Spécificité et Complémentarité des méthodes de la biologie structurale

Study of the type VI secretion system : an example of an integrated structural biology approach - Alain Roussel (AFMB, Marseille) - Présentation au format pdf

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


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) - Présentation au format pdf

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.


Topoisomérases de type II: éudes structurales d’une famille de nanomachines flexibles - Claudine Mayer (Institut Pasteur, Paris) - Présentation au format pdf

 Les topoisomérases de type II sont des nanomachines flexibles d’environ 400 kDa constituées de quatre domaines et présentant une symétrie d’ordre 2. Elles sont responsables de la manipulation de la topologie de l’ADN lors des processus biologiques fondamentaux (transcription, réplication, recombinaison). L’objectif est de montrer comment la combinaison d’informations structurales obtenues par plusieurs techniques (RX, SAXS, cryo-EM mais aussi smFRET) sur une période de plus de 30 ans permet de proposer un modèle structural presque complet du mode de fonctionnement de cette famille de nanomachines.


RMN et intégration des données - Bruno Kieffer (ESBS-IGBMC, Illkirch) - Présentation au format pdf

 Les macromolécules biologiques sont des polymères complexes qui peuvent adopter plusieurs états differents en solution. La compréhension que nous pouvons avoir de certains mécanismes biologiques est conditionné par la possibilité d'élaborer des modèles permettant de rendre compte des multiples états des systèmes moléculaires en jeu, ainsi que de la dynamique de ces états. Après avoir précisé les notions de modèle et de mesure, l'exposé s'attachera à montrer comment notre vision du fonctionnement des systèmes biologiques a pu évoluer grâce à la combinaison des données de la biologie structurale et des approches biophysiques complémentaires.


How scarce sequence elements control the function of single β-thymosin/WH2 intrinsically disordered domains in actin assembly - Carine van Heijenoort (ICSN, Gif sur Yvette) - Présentation au format pdf

Intrinsically Disordered Domains/Regions (IDRs) appear more and more widespread in eukaryotic proteins, especially those implied in regulatory and signaling processes [1]. We will here focus on the case of actin-binding proteins (ABPs), which often exhibit complex multi-domain architectures integrating and coordinating multiple signals and interactions with the dynamic remodeling of actin cytoskeleton [2]. In most modular ABPs, IDRs relay labile interactions with multiple partners and act as interaction hubs in inter-domain and protein–protein interfaces. They thereby control multiple conformational transitions between inactive and active states and play an important role to coordinate the high turnover of interactions in actin self-assembly dynamics. Understanding the functional versatility of IDRs, here in actin assembly, requires deciphering their conformational plasticity and dynamics by multiple structural and functional approaches.

β-thymosin (βT) and WH2 domains are archetypal intrinsically disordered domains that fold upon binding actin. They display significant sequence variability associated to versatile regulations of actin assembly in motile processes [3]. Here we reveal the structural basis by which, in their basic 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers (Thymosin-β4), or enhance motility by directing polarized filament assembly (Ciboulot βT or WASP/WAVE WH2 domains). Combined mutational, functional, and structural analysis by X-ray crystallography, SAXS and NMR on Thymosin-β4, Ciboulot and the WH2 domain of WASP-interacting protein (WIP) allowed us to show that functionally different βT/WH2 domains do not target alternative actin binding sites but rather differ by alternative dynamics of their C-terminal half interactions with G-actin pointed face. We decipher how this interaction dynamics can be controlled by various subtle variations along the whole sequence of these small intrinsically disordered domains. In particular, it depends in specific cases on the presence of a unique ionic interaction in the central part of the sequence [4]. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins and enlighten how intrinsic structural disorder can lead to a novel mode of functional versatility.

[1] Johnny Habchi, Peter Tompa, Sonia Longhi, and Vladimir N. Uversky, Chemical Review 2014, 114, 6561

[2] Louis Renault, Célia Deville and Carine van Heijenoort, Cytoskeleton 2013, 70:686.

[3] Husson et al., Ann. N. Y. Acad. Sci. 2010, 1194:44

[4] Didry et al., EMBO Journal 2012, 31:1000


Towards the characterization of the plasticity of a Retinoic Acid nuclear Receptor heterodimer complexes involving an IDP - Nathalie Sibille (CBS, Montpellier) - (La présentation au format pdf sera disponible ultérieurment)

Intrinsically Disordered Proteins (IDPs) perform a large variety of functions that are crucial for cell regulation and maintenance. Functions performed by IDPs are complementary to these executed by globular ones indicating that the biophysical properties of disordered proteins dictate their functional mechanisms. Conformational plasticity, large solvent accessibility, and transient structuration are inherent characteristics of IDPs that are ideal to modulate signalling processes. As a consequence, the characterization of the structural features of IDPs in their free state and in complex with the relevant biological partners are crucial to reveal the molecular bases of signalling and cell control. The biological versatility of IDPs will be exemplified with a recent study from our laboratory on a protein involved in the regulation of gene transcription. The structural and dynamic characterization of these flexible biological systems requires the integration of experimental data containing complementary information. To achieve this aim Nuclear Magnetic Resonance (NMR), Small-Angle X-ray Scattering (SAXS) and X-ray crystallography have been integrated to derive complete structural models of these multifaceted biomolecular systems.