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During host cell invasion,Shigellaescapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with autophagy to targetShigellato destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to studyShigellaseptin cage entrapment in its near native state. Quantitative cryo-SXT showed thatShigellafragments mitochondria and enabled visualization of X-ray dense structures (∼30 nm resolution) surroundingShigellaentrapped in septin cages. Using Airyscan confocal microscopy, we observed Lysine 63 (K63)-linked ubiquitin chains decorating septin caged entrappedShigella. Remarkably, septins and K63 chains form separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy ofShigella. Together, these findings demonstrate how septin cagedShigellaare targeted to autophagy and provide fundamental insights into autophagy-cytoskeleton interactions.

The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.