Structural mechanism for guanylate-binding proteins (GBPs) targeting by the Shigella E3 ligase IpaH9.8.

The guanylate-binding proteins (GBPs) belong to the dynamin superfamily of GTPases and function in cell-autonomous defense against intracellular pathogens. IpaH9.8, an E3 ligase from the pathogenic bacterium Shigella flexneri, ubiquitinates a subset of GBPs and leads to their proteasomal degradation. Here we report the structure of a C-terminally truncated GBP1 in complex with the IpaH9.8 Leucine-rich repeat (LRR) domain. IpaH9.8(LRR) engages the GTPase domain of GBP1, and differences in the Switch II and alpha 3 helix regions render some GBPs such as GBP3 and GBP7 resistant to IpaH9.8. Comparisons with other IpaH structures uncover interaction hot spots in their LRR domains. The C-terminal region of GBP1 undergoes a large rotation compared to previously determined structures. We further show that the C-terminal farnesylation modification also plays a role in regulating GBP1 conformation. Our results suggest a general mechanism by which the IpaH proteins target their cellular substrates and shed light on the structural dynamics of the GBPs. Author summary Shigella flexneri is a Gram-negative bacteria that causes diarrhea in humans and leads to a million deaths every year. Once inside the cell, S. flexneri injects the host cell cytoplasm with effector proteins to suppress host defense. The guanylate-binding proteins (GBPs) have potent antimicrobial functions against a number of pathogens including S. flexneri. For successful infection, S. flexneri relies on an effector protein known as IpaH9.8, a unique ubiquitin E3 ligase to target a subset of GBPs for proteasomal degradation. How these GBPs are specifically recognized by IpaH9.8 was unclear. Here, using a combination of structural and biochemical approaches, we reveal the molecular basis of GBP-IpaH9.8 interaction, and show that subtle differences in the seven human GBPs can significantly impact the targeting specificity of IpaH9.8. We also show that the GBPs have considerable structural flexibility, which is likely important for their function. Our results provide further insights into S. flexneri pathogenesis, and laid the groundwork for future biophysical and biochemical studies to investigate the functional mechanism of GBPs.