摘要：SummaryRab GTPases are central regulators of intracellular vesicular trafficking. They are frequently targeted by bacterial pathogens through post-translational modifications.Salmonella typhimuriumsecretes the cysteine protease GtgE during infection, leading to a regioselective proteolytic cleavage of the regulatory switch I loop in the small GTPases of the Rab32 subfamily. Here, using a combination of biochemical methods, molecular dynamics simulations, NMR spectroscopy, and single-pair Förster resonance energy transfer, we demonstrate that the cleavage of Rab32 causes a local increase of conformational flexibility in both switch regions. Cleaved Rab32 maintains its ability to interact with the GDP dissociation inhibitor (GDI). Interestingly, the Rab32 cleavage enables GDI binding also with an active GTP-bound Rab32in vitro. Furthermore, the Rab32 proteolysis provokes disturbance in the interaction with its downstream effector VARP. Thus, the proteolysis of Rab32 is not a globally degradative mechanism but affects various biochemical and structural properties of the GTPase in a diverse manner.Graphical abstractDisplay OmittedHighlights•Cleavage by GtgE disrupts Rab32 interswitch region and blunts its binding to VARP•Cleaved Rab32 can bind GDI in both nucleotide states•Proteolytic PTM of Rab32 results in increased flexibility of the switch regions•Proteolysis forces Rab32 to its inactive structural state even upon GTP bindingMolecular Structure; Microbiology
