Atomic structure of a thermostable subdomain of HIV-1 gp41

Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell–surface receptors, and gp41 then promotes viral–cell membrane fusion. A soluble, α-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.

Fish and mammals in the economy of an ancient Peruvian kingdom

Fish and mammal bones from the coastal site of Cerro Azul, Peru shed light on economic specialization just before the Inca conquest of A.D. 1470. The site devoted itself to procuring anchovies and sardines in quantity for shipment to agricultural communities. These small fish were dried, stored, and eventually transported inland via caravans of pack llamas. Cerro Azul itself did not raise llamas but obtained charqui (or dried meat) as well as occasional whole adult animals from the caravans. Guinea pigs were locally raised. Some 20 species of larger fish were caught by using nets; the more prestigious varieties of these show up mainly in residential compounds occupied by elite families.

The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history

Classical familial adenomatous polyposis (FAP) is a high-penetrance autosomal dominant disease that predisposes to hundreds or thousands of colorectal adenomas and carcinoma and that results from truncating mutations in the APC gene. A variant of FAP is attenuated adenomatous polyposis coli, which results from germ-line mutations in the 5′ and 3′ regions of the APC gene. Attenuated adenomatous polyposis coli patients have “multiple” colorectal adenomas (typically fewer than 100) without the florid phenotype of classical FAP. Another group of patients with multiple adenomas has no mutations in the APC gene, and their phenotype probably results from variation at a locus, or loci, elsewhere in the genome. Recently, however, a missense variant of APC (I1307K) was described that confers an increased risk of colorectal tumors, including multiple adenomas, in Ashkenazim. We have studied a set of 164 patients with multiple colorectal adenomas and/or carcinoma and analyzed codons 1263–1377 (exon 15G) of the APC gene for germ-line variants. Three patients with the I1307K allele were detected, each of Ashkenazi descent. Four patients had a germ-line E1317Q missense variant of APC that was not present in controls; one of these individuals had an unusually large number of metaplastic polyps of the colorectum. There is increasing evidence that there exist germ-line variants of the APC gene that predispose to the development of multiple colorectal adenomas and carcinoma, but without the florid phenotype of classical FAP, and possibly with importance for colorectal cancer risk in the general population.

Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target

Synthetic C peptides, corresponding to the C helix of the HIV type 1 (HIV-1) gp41 envelope protein, are potent inhibitors of HIV-1 membrane fusion. One such peptide is in clinical trials. The crystal structure of the gp41 core, in its proposed fusion-active conformation, is a trimer of helical hairpins in which three C helices pack against a central coiled coil. Each C helix shows especially prominent contacts with one of three symmetry-related, hydrophobic cavities on the surface of the coiled coil. We show that the inhibitory activity of the C peptide C34 depends on its ability to bind to this coiled-coil cavity. Moreover, examining a series of C34 peptide variants with modified cavity-binding residues, we find a linear relationship between the logarithm of the inhibitory potency and the stability of the corresponding helical-hairpin complexes. Our results provide strong evidence that this coiled-coil cavity is a good drug target and clarify the mechanism of C peptide inhibition. They also suggest simple, quantitative assays for the identification and evaluation of analogous inhibitors of HIV-1 entry.

Inhibition of HIV type 1 infectivity by constrained α-helical peptides: Implications for the viral fusion mechanism

Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.

Reverse engineering the (β/α)8 barrel fold

The (β/α)8 barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (β/α)8 barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, α-helix capping and stop motifs, and residues that pack the interface between β-strands and α-helices are highly mutable. Conversely, any mutation of residues in the central core of the β-barrel, β-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that (β/α)8 barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (β/α)8 barrel design.

The trimer-of-hairpins motif in membrane fusion: Visna virus

Structural studies of viral membrane fusion proteins suggest that a “trimer-of-hairpins” motif plays a critical role in the membrane fusion process of many enveloped viruses. In this motif, a coiled coil (formed by homotrimeric association of the N-terminal regions of the protein) is surrounded by three C-terminal regions that pack against the coiled coil in an oblique antiparallel manner. The resulting trimer-of-hairpins structure serves to bring the viral and cellular membranes together for fusion. learncoil-vmf, a computational program developed to recognize coiled coil-like regions that form the trimer-of-hairpins motif, predicts these regions in the membrane fusion protein of the Visna virus. Peptides corresponding to the computationally identified sequences were synthesized, and the soluble core of the Visna membrane fusion protein was reconstituted in solution. Its crystal structure at 1.5-Å resolution demonstrates that a trimer-of-hairpins structure is formed. Remarkably, despite less than 23% sequence identity, the ectodomains in Visna and HIV-1 envelope glycoproteins show detailed structural conservation, especially within the area of a hydrophobic pocket in the central coiled coil currently being targeted for the development of new anti-HIV drugs.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

X-ray structure of a vanadium-containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis.

The chloroperoxidase (EC 1.11.1.-) from the fungus Curvularia inaequalis belongs to a class of vanadium enzymes that oxidize halides in the presence of hydrogen peroxide to the corresponding hypohalous acids. The 2.1 A crystal structure (R = 20%) of an azide chloroperoxidase complex reveals the geometry of the catalytic vanadium center. Azide coordinates directly to the metal center, resulting in a structure with azide, three nonprotein oxygens, and a histidine as ligands. In the native state vanadium will be bound as hydrogen vanadate(V) in a trigonal bipyramidal coordination with the metal coordinated to three oxygens in the equatorial plane, to the OH group at one apical position, and to the epsilon 2 nitrogen of a histidine at the other apical position. The protein fold is mainly alpha-helical with two four-helix bundles as main structural motifs and an overall structure different from other structures. The helices pack together to a compact molecule, which explains the high stability of the protein. An amino acid sequence comparison with vanadium-containing bromoperoxidase from the seaweed Ascophyllum nodosum shows high similarities in the regions of the metal binding site, with all hydrogen vanadate(V) interacting residues conserved except for lysine-353, which is an asparagine.

El gobierno económico de la Unión Europea y los principios de justicia del gasto público en una hacienda plural

A partir de 2011 se ha reforzado el gobierno económico de la UE a través de seis instrumentos legislativos, el llamado Six Pack, que supone fundamentalmente una reforma de la supervisión de la política presupuestaria de los Estados miembros. Más recientemente el Tratado de estabilidad, coordinación y gobernanza de UE de marzo de 2012 (TECGUE) establece un conjunto de normas destinadas a promover la disciplina presupuestaria a través de un pacto presupuestario; a reforzar la coordinación de sus políticas económicas; y a mejorar la gobernanza de la zona del euro. En el presente trabajo se analiza si este modelo basado en una estricta disciplina presupuestaria es compatible con los postulados del Estado social, y más concretamente con los principios de justicia del gasto público. En efecto, a partir de la reforma del art. 135 de la Constitución Española, el principio de estabilidad presupuestaria debe ser interpretado coordinadamente con otros principios constitucionales que en el momento presente están plenamente vigentes y pueden adquirir una nueva función: la de actuar como límite y medida del objetivo de estabilidad presupuestaria. Del mismo modo se analizan los principios de coordinación entre las políticas presupuestarias y de endeudamiento de los Estados miembros en un Estado con una pluralidad de Haciendas, como es el caso español.

O marca-passo das emoções no ritmo de crianças portuguesas com necessidades educativas especiais (estudo de caso)

Estudo de caso (metodologia qualitativa) realizado em 2013sobre inteligência emocional em alunos portugueses, frequentando 1.º ciclo educação básica, com idades de 6-7 anos de 2 escolas urbanas:2 crianças (M=microcefalia, A=autismo atípico); 2 crianças (N= criança normal, H= perturbação de hiperatividade com défice de atenção). Os objetivos pretenderam: Demonstrar a importância das emoções na aprendizagem; identificar e lidar com as emoções em situações; propor estratégias para cada criança, aquando dos resultados obtidos com aplicação do teste projetivo e utilização material didático. Metodologia: atividades com ―Uma caixa cheia de emoções‟; prova projetiva ―Era uma vez…‖ Teresa Fagulha; ficha de anamnese aos pais; análise documental aos processos; observação participante; notas de campo e triangulação. Os resultados permitiram estabelecer estratégias para diminuir os fatores de distratibilidade, facilitar a atenção com material visualmente atrativo e manuseável e fornecer instruções acompanhadas de observação. Houve dificuldades nos sujeitos em identificar a ‘ira’ e ‘raiva‘.

Gakara region, Burundi, 1994, Defense Mapping Agency (DMA) Series Z724, Sheet 4673-I (Raster Image)

This layer is a georeferenced raster image of the United States Defense Mapping Agency (DMA) Series Z724, Burundi, 1:50,000 Topographic Line Map (TLM) Series sheet map entitled: Gakara. Printed in: 1994. Covers portions of Gakara region, Burundi. Sheet: 4673-I. Edition statement: Ed. 1 - DMA. The image inside the map neatline is georeferenced to the surface of the earth and fit to World Geodetic System (1984) coordinates. All map collar information is also available as part of the raster image. Burundi 1:50:000 Series Z724 maps are in English and French (legends also include Rundi). Each source map in the series is printed in color at a scale of 1:50,000. Series source sheets were published in 1994-1995 by the United States Defense Mapping Agency, Hydrographic/Topographic Center. The source map was scanned and georeferenced for Harvard University's Center for Geographic Analysis' AfricaMap project by East View Cartographic. Individual TLM sheets covering Burundi (40 sheets in total) were selected from the TLM worldwide series. DMA Topographic Line Map series maps are typical topographic maps portraying both natural and manmade features. They show and name works of nature, such as mountains, valleys, lakes, rivers, vegetation, etc. They also identify the principal works of humans, such as roads, railroads, boundaries, transmission lines, major buildings, etc. Relief is shown with standard contour intervals of 20 meters, with some sheets having supplemental meter contours, form lines, hachures, shading, and/or spot heights. Depths shown by bathymetric isolines. Please pay close attention to map collar information on projections, spheroid, compilation dates, legend information, and keys to grid numbering and other numbers which appear inside the neatline.

Bubanza region, Burundi, 1994, Defense Mapping Agency (DMA) Series Z724, Sheet 4674-I (Raster Image)

This layer is a georeferenced raster image of the United States Defense Mapping Agency (DMA) Series Z724, Burundi, 1:50,000 Topographic Line Map (TLM) Series sheet map entitled: Bubanza. Printed in: 1994. Covers portions of Bubanza region, Burundi. Sheet: 4674-I. Edition statement: Ed. 1 - DMA. The image inside the map neatline is georeferenced to the surface of the earth and fit to World Geodetic System (1984) coordinates. All map collar information is also available as part of the raster image. Burundi 1:50:000 Series Z724 maps are in English and French (legends also include Rundi). Each source map in the series is printed in color at a scale of 1:50,000. Series source sheets were published in 1994-1995 by the United States Defense Mapping Agency, Hydrographic/Topographic Center. The source map was scanned and georeferenced for Harvard University's Center for Geographic Analysis' AfricaMap project by East View Cartographic. Individual TLM sheets covering Burundi (40 sheets in total) were selected from the TLM worldwide series. DMA Topographic Line Map series maps are typical topographic maps portraying both natural and manmade features. They show and name works of nature, such as mountains, valleys, lakes, rivers, vegetation, etc. They also identify the principal works of humans, such as roads, railroads, boundaries, transmission lines, major buildings, etc. Relief is shown with standard contour intervals of 20 meters, with some sheets having supplemental meter contours, form lines, hachures, shading, and/or spot heights. Depths shown by bathymetric isolines. Please pay close attention to map collar information on projections, spheroid, compilation dates, legend information, and keys to grid numbering and other numbers which appear inside the neatline.

Bujumbura region, Burundi, 1994, Defense Mapping Agency (DMA) Series Z724, Sheet 4674-II (Raster Image)

This layer is a georeferenced raster image of the United States Defense Mapping Agency (DMA) Series Z724, Burundi, 1:50,000 Topographic Line Map (TLM) Series sheet map entitled: Bujumbura. Printed in: 1994. Covers portions of Bujumbura region, Burundi. Sheet: 4674-II. Edition statement: Ed. 1 - DMA. The image inside the map neatline is georeferenced to the surface of the earth and fit to World Geodetic System (1984) coordinates. All map collar information is also available as part of the raster image. Burundi 1:50:000 Series Z724 maps are in English and French (legends also include Rundi). Each source map in the series is printed in color at a scale of 1:50,000. Series source sheets were published in 1994-1995 by the United States Defense Mapping Agency, Hydrographic/Topographic Center. The source map was scanned and georeferenced for Harvard University's Center for Geographic Analysis' AfricaMap project by East View Cartographic. Individual TLM sheets covering Burundi (40 sheets in total) were selected from the TLM worldwide series. DMA Topographic Line Map series maps are typical topographic maps portraying both natural and manmade features. They show and name works of nature, such as mountains, valleys, lakes, rivers, vegetation, etc. They also identify the principal works of humans, such as roads, railroads, boundaries, transmission lines, major buildings, etc. Relief is shown with standard contour intervals of 20 meters, with some sheets having supplemental meter contours, form lines, hachures, shading, and/or spot heights. Depths shown by bathymetric isolines. Please pay close attention to map collar information on projections, spheroid, compilation dates, legend information, and keys to grid numbering and other numbers which appear inside the neatline.