Actin and AnkJ reciprocally regulate the catalytic state of the Legionella effector Ceg14

Ceg14 is a Legionella pneumophila effector whose enzymatic activity is activated by host actin and inhibited by the cognate metaeffector AnkJ. Recent studies established that Ceg14 cleaves ATP to AMP and pyrophosphate and can transfer AMP to 3-phosphoglycerate, but how actin and AnkJ regulate these catalytic states remained unclear. Here, we show that Ceg14 is selectively activated by G-actin and determine the cryo-EM structure of the Ceg14-actin-AnkJ complex. Structure-guided mutagenesis identifies two actin-binding elements, a C-terminal anchor and an extended sensor region, that are required for Ceg14 activation and toxicity. Structural modelling and molecular dynamics simulations reveal that the catalytic center is assembled from three spatially distinct components: an N-terminal nucleoside-binding region, a phosphate-coordinating region within the actin sensor, and the catalytic triad. G-actin constrains the sensor and thereby organizes the phosphate-binding component of the active site. AnkJ binds at a distinct surface and allosterically repositions the N-terminal domain, disrupting nucleoside coordination while leaving part of the catalytic machinery intact. Consistent with this mechanism, AnkJ strongly suppresses AMP production and 3-phosphoglycerate-stimulated activity, yet the ternary complex retains weak Ceg14-dependent ATP-to-ADP activity. Thus, our results show that actin and AnkJ do not simply switch Ceg14 on and off, but reciprocally remodel its multipart catalytic center to determine its catalytic state and reaction output.