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Biochemistry, Biophysics and Structural Biology department

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Agenda

  • Wednesday 29 November 11:30-12:30 - Dr Sonia LONGHI - Lab. AFMB, CNRS & Aix-Marseille University, Marseille, France

    The interplay between order and disorder in the replicative complex of paramyxoviruses

    Résumé : In the course of the structural characterization of the nucleoproteins (N) and phosphoproteins (P) from three paramyxoviruses (e.g. measles, Nipah and Hendra virus) we discovered that they contain long disordered regions. The N and P proteins from these viruses thus provide an excellent model system to study the functional impact of disordered motifs. The non-segmented, single-stranded RNA genome of these paramyxoviruses is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Transcription and replication are carried out onto this ribonucleoproteic complex by the viral RNA dependent RNA polymerase that consists of a complex between the large protein (L) and the phosphoprotein (P). The P protein serves as an essential polymerase co-factor as it allows recruitment of L onto the nucleocapsid template. Tethering of L relies on the interaction between the C-terminal X domain (PXD) of the P protein and the C- terminal, intrinsically disordered domain (NTAIL) of N. This latter is disordered not only in isolation but also in the context of the nucleocapsid, being partly exposed at the surface of this latter. Within NTAIL, a short motif, serving as molecular recognition element (MoRE), has been identified and the mechanisms of its interaction with PXD thoroughly investigated. In its free from, the MoRE is partly pre-configured as an α-helix. Binding to PXD triggers stable α-helical folding of this motif, while the majority of NTAIL remains “fuzzy”. Beyond PXD, measles virus NTAIL also binds the major inducible heat shock protein 70 (hsp70). Although NTAIL binds hsp70 through the same motif involved in binding to PXD, the binding mechanisms are not the same, which constitutes an illustrative example of partner-mediated polymorphism of an intrinsically disordered protein and of the relative insensitiveness of the bound structure to the pre-recognition state.
    In my talk, I will focus on measles virus NTAIL and will summarize the main results we obtained so far. In particular, I will focus on the mechanistic and functional aspects of the interactions established by NTAIL and will highlight the functional implications of disorder for viral transcription and replication.
    Choice of five selected references
    1. Longhi et al (2017) Cell. Mol. Life Sci. 74, 3091–3118
    2. Bloyet LM et al (2016) Plos Pathogens 12(12):e1006058.
    3. Longhi S. (2015) FEBS Lett. 589, 2649-59.
    4. Dosnon et al (2015) ACS Chem. Biol. 10, 795−802
    5. Habchi J et al (2014) Chem. Rev. 114, 6561-88.

    Lieu : Bibliothèque - Bâtiment 34, Campus de Gif


  • Monday 16 October 14:00-17:00 - The Quyen Nguyen - team : Structural Biochemistry of Microtubules, Kinesins and their Cargos

    Structural characterization of JIP1 recruitment by kinesin1 light chain (KLC)

    Résumé : Kinesins are molecular motors involved in the intracellular transport of many cargos within the cell. Although the motility of kinesins is well understood, the molecular mechanisms underlying cargo recruitment are much less so. Kinesin1 plays various roles in neuronal cells, where it contributes to the spatial and temporal organization of many cellular components. It would play a role in various neurological pathologies, such as Alzheimer’s disease. Understanding how kinesin1 recognizes and interacts with its cargos is important to decorticate its role, as well as that of its cargos, in normal and pathological cells. Kinesin1 is a heterotetramer consisting of two heavy chains (KHC) and two light chains (KLC), both of which are capable of recruiting cargo proteins. One of the first cargo proteins to have been identified is JNK-interacting protein 1 (JIP1) which is: (i) a scaffold protein for the signaling pathway of MAP kinases and (ii) an adaptor protein for transporting amyloid precursor protein (APP) responsible for Alzheimer’s disease. In both cases, JIP1 regulates critical processes at the cell level, making it an interesting protein to study. Early studies have led to a better understanding of how JIP1 is recruited and transported by kinesin1. However, the detail of the interaction between KLC and JIP1 is not yet fully described and therefore understood.
    Objectives: My doctoral work aims at characterizing at the molecular level the interaction between KLC and JIP1. To do this, I had the following objectives: 1) to characterize the interaction domains of the two proteins alone, 2) to study the formation of the complex in solution by biophysical approaches, and 3) to determine the 3D structure of the complex by crystallography.
    Results: Initially, I characterized the TPR domain of KLC alone, contributing among others to the development of a molecular toolbox. I also participated in the determination of two crystallographic structures of the TPR domain of KLC1/2 that highlights the structural plasticity of the first helix of this domain (Nguyen et al, submitted). In a second step, I set up the conditions for the expression and purification of the PTB domain of JIP1 and carry out the structural characterization of this domain in solution. Although this domain of JIP1 is not necessary for interaction with KLC, I studied the impact of its presence on recruitment by KLC. Finally, I characterized the recruitment of JIP1 by KLC by confirming a number of information on the interaction between the KLC-TPR and the C-terminal region (Cter) of JIP1 at the molecular level. The numerous crystallization tests that I carried out did not make it possible to obtain crystals of the KLC: JIP1 complex. However, I was able to precisely map the interaction zone of JIP1-Cter with the KLC-TPR domain using the various KLC tools available by determining by ITC their affinity with JIP1-Cter (Nguyen et al., In preparation).
    Conclusion: Thus, my PhD work allowed to better understand 1) the structural versatility of the KLC-TPR domain, 2) the impact of the JIP1-PTB domain for its KLC recruitment, and 3) the interaction mode of JIP1 by KLC.
    Keywords : Kinesin1, KLC-TPR, JIP1
    Co-responsables : Paola Llinas et Julie Ménétrey

    Lieu : Bibliothèque - Bâtiment 34, Campus de Gif


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