Spécificité et Complémentarité des méthodes de la biologie structurale

Biologie Structurale Intégrative

Présentation Générale de la 4ème école d'Oléron - Jean Cavarelli (IGBMC, Illkirch)

Enjeux et défis d'un formation en Biologie Structurale Intégrative

 Les Infrastructures de recherche

Conférence I - Study of the type VI secretion system : an example of an integrated structural biology approach - Alain Roussel (AFMB, Marseille)

Bacteria do not live alone. When thriving in their environment, bacteria have to cope with many other species and therefore should collaborate or compete to access nutrients or to colonize more efficiently the ecological niche. We are using as model the Gram-negative bacterium “enteroaggregative Escherichia coli”, an inhabitant of the digestive tract. The strain under study is a close relative to the strain responsible for the German outbreak in 2011 (the so-called “cucumber disease”). This bacterium is able to kill other bacteria from the Escherichia or related genus. This ability to target and kill prey cells lies on a macromolecular system produced in the predator cell, the Type VI secretion system (T6SS). The T6SS is a macromolecular system anchored into the cell envelope and acting as a micro-syringe to deliver toxins into target bacteria or eukaryotic cells. Biochemical and structural studies support a general model in which the 13 T6SS core-components form two sub-assemblies: a cytoplasmic tubular structure and a membrane complex.

The membrane complex is composed of three proteins: the TssM and TssL inner membrane components and the TssJ outer membrane lipoprotein. The TssM protein, a 1129-amino-acid protein, is central as it interacts with both TssL and TssJ, therefore linking the membranes.  We have raised camelid antibodies (nanobodies) against the purified TssM periplasmic domain and we got the crystal structure of a 32.4 kDa C-terminal fragment of TssM in complex with a nanobody. From this structure we were able to design a shorter fragment, which still binds to TssJ, and we have succeeded in getting crystals of this complex. In parallel, fluorescence microscopy has been used in the team of Eric Cascales in Marseille to follow the dynamics of the formation of the membrane complex and an envelop of the membrane complex was determined by negative stainning electron microscopy in the team of Remy Fronzes in Paris. At the end, the results obtained by our three teams were combined to reveal the biogenesis and overall architecture of the type VI secretion membrane core complex.

All the steps of this study will be presented with a special emphasis on the technologies available in the platform for structural biology of the AFMB laboratory in Marseille.

Related publications :

- Durand et al. (2015) Biogenesis and structure of a type VI secretion membrane core complex. Nature 523, 555-560

- Nguyen et al. (2015) Inhibition of Type VI Secretion by an Anti-TssM Llama Nanobody. PLoS One 10, e0122187

- Nguyen et al. (2015) Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody. Acta crystallographica. Section F, Structural biology communications 71, 266-271

- Desmyter et al. (2015) Camelid nanobodies: killing two birds with one stone. Current opinion in structural biology 32C, 1-8


Conférence II - Relations structures fonctions des complexes participant à l’intégration du génome du VIH-1 dans le génome de la cellule cible - Marc Ruff (IGBMC, Illkirch)

Des techniques de biologie structurale intégrées (Cristallographie, RMN et Cryoelectromicroscopie pour les études structurales ainsi que des techniques d’analyses biophysiques pour les études fonctionnelles) sont utilisés pour  étudier les relations structures fonctions des complexes participant à l’intégration du génome du VIH-1 dans le génome de la cellule cible.  L’intégration de plusieurs techniques structurales et biophysiques a permis des avancées significatives dans la compréhension des mécanismes d’intégration du génome du VIH-1 dans le génome de la cellule cible et seront décrites dans ce cours.

 Document de présentation

Conférence III - Functional and structural plasticity at telomeres - Marie-Hélène LeDu (I2BC, Saclay)

Telomeres are nucleoprotein complexes that protect the extremities of linear chromosomes from degradation or illicit repair. At each cell division, telomeres shorten due to incomplete end replication until they reach a critical length at which cells enter senescence.

One major aspect of telomere architecture comes from the DNA telomere repeats observed in all eukaryotic species, which induces subnuclear compartment with high local concentration of telomere proteins. This elevated local concentration implies that the description of high affinity interactions is not sufficient to properly understand the regulatory processes involved in telomere maintenance. It is known that weak-affinity (KD > 10-4 M) and transient interactions are equally important than high-affinity interactions (KD < 10-6 M) in the regulation of many cellular pathways. In telomeres biology, shortening is a crucial issue with different functions associated to short versus long telomere states, which also induces variation of local concentrations that may therefore be linked to critical functional switches. However, the available technical tools to study short-lived interactions limit our understanding of these regulatory processes. Weak and transient interactions are particularly difficult to study in multifunctional proteins, which interact with numerous partners often through a common or similar interface.

The protein Rap1 is the only telomeric protein found physically and functionally associated to telomere from yeast to human. In the yeast Saccharomyces cerevisiae it directly binds telomeric DNA and plays a central role, in Human it is recruited through its interaction with the protein TRF2 and its telomeric function remains controversial.

Rap1 structure includes three globular domains connected by flexible unstructured regions. Its interaction with DNA in yeast or with TRF2 in Human is associated with large conformational adjustment. Our extensive studies of Rap1 proteins reveal an essential conserved structural plasticity. This plasticity allows the formation of different entities with its various partners, which are sensitive to local concentrations and therefore to telomere state.


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