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Structure et fonction des toxines bactériennes à domaine FIC

Abstract : FIC (Filamentation induced by cAMP ) domain containing proteins are widespread in bacteria where they use different substrate such ATP to modify a target protein with a phosphate containing post-translational modification. Some of those proteins are secreted toxins from pathogens but the function of the majority stay unknown. Some recently resolved structures explain the catalytic mechanism. A subfamily of FIC proteins was proposed to be auto-inhibited for ATP binding by a glutamate in their active site. In my thesis, I lead a structural and biochemical study of two FIC proteins family: the auto-inhibited by a glutamate FIC proteins and the Legionella pneumophila toxin AnkX.For the first study, I focused on the pathogenic bacteria Enterococcus faecalis protein EfFIC that is an auto-inhibited FIC protein. I solved several crystallographic structures to characterize the active site and the AMP and ADP binding. Using the classic auto-AMPylation (modification with an AMP molecule) mechanism and radioactive ATP, I showed that EfFIC is active and I identified a new de-AMPylation activity. Using metals found in my crystallographic structures, I showed that the AMPylation and de-AMPylation switch is controlled by the nature of the metal bound in the active site and that this switch is inhibitory glutamate-dependent. This glutamate is found in human HYPE that shows a double AMPyaltion and de-AMPylation activity of the ER chaperone BIP. Using fluorescence assays, I showed that those two activities are alors regulated by metals as in EfFIC. Those results point on a new regulation model shared between FIC proteins from bacteria to human.The second study focused on Legionella pneumophila toxin AnkX that modifies small GTPases Rab1 and Rab35 with a phosphocholine (PC) molecule. Using controlled composition liposomes, I showed that AnkX interact with membranes and mapped the interaction domain by mutagenesis. With artificially anchored to nickel containing liposomes surface Rab GTPases, I demonstrated the stimulation of AnkX activity by the membranes. Preliminary results also suggest that Rab35 is a better substrate than Rab1a, giving information on AnkX function and localization during infection. I lead a small angle X-ray scattering (SAXS) study on AnkX that gave low-resolution structural information on AnkX in solution. The analyses of SAXS results show that AnkX is horseshoe shaped, suggesting an association with the membrane and Rab of AnkX model. In this model, membranes spatially regulate AnkX, allowing a targeting of Rab and cellular compartment targeting.
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Simon Veyron. Structure et fonction des toxines bactériennes à domaine FIC. Biochimie, Biologie Moléculaire. Université Paris-Saclay, 2017. Français. ⟨NNT : 2017SACLS452⟩. ⟨tel-01865817⟩

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