Browsing by Author "Silva, Leslie P."

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    Assembly of Ebola Virus Matrix Protein VP40 Is Regulated by Latch-Like Properties of N and C Terminal Tails
    (PLOS One, 2012-7-5) Silva, Leslie P.; Vanzile, Michael; Bavari, Sina; Aman, J. M. Javad; Schriemer, David C.
    The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers) but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.
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    Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens
    (Journal of Biological Chemistry, 2011-06-17) Silva, Leslie P.; Yu, Ronghua; Calmettes, Charles; Yang, Xue; Moraes, Trevor F.; Schryvers, Anthony B.; Schriemer, David C.
    Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.
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    Structural Variations within the Transferrin Binding Site on Transferrin-binding Protein B, TbpB
    (Journal of Biological Chemistry, 2011-04-08) Calmettes, Charles; Yu, Rong-hua; Silva, Leslie P.; Curran, Dave; Schriemer, David C.; Schryvers, Anthony B.; Moraes, Trevor F.
    Pathogenic bacteria acquire the essential element iron through specialized uptake pathways that are necessary in the iron-limiting environments of the host. Members of the Gram-negative Neisseriaceae and Pasteurellaceae families have adapted to acquire iron from the host iron binding glycoprotein, transferrin (Tf), through a receptor complex comprised of transferring-binding protein (Tbp) A and B. Because of the critical role they play in the host, these surface-exposed proteins are invariably present in clinical isolates and thus are considered prime vaccine targets. The specific interactions between TbpB and Tf are essential and ultimately might be exploited to create a broad-spectrum vaccine. In this study, we report the structure of TbpBs from two porcine pathogens, Actinobacillus pleuropneumoniae and suis. Paradoxically, despite a common Tf target, these swine related TbpBs show substantial sequence variation in their Tf-binding site. The TbpB structures, supported by docking simulations, surface plasmon resonance and hydrogen/deuterium exchange experiments with wild-type and mutant TbpBs, explain why there are structurally conserved elements within TbpB homologs despite major sequence variation that are required for binding Tf.