiharu; Teranishi, Mika; Hirouchi, Tokuhisa; Yamamoto, Kazuo; Iwai, Shigenori; Tainer, John A; Hidema, Jun; Getzoff, Elizabeth D

    2012-04-01

    Ozone depletion increases terrestrial solar ultraviolet B (UV-B; 280-315 nm) radiation, intensifying the risks plants face from DNA damage, especially covalent cyclobutane pyrimidine dimers (CPD). Without efficient repair, UV-B destroys genetic integrity, but plant breeding creates rice cultivars with more robust photolyase (PHR) DNA repair activity as an environmental adaptation. So improved strains of Oryza sativa (rice), the staple food for Asia, have expanded rice cultivation worldwide. Efficient light-driven PHR enzymes restore normal pyrimidines to UV-damaged DNA by using blue light via flavin adenine dinucleotide to break pyrimidine dimers. Eukaryotes duplicated the photolyase gene, producing PHRs that gained functions and adopted activities that are distinct from those of prokaryotic PHRs yet are incompletely understood. Many multicellular organisms have two types of PHR: (6-4) PHR, which structurally resembles bacterial CPD PHRs but recognizes different substrates, and Class II CPD PHR, which is remarkably dissimilar in sequence from bacterial PHRs despite their common substrate. To understand the enigmatic DNA repair mechanisms of PHRs in eukaryotic cells, we determined the first crystal structure of a eukaryotic Class II CPD PHR from the rice cultivar Sasanishiki. Our 1.7 Å resolution PHR structure reveals structure-activity relationships in Class II PHRs and tuning for enhanced UV tolerance in plants. Structural comparisons with prokaryotic Class I CPD PHRs identified differences in the binding site for UV-damaged DNA substrate. Convergent evolution of both flavin hydrogen bonding and a Trp electron transfer pathway establish these as critical functional features for PHRs. These results provide a paradigm for light-dependent DNA repair in higher organisms. PMID:22170053

  10. Structures of human phosphofructokinase-1 and atomic basis of cancer-associated mutations

    PubMed Central

    Webb, Bradley A.; Forouhar, Farhad; Szu, Fu-En; Seetharaman, Jayaraman; Tong, Liang; Barber, Diane L.

    2015-01-01

    Phosphofructokinase-1 (PFK1), the “gatekeeper” of glycolysis, catalyses the committed step of the glycolytic pathway by converting fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate. Allosteric activation and inhibition of PFK1 by over 10 metabolites and in response to hormonal signaling fine-tune glycolytic flux to meet energy requirements1. Mutations inhibiting PFK1 activity cause glycogen storage disease type VII, also known as Tarui disease2, and mice deficient in muscle PFK1 have decreased fat stores3. Additionally, PFK1 is suggested to have important roles in metabolic reprograming in cancer4,5. Despite its critical role in glucose flux, the biologically relevant crystal structure of the mammalian PFK1 tetramer has not been determined. We report here the first structures of the mammalian PFK1 tetramer, for the human platelet isoform (PFKP), in complex with ATP-Mg2+ and ADP at 3.1 and 3.4 Å, respectively. The structures reveal substantial conformational changes in the enzyme upon nucleotide hydrolysis as well as a unique tetramer interface. Mutations of residues in this interface can affect tetramer formation, enzyme catalysis and regulation, indicating the functional importance of the tetramer. With altered glycolytic flux being a hallmark of cancers6, these new structures allow a molecular understanding of the functional consequences of somatic PFK1 mutations identified in human cancers. We characterized three of these mutations and show they have distinct effects on allosteric regulation of PFKP activity and lactate production. The PFKP structural blueprint for somatic mutations as well as the catalytic site can guide therapeutic targeting of PFK1 activity to control dysregulated glycolysis in disease. PMID:25985179

  11. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    SciTech Connect

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P.

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

  12. Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase

    SciTech Connect

    Kirouac, Kevin N.; Ling, Hong

    2009-06-30

    Human DNA polymerase iota (pol iota) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of pol complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by pol. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by pol and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for pol's high error rates on pyrimidines and determines the incorporation specificity.

  13. Crystallographic analysis of NHERF1–PLCβ3 interaction provides structural basis for CXCR2 signaling in pancreatic cancer

    SciTech Connect

    Jiang, Yuanyuan; Wang, Shuo; Holcomb, Joshua; Trescott, Laura; Guan, Xiaoqing; Hou, Yuning; Brunzelle, Joseph; Sirinupong, Nualpun; Li, Chunying; Yang, Zhe

    2014-04-04

    Highlights: • CXCR2–NHERF1–PLCβ3 complex regulates CXCR2 signaling in pancreatic cancer. • The crystal structure of the NHERF1 PDZ1 domain in complex with PLCβ3. • The structure reveals specificity determinants of PDZ1–PLCβ3 interaction. • Endogenous PLCβ3 in pancreatic cancer cells interacts with both PDZ1 and PDZ2. • Structural basis of the PDZ1–PLCβ3 interaction is valuable in selective drug design. - Abstract: The formation of CXCR2–NHERF1–PLCβ3 macromolecular complex in pancreatic cancer cells regulates CXCR2 signaling activity and plays an important role in tumor proliferation and invasion. We previously have shown that disruption of the NHERF1-mediated CXCR2–PLCβ3 interaction abolishes the CXCR2 signaling cascade and inhibits pancreatic tumor growth in vitro and in vivo. Here we report the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal PLCβ3 sequence. The structure reveals that the PDZ1–PLCβ3 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four PLCβ3 residues contributing to specific interactions. We also show that PLCβ3 can bind both NHERF1 PDZ1 and PDZ2 in pancreatic cancer cells, consistent with the observation that the peptide binding pockets of these PDZ domains are highly structurally conserved. This study provides an understanding of the structural basis for the PDZ-mediated NHERF1–PLCβ3 interaction that could prove valuable in selective drug design against CXCR2-related cancers.

  14. Structuring the documentation of nursing care on the basis of a theoretical process model.

    PubMed

    Kärkkäinen, Oili; Eriksson, Katie

    2004-06-01

    The aim of this study was to develop the documentation of the substance of nursing care on the basis of a theoretical caring process model. The theory is Eriksson's caring process model and her theory of health, suffering and caring. The approach of the research task was dialogue. As a research method Koski's adaptation of Gadamer's theory of hermeneutic experience was used, in which Gadamer's hermeneutic text interpretation is divided into four phases. The phases are the explicit analysis of preunderstanding, hermeneutic dialogue, the merging of horizons and active application. The dialogue is carried on between Eriksson's theory of health, suffering and caring and clinical nursing practice and between the caring process model and nursing practice. The goal is to achieve a new scientific view on which to base the documentation of nursing care. As a result of the dialogue a classification in accordance with Eriksson's caring-process model is presented. In the next phase of the study the classification is piloted in a clinical context. The purpose is to