The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change

Myosin VI Must Dimerize and Deploy Its Unusual Lever Arm in Order to Perform Its Cellular Roles

It is unclear whether the reverse-direction myosin (myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions

Cryo-EM structure of the folded-back state of human β-cardiac myosin

Abstract To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an ‘off’ state that can be converted to an ‘on’ state when exertion is increased. The ‘off’ state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human β-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.6 Å resolution, providing details of all the interfaces stabilizing the ‘off’ state. The structure shows that these interfaces are hot spots of hypertrophic cardiomyopathy mutations that are thought to cause hypercontractility by destabilizing the ‘off’ state. Importantly, the cardiac and smooth muscle myosin IHM structures dramatically differ, providing structural evidence for the divergent physiological regulation of these muscle types. The cardiac IHM structure will facilitate development of clinically useful new molecules that modulate IHM stability

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

International audienceThe four serotypes of dengue virus (DENV-1 to-4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease

Nucleotide-free structures of KIF20A illuminate atypical mechanochemistry in this kinesin-6

KIF20A is a critical kinesin for cell division and a promising anti-cancer drug target. The mechanisms underlying its cellular roles remain elusive. Interestingly, unusual coupling between the nucleotide- and microtubule-binding sites of this kinesin-6 has been reported, but little is known about how its divergent sequence leads to atypical motility properties. We present here the first high-resolution structure of its motor domain that delineates the highly unusual structural features of this motor, including a long L6 insertion that integrates into the core of the motor domain and that drastically affects allostery and ATPase activity. Together with the high-resolution cryo-electron microscopy microtubule-bound KIF20A structure that reveals the microtubule-binding interface, we dissect the peculiarities of the KIF20A sequence that influence its mechanochemistry, leading to low motility compared to other kinesins. Structural and functional insights from the KIF20A pre-power stroke conformation highlight the role of extended insertions in shaping the motor's mechanochemical cycle. Essential for force production and processivity is the length of the neck linker in kinesins. We highlight here the role of the sequence preceding the neck linker in controlling its backward docking and show that a neck linker four times longer than that in kinesin-1 is required for the activity of this motor

Influenza-like illness in an urban community of Salvador, Brazil: incidence, seasonality and risk factors

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2016-09-01T13:36:03Z No. of bitstreams: 1 Oliveira CR Influenza-like illness....pdf: 1335497 bytes, checksum: 6d494f040966fcf902b40625771d4f90 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2016-09-01T14:09:47Z (GMT) No. of bitstreams: 1 Oliveira CR Influenza-like illness....pdf: 1335497 bytes, checksum: 6d494f040966fcf902b40625771d4f90 (MD5)Made available in DSpace on 2016-09-01T14:09:47Z (GMT). No. of bitstreams: 1 Oliveira CR Influenza-like illness....pdf: 1335497 bytes, checksum: 6d494f040966fcf902b40625771d4f90 (MD5) Previous issue date: 2016National Council for Scientific and Technological Development - CNPqYale School of Public Health. Department of Pediatrics. School of Medicine. New Haven, Connecticut, USAFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Instituto de Ciências da Saúde. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Instituto de Ciências da Saúde. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Instituto de Ciências da Saúde. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Yale School of Public Health. Department of Epidemiology of Microbial Diseases. School of Medicine. New Haven, Connecticut, USA / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilYale School of Public Health. Department of Epidemiology of Microbial Diseases. School of Medicine. New Haven, Connecticut, USAYale School of Public Health. Department of Pediatrics. School of Medicine. New Haven, Connecticut, USA / Yale School of Public Health. Department of Epidemiology of Microbial Diseases. School of Medicine. New Haven, Connecticut, USA / Yale School of Public Health. Department of Investigative of Medicine. New Haven, Connecticut, USAFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Yale School of Public Health. Department of Epidemiology of Microbial Diseases. School of Medicine. New Haven, Connecticut, USAFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Instituto de Ciências da Saúde. Salvador, BA, Brasil / Yale School of Public Health. Department of Epidemiology of Microbial Diseases. School of Medicine. New Haven, Connecticut, USAOur understanding of the epidemiology of influenza is limited in tropical regions, which in turn has hampered identifying optimal region-specific policy to diminish disease burden. Influenza-like illness (ILI) is a clinical diagnosis that can be used as a surrogate for influenza. This study aimed to define the incidence and seasonality of ILI and to assess its association with climatic variables and school calendar in an urban community in the tropical region of Salvador, Brazil