Signalisation par des ATPases STAND : études fonctionnelles et structurales – STAND

The STAND (Signal Transduction ATPases with Numerous Domains) class of signaling proteins represents a novel paradigm in signal transduction. These widespread proteins function as regulatory nexus that integrate positive and negative regulatory signals, and respond by downstream signaling. Their role as signaling hubs is reflected by their complex modular architecture. The STAND proteins characteristically comprise a conserved core of ~ 35 kDa, the NOD module, which carries the ATPase activity. Via a connector domain, NOD is fused to a sensor domain which binds the inducer and which is most often made of repeated amino-acid motifs. Downstream signaling is mediated by one or several effector domains that are localized at either end of the polypeptide. Many of these proteins play key roles in human physiology (the apoptose triggering factor Apaf-1, the innate immunity NLR proteins, the transcription factor CIITA, …), as highlighted by the existence of severe pathologies that are conferred by mutations in STAND protein encoding genes (e.g., the Bare Lymphocyte Syndrome, Crohn's disease, the NALP3-associated auto-inflammatory disorders...). Despite their importance, rare are the human STAND proteins that have subjected to in vitro studies, and little is known on the molecular details of the mechanism whereby STAND proteins function as signaling hubs. Yet, available genetical, biochemical and structural data strongly suggest that the mechanism underlying signal tranduction is conserved throughout the STAND class. The emerging picture is that of a regulated molecular switch, with the OFF position corresponding to a resting, ADP-bound, monomeric form stabilized by negative effectors, and the ON position corresponding to an ATP-bound, oligomeric form, competent for downstream signaling. Conversion of the resting form into the active form involves an ADP/ATP exchange triggered by the inducer. Signal extinction and resetting to the resting state relies on ATP hydrolysis that ensues multimerization. However, little is known so far regarding the structures of the resting and active forms of STAND proteins and the reasons why the nucleotide exchange step so critically depends on the presence of the inducer. In this project, we propose to develop genetical, biochemical and structural approaches to address this question and obtain insight into the molecular mechanisms underlying signal transduction by a STAND ATPase. We will use bacterial STAND proteins as model systems, with focus on MalT, an E. coli transcription factor and one of the best characterized STAND proteins. We will thoroughly characterize a panel of MalT variants that are altered in a specific step of the activation process. Comparison of their properties (conformations, ligand binding constants, intramolecular interdomain contacts,...) with those of the wild-type protein will allow us to delineate the steps and the conformational changes governing conversion of the resting form of MalT into the active form. In particular, this study will allow us to uncover why the ADP/ATP exchange step critically depends on the inducer. Additionally, we will develop a structural approach to determine the X-ray structures of the resting and the active forms of a full-length bacterial STAND protein. These approaches should provide a deep insight into the molecular events underlying signal transduction by STAND ATPases, which will represent a precious framework for studies of physiologically important STAND proteins. In this project, we also propose to develop an in vitro approach to study signaling by NOD1, a human cytosolic receptor that triggers innate immune responses when sensing the presence of specific peptidoglycan motifs. This approach implies the development of specific protein production and purification procedures, the in vitro reconstitution of NOD1 activation by peptidoglycan motifs as well as the characterization of the process. In vitro study of signaling by NOD1 will open new avenues for investigating the control of innate immnunity responses.