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Marc Jamin - One of the best experts on this subject based on the ideXlab platform.
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structural description of the nipah Virus Phosphoprotein and its interaction with stat1
Biophysical Journal, 2020Co-Authors: Malene Ringkjøbing Jensen, Jean-marie Bourhis, Guillaume Communie, Martin Blackledge, Nicolas Tarbouriech, Filip Yabukarski, Eric Condamine, Valentina A Volchkova, Viktor E Volchkov, Marc JaminAbstract:Abstract The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah Virus (NiV) Phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the Phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoViruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV Phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the Phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the Phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.
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Vesicular Stomatitis Virus Phosphoprotein Dimerization Domain Is Dispensable for Virus Growth.
Journal of Virology, 2019Co-Authors: Marc Jamin, Danielle Blondel, Martin Blackledge, Jean-marie BourhisAbstract:The Phosphoprotein (P) of the nonsegmented negative-sense RNA Viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (PMD), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis Virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, PMD, for viral gene expression and Virus growth. A truncated P protein lacking the central dimerization domain (PΔMD) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant Virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type Virus, showing that the dimerization domain of P is dispensable for viral gene expression and Virus replication in cell culture. Because RNA Viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new Viruses.IMPORTANCE The Phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA Viruses, including some major human pathogens (e.g., rabies Virus, measles Virus, respiratory syncytial Virus [RSV], Ebola Virus, and Nipah Virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different Viruses and, in some cases, hijacks cell components to the advantage of the Virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for Virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA Viruses.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating LyssaVirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Danielle Blondel, Marc Jamin, Francine C. Gérard, Jean-marie Bourhis, Elisar BarbarAbstract:The rabies and Ebola Viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies Virus Phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related Virus Phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in LyssaVirus Phosphoproteins and its presence in other analogous proteins such as the Ebola Virus VP35 evinces a broader purpose for LC8 in regulating downstream Phosphoprotein functions vital for viral replication.
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Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein
Journal of Virology, 2013Co-Authors: Nicolas Martinez, Cedric Leyrat, Nicolas Tarbouriech, Marc JaminAbstract:Lettuce necrotic yellows Virus (LNYV) is a prototype of the plant-adapted cytorhabdoViruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its Phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules.
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Ensemble Structure of the Modular and Flexible Full-Length Vesicular Stomatitis Virus Phosphoprotein
Journal of Molecular Biology, 2012Co-Authors: Cedric Leyrat, Malene Ringkjøbing Jensen, Martin Blackledge, Filip Yabukarski, Robert Schneider, Mingxi Yao, Marc JaminAbstract:Abstract The Phosphoprotein (P) is an essential component of the viral replication machinery of non-segmented negative‐strand RNA Viruses, connecting the viral polymerase to its nucleoprotein–RNA template and acting as a chaperone of the nucleoprotein by preventing nonspecific encapsidation of cellular RNAs. The Phosphoprotein of vesicular stomatitis Virus (VSV) forms homodimers and possesses a modular organization comprising two stable, well-structured domains concatenated with two intrinsically disordered regions. Here, we used a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering to depict VSV P as an ensemble of continuously exchanging conformers that captures the dynamic character of this protein. We discuss the implications of the dynamics and the large conformational space sampled by VSV P in the assembly and functioning of the viral transcription/replication machinery.
Danielle Blondel - One of the best experts on this subject based on the ideXlab platform.
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Vesicular Stomatitis Virus Phosphoprotein Dimerization Domain Is Dispensable for Virus Growth.
Journal of Virology, 2019Co-Authors: Marc Jamin, Danielle Blondel, Martin Blackledge, Jean-marie BourhisAbstract:The Phosphoprotein (P) of the nonsegmented negative-sense RNA Viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (PMD), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis Virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, PMD, for viral gene expression and Virus growth. A truncated P protein lacking the central dimerization domain (PΔMD) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant Virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type Virus, showing that the dimerization domain of P is dispensable for viral gene expression and Virus replication in cell culture. Because RNA Viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new Viruses.IMPORTANCE The Phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA Viruses, including some major human pathogens (e.g., rabies Virus, measles Virus, respiratory syncytial Virus [RSV], Ebola Virus, and Nipah Virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different Viruses and, in some cases, hijacks cell components to the advantage of the Virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for Virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA Viruses.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating LyssaVirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Danielle Blondel, Marc Jamin, Francine C. Gérard, Jean-marie Bourhis, Elisar BarbarAbstract:The rabies and Ebola Viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies Virus Phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related Virus Phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in LyssaVirus Phosphoproteins and its presence in other analogous proteins such as the Ebola Virus VP35 evinces a broader purpose for LC8 in regulating downstream Phosphoprotein functions vital for viral replication.
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Role of interferon antagonist activity of rabies Virus Phosphoprotein in viral pathogenicity.
Journal of Virology, 2010Co-Authors: Naoto Ito, Danielle Blondel, David A. Jans, Gregory W. Moseley, Tatsunori Masatani, Keisuke Nakagawa, Kenta Shimizu, Caitlin Lorraine Rowe, Yuki Ito, Makoto SugiyamaAbstract:The fixed rabies Virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the Phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.
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modular organization of rabies Virus Phosphoprotein
Journal of Molecular Biology, 2009Co-Authors: Cedric Leyrat, Danielle Blondel, Ivan Ivanov, Sonia Longhi, Marc JaminAbstract:A Phosphoprotein (P) is found in all Viruses of the Mononegavirales order. These proteins form homo-oligomers, fulfil similar roles in the replication cycles of the various Viruses, but differ in their length and oligomerization state. Sequence alignments reveal no sequence similarity among proteins from Viruses belonging to the same family. Sequence analysis and experimental data show that Phosphoproteins from Viruses of the Paramyxoviridae contain structured domains alternating with intrinsically disordered regions. Here, we used predictions of disorder of secondary structure, and an analysis of sequence conservation to predict the domain organization of the Phosphoprotein from Sendai Virus, vesicular stomatitis Virus (VSV) and rabies Virus (RV P). We devised a new procedure for combining the results from multiple prediction methods and locating the boundaries between disordered regions and structured domains. To validate the proposed modular organization predicted for RV P and to confirm that the putative structured domains correspond to autonomous folding units, we used two-hybrid and biochemical approaches to characterize the properties of several fragments of RV P. We found that both central and C-terminal domains can fold in isolation, that the central domain is the oligomerization domain, and that the C-terminal domain binds to nucleocapsids. Our results suggest a conserved organization of P proteins in the Rhabdoviridae family in concatenated functional domains resembling that of the P proteins in the Paramyxoviridae family.
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solution structure of the c terminal nucleoprotein rna binding domain of the vesicular stomatitis Virus Phosphoprotein
Journal of Molecular Biology, 2008Co-Authors: Euripedes A Ribeiro, Cedric Leyrat, Danielle Blondel, Francine C A Gerard, Rob W H Ruigrok, Martin Blackledge, Adrien Favier, Bernhard Brutscher, Marc JaminAbstract:Abstract Beyond common features in their genome organization and replication mechanisms, the evolutionary relationships among Viruses of the Rhabdoviridae family are difficult to decipher because of the great variability in the amino acid sequence of their proteins. The Phosphoprotein (P) of vesicular stomatitis Virus (VSV) is an essential component of the RNA transcription and replication machinery; in particular, it contains binding sites for the RNA-dependent RNA polymerase and for the nucleoprotein. Here, we devised a new method for defining boundaries of structured domains from multiple disorder prediction algorithms, and we identified an autonomous folding C-terminal domain in VSV P (P CTD ). We show that, like the C-terminal domain of rabies Virus (RV) P, VSV P CTD binds to the viral nucleocapsid (nucleoprotein–RNA complex). We solved the three-dimensional structure of VSV P CTD by NMR spectroscopy and found that the topology of its polypeptide chain resembles that of RV P CTD . The common part of both proteins could be superimposed with a backbone RMSD from mean atomic coordinates of 2.6 A. VSV P CTD has a shorter N-terminal helix (α 1 ) than RV P CTD ; it lacks two α-helices (helices α 3 and α 6 of RV P), and the loop between strands β 1 and β 2 is longer than that in RV. Dynamical properties measured by NMR relaxation revealed the presence of fast motions (below the nanosecond timescale) in loop regions (amino acids 209–214) and slower conformational exchange in the N- and C-terminal helices. Characterization of a longer construct indicated that P CTD is preceded by a flexible linker. The results presented here support a modular organization of VSV P, with independent folded domains separated by flexible linkers, which is conserved among different genera of Rhabdoviridae and is similar to that proposed for the P proteins of the Paramyxoviridae .
Martin Blackledge - One of the best experts on this subject based on the ideXlab platform.
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structural description of the nipah Virus Phosphoprotein and its interaction with stat1
Biophysical Journal, 2020Co-Authors: Malene Ringkjøbing Jensen, Jean-marie Bourhis, Guillaume Communie, Martin Blackledge, Nicolas Tarbouriech, Filip Yabukarski, Eric Condamine, Valentina A Volchkova, Viktor E Volchkov, Marc JaminAbstract:Abstract The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah Virus (NiV) Phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the Phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoViruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV Phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the Phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the Phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.
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Vesicular Stomatitis Virus Phosphoprotein Dimerization Domain Is Dispensable for Virus Growth.
Journal of Virology, 2019Co-Authors: Marc Jamin, Danielle Blondel, Martin Blackledge, Jean-marie BourhisAbstract:The Phosphoprotein (P) of the nonsegmented negative-sense RNA Viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (PMD), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis Virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, PMD, for viral gene expression and Virus growth. A truncated P protein lacking the central dimerization domain (PΔMD) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant Virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type Virus, showing that the dimerization domain of P is dispensable for viral gene expression and Virus replication in cell culture. Because RNA Viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new Viruses.IMPORTANCE The Phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA Viruses, including some major human pathogens (e.g., rabies Virus, measles Virus, respiratory syncytial Virus [RSV], Ebola Virus, and Nipah Virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different Viruses and, in some cases, hijacks cell components to the advantage of the Virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for Virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA Viruses.
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Structure of the tetramerization domain of measles Virus Phosphoprotein.
Journal of Virology, 2013Co-Authors: Guillaume Communie, Thibaut Crépin, Damien Maurin, Malene Ringkjøbing Jensen, Martin BlackledgeAbstract:The atomic structure of the stable tetramerization domain of the measles Virus Phosphoprotein shows a tight four-stranded coiled coil. Although at first sight similar to the tetramerization domain of the Sendai Virus Phosphoprotein, which has a hydrophilic interface, the measles Virus domain has kinked helices that have a strongly hydrophobic interface and it lacks the additional N-terminal three helical bundles linking the long helices.
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Ensemble Structure of the Modular and Flexible Full-Length Vesicular Stomatitis Virus Phosphoprotein
Journal of Molecular Biology, 2012Co-Authors: Cedric Leyrat, Malene Ringkjøbing Jensen, Martin Blackledge, Filip Yabukarski, Robert Schneider, Mingxi Yao, Marc JaminAbstract:Abstract The Phosphoprotein (P) is an essential component of the viral replication machinery of non-segmented negative‐strand RNA Viruses, connecting the viral polymerase to its nucleoprotein–RNA template and acting as a chaperone of the nucleoprotein by preventing nonspecific encapsidation of cellular RNAs. The Phosphoprotein of vesicular stomatitis Virus (VSV) forms homodimers and possesses a modular organization comprising two stable, well-structured domains concatenated with two intrinsically disordered regions. Here, we used a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering to depict VSV P as an ensemble of continuously exchanging conformers that captures the dynamic character of this protein. We discuss the implications of the dynamics and the large conformational space sampled by VSV P in the assembly and functioning of the viral transcription/replication machinery.
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the n0 binding region of the vesicular stomatitis Virus Phosphoprotein is globally disordered but contains transient α helices
Protein Science, 2011Co-Authors: Cedric Leyrat, Francine C A Gerard, Euripedes A Ribeiro, Rob W H Ruigrok, Malene Ringkjøbing Jensen, Martin Blackledge, Marc JaminAbstract:The Phosphoprotein (P) of vesicular stomatitis Virus (VSV) interacts with nascent nucleoprotein (N), forming the N0–P complex that is indispensable for the correct encapsidation of newly synthesized viral RNA genome. In this complex, the N-terminal region (PNTR) of P prevents N from binding to cellular RNA and keeps it available for encapsidating viral RNA genomes. Here, using nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS), we show that an isolated peptide corresponding to the 60 first N-terminal residues of VSV P (P60) and encompassing PNTR has overall molecular dimensions and a dynamic behavior characteristic of a disordered protein but transiently populates conformers containing α-helices. The modeling of P60 as a conformational ensemble by the ensemble optimization method using SAXS data correctly reproduces the α-helical content detected by NMR spectroscopy and suggests the coexistence of subensembles of different compactness. The populations and overall dimensions of these subensembles are affected by the addition of stabilizing (1M trimethylamine-N-oxide) or destabilizing (6M guanidinium chloride) cosolvents. Our results are interpreted in the context of a scenario whereby VSV PNTR constitutes a molecular recognition element undergoing a disorder-to-order transition upon binding to its partner when forming the N0–P complex.
Sonia Longhi - One of the best experts on this subject based on the ideXlab platform.
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Insights into the coiled-coil organization of the Hendra Virus Phosphoprotein from combined biochemical and SAXS studies
Virology, 2015Co-Authors: Matilde Beltrandi, Sonia Longhi, David Blocquel, Jenny Erales, Pascale Barbier, Andrea CavalliAbstract:Nipah and Hendra Viruses are recently emerged paramyxoViruses belonging to the HenipaVirus genus. The HenipaVirus Phosphoprotein (P) consists of a large intrinsically disordered domain and a C-terminal domain (PCT) containing alternating disordered and ordered regions. Among these latter is the P multimerization domain (PMD). Using biochemical, analytical ultracentrifugation and small-angle X-ray scattering (SAXS) studies, we show that Hendra Virus (HeV) PMD forms an elongated coiled-coil homotrimer in solution, in agreement with our previous findings on Nipah Virus (NiV) PMD. However, the orientation of the N-terminal region differs from that observed in solution for NiV PMD, consistent with the ability of this region to adopt different conformations. SAXS studies provided evidence for a trimeric organization also in the case of PCT, thus extending and strengthening our findings on PMD. The present results are discussed in light of conflicting reports in the literature pointing to a tetrameric organization of paramyxoviral P proteins.
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Coiled-Coil Deformations in Crystal Structures: The Measles Virus Phosphoprotein Multimerization Domain as an Illustrative Example.
Acta Crystallographica Section D Biological Crystallography, 2014Co-Authors: David Blocquel, Sonia Longhi, Johnny Habchi, Eric Durand, Marion Sevajol, François Ferron, Jenny Erales, Nicolas PapageorgiouAbstract:The structures of two constructs of the measles Virus (MeV) Phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communie et al. (2013), J. Virol. 87, 7166-7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.
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multiscaled exploration of coupled folding and binding of an intrinsically disordered molecular recognition element in measles Virus nucleoprotein
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Yong Wang, Sonia Longhi, Xiakun Chu, Philippe Roche, Wei Han, Erkang Wang, Jin WangAbstract:Numerous relatively short regions within intrinsically disordered proteins (IDPs) serve as molecular recognition elements (MoREs). They fold into ordered structures upon binding to their partner molecules. Currently, there is still a lack of in-depth understanding of how coupled binding and folding occurs in MoREs. Here, we quantified the unbound ensembles of the α-MoRE within the intrinsically disordered C-terminal domain of the measles Virus nucleoprotein. We developed a multiscaled approach by combining a physics-based and an atomic hybrid model to decipher the mechanism by which the α-MoRE interacts with the X domain of the measles Virus Phosphoprotein. Our multiscaled approach led to remarkable qualitative and quantitative agreements between the theoretical predictions and experimental results (e.g., chemical shifts). We found that the free α-MoRE rapidly interconverts between multiple discrete partially helical conformations and the unfolded state, in accordance with the experimental observations. We quantified the underlying global folding–binding landscape. This leads to a synergistic mechanism in which the recognition event proceeds via (minor) conformational selection, followed by (major) induced folding. We also provided evidence that the α-MoRE is a compact molten globule-like IDP and behaves as a downhill folder in the induced folding process. We further provided a theoretical explanation for the inherent connections between “downhill folding,” “molten globule,” and “intrinsic disorder” in IDP-related systems. Particularly, we proposed that binding and unbinding of IDPs proceed in a stepwise way through a “kinetic divide-and-conquer” strategy that confers them high specificity without high affinity.
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solution structure of the c terminal x domain of the measles Virus Phosphoprotein and interaction with the intrinsically disordered c terminal domain of the nucleoprotein
Journal of Molecular Recognition, 2010Co-Authors: Stéphane Gely, Jean-marie Bourhis, Malene Ringkjøbing Jensen, Martin Blackledge, David F. Lowry, Cédric Bernard, Stéphanie Costanzo, Hervé Darbon, Gary W. Daughdrill, Sonia LonghiAbstract:In this report, the solution structure of the nucleocapsid-binding domain of the measles Virus Phosphoprotein (XD, aa 459–507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of thenucleocapsid protein, (NTAIL,aa401–525), isalsopresented.XDisan all aprotein consisting ofathree-helix bundle with an up-down-up arrangement of the helices. Thesolution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489–491, which is involved in the embedding of the a-helical XD-binding region of NTAIL. Secondary chemical shift values for full-length NTAIL were used to define the precise boundaries of a transient helical segment that coincides withtheXD-bindingdomain,thussheddinglightonthepre-recognitionstateofNTAIL.Titrationexperiments with unlabeled XD showed that the transient a-helical conformation of NTAIL is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483–506 of NTAIL are in intermediate exchange with XD, while the 475–482 and 507–525 regions are in fast exchange. The NTAIL resonance behavior in the titration experiments is consistent with a complex binding model with more than two states. Copyright 2010 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this paper.
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solution structure of the c terminal x domain of the measles Virus Phosphoprotein and interaction with the intrinsically disordered c terminal domain of the nucleoprotein
Journal of Molecular Recognition, 2010Co-Authors: Stéphane Gely, Jean-marie Bourhis, Malene Ringkjøbing Jensen, Martin Blackledge, David F. Lowry, Cédric Bernard, Stéphanie Costanzo, Hervé Darbon, Gary W. Daughdrill, Sonia LonghiAbstract:In this report, the solution structure of the nucleocapsid-binding domain of the measles Virus Phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.
Jean-marie Bourhis - One of the best experts on this subject based on the ideXlab platform.
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structural description of the nipah Virus Phosphoprotein and its interaction with stat1
Biophysical Journal, 2020Co-Authors: Malene Ringkjøbing Jensen, Jean-marie Bourhis, Guillaume Communie, Martin Blackledge, Nicolas Tarbouriech, Filip Yabukarski, Eric Condamine, Valentina A Volchkova, Viktor E Volchkov, Marc JaminAbstract:Abstract The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah Virus (NiV) Phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the Phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoViruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV Phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the Phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the Phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.
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Vesicular Stomatitis Virus Phosphoprotein Dimerization Domain Is Dispensable for Virus Growth.
Journal of Virology, 2019Co-Authors: Marc Jamin, Danielle Blondel, Martin Blackledge, Jean-marie BourhisAbstract:The Phosphoprotein (P) of the nonsegmented negative-sense RNA Viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (PMD), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis Virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, PMD, for viral gene expression and Virus growth. A truncated P protein lacking the central dimerization domain (PΔMD) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant Virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type Virus, showing that the dimerization domain of P is dispensable for viral gene expression and Virus replication in cell culture. Because RNA Viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new Viruses.IMPORTANCE The Phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA Viruses, including some major human pathogens (e.g., rabies Virus, measles Virus, respiratory syncytial Virus [RSV], Ebola Virus, and Nipah Virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different Viruses and, in some cases, hijacks cell components to the advantage of the Virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for Virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA Viruses.
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The LC8-RavP ensemble structure evinces a role for LC8 in regulating LyssaVirus polymerase functionality
Journal of Molecular Biology, 2019Co-Authors: Nathan E. Jespersen, Cedric Leyrat, Danielle Blondel, Marc Jamin, Francine C. Gérard, Jean-marie Bourhis, Elisar BarbarAbstract:The rabies and Ebola Viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies Virus Phosphoprotein (RavP) result in completely non-lethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 co-localize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related Virus Phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in LyssaVirus Phosphoproteins and its presence in other analogous proteins such as the Ebola Virus VP35 evinces a broader purpose for LC8 in regulating downstream Phosphoprotein functions vital for viral replication.
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solution structure of the c terminal x domain of the measles Virus Phosphoprotein and interaction with the intrinsically disordered c terminal domain of the nucleoprotein
Journal of Molecular Recognition, 2010Co-Authors: Stéphane Gely, Jean-marie Bourhis, Malene Ringkjøbing Jensen, Martin Blackledge, David F. Lowry, Cédric Bernard, Stéphanie Costanzo, Hervé Darbon, Gary W. Daughdrill, Sonia LonghiAbstract:In this report, the solution structure of the nucleocapsid-binding domain of the measles Virus Phosphoprotein (XD, aa 459–507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of thenucleocapsid protein, (NTAIL,aa401–525), isalsopresented.XDisan all aprotein consisting ofathree-helix bundle with an up-down-up arrangement of the helices. Thesolution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489–491, which is involved in the embedding of the a-helical XD-binding region of NTAIL. Secondary chemical shift values for full-length NTAIL were used to define the precise boundaries of a transient helical segment that coincides withtheXD-bindingdomain,thussheddinglightonthepre-recognitionstateofNTAIL.Titrationexperiments with unlabeled XD showed that the transient a-helical conformation of NTAIL is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483–506 of NTAIL are in intermediate exchange with XD, while the 475–482 and 507–525 regions are in fast exchange. The NTAIL resonance behavior in the titration experiments is consistent with a complex binding model with more than two states. Copyright 2010 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this paper.
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solution structure of the c terminal x domain of the measles Virus Phosphoprotein and interaction with the intrinsically disordered c terminal domain of the nucleoprotein
Journal of Molecular Recognition, 2010Co-Authors: Stéphane Gely, Jean-marie Bourhis, Malene Ringkjøbing Jensen, Martin Blackledge, David F. Lowry, Cédric Bernard, Stéphanie Costanzo, Hervé Darbon, Gary W. Daughdrill, Sonia LonghiAbstract:In this report, the solution structure of the nucleocapsid-binding domain of the measles Virus Phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.
