RESUMO
Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double-stranded DNA virus belonging to the poxviridae that causes mild monkeypox-like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl-2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl-2 fold comprising 7 α-helices and utilizes the canonical Bcl-2 binding groove to engage proapoptotic host cell Bcl-2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain-swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain-swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus-mediated inhibition of host cell apoptosis and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. DATABASE: Structural data are available in the Protein Data Bank (PDB) under the accession numbers 6TPQ, 6TQQ and 6TRR.
Assuntos
Proteínas Reguladoras de Apoptose/química , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas não Estruturais Virais/química , Yatapoxvirus/fisiologia , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/fisiologiaRESUMO
Fullerene (C60) and multilayer graphene hybrid devices were fabricated using electrophoretic deposition, where the C60 clusters are electrically charged upon the application of an external bias in a polar solvent, acetonitrile, mixed with toluene, which facilitates their deposition on the graphene membranes. Raman spectroscopy unveiled the unique vibrational fingerprints associated with the A2g mode of the C60 molecules at â¼1453 cm-1, while blue shifts of â¼6 and â¼17 cm-1 were also attributed to the G- and 2D-bands of the hybrids relative to bare graphene, suggestive of p-doped graphene. The intensity ratio of the G- and the 2D-bands I2D/IG (hybrid) dropped to â¼0.18 from â¼0.3 (bare graphene), and this reduction in I2D/IG is also a signature of hole-doped graphene, consistent with the relatively strong electron accepting nature of C60. The electronic conductance of the two-terminal hybrid devices increased relative to bare graphene at room temperature which was attributed to the increased carrier density, and temperature-dependent electronic transport measurements were also conducted from ambient down to â¼5.8 K. Additionally, a low energy shift in the Fermi level, EF ≈ 140 meV, was calculated for the hybrids. When the hybrid devices were irradiated with a broadband white light source and a tunable laser source (with a wavelength λ ranging from â¼400-1100 nm), a strong photoresponse was evident, in contrast to the bare graphene devices which appeared unresponsive. The responsivity R of the hybrids was measured to be â¼109 A/W at λ ≈ 400 nm and â¼298 K, while the detectivity and external quantum efficiency were also exceptional, â¼1015 jones and â¼109%, respectively, at â¼1 V and a light power density of â¼3 mW/cm2. The R values are â¼10 times higher compared to other hybrid devices derived from graphene reported previously, such as quantum dot-graphene and few-layer MoS2-graphene heterostructures. The strong photoresponse of the C60-graphene hybrids reported here is attributed to the doping enhancement arising in graphene upon the adsorption of C60. This work demonstrates the exceptional potential of such hybrid nanocarbon-based structures for optoelectronics.
RESUMO
BHRF1 is a pro-survival Bcl-2 homologue encoded by Epstein-Barr virus (EBV) that plays a key role in preventing premature host cell death during viral infection and may contribute to the development of malignancies associated with chronic EBV infections. The anti-apoptotic action of BHRF1 is based on its ability to sequester pro-apoptotic Bcl-2 family proteins, in particular Bim and Bak. These interactions have been previously studied in three dimensions by determining crystal structures of BHRF1 in complex with both Bim and Bak BH3 domains. Screening of a library of peptidomimetic compounds based on the benzoylurea scaffold that mimics critical Bim BH3 domain side chains against BHRF1 led to the identification of an inhibitor of BHRF1 that displays micromolar affinity. Single crystals were obtained from the co-crystallization of recombinant BHRF1 protein with this peptidomimetic compound. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=66.8, b=91.1, c=151.9â Å. Diffraction data were collected to 2.11â Å resolution on the MX2 beamline at the Australian Synchrotron.