Biology subjects

Shatskiy, D.

Publications and source records attributed to Shatskiy, D..

3 recordsLinked to original sources

bioRxiv2026

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.

biochemistry↗

bioRxiv2025

Cryo-EM structure of a type VI secretion system delivered membrane-depolarizing toxin involved in bacterial antagonism

Many Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring competitor cells. Members of the VasX protein family, such as VasX from Vibrio cholerae and Tke5 from Pseudomonas putida, disrupt the inner membrane of target cells by forming ion-permeable channels that dissipate the proton motive force, thereby interfering with essential physiological processes. However, the molecular structure of any VasX family effector has remained unknown. Here, we present a cryo-EM structure of Ptx2, a recently identified VasX family effector exported by a T6SS of Pseudomonas aeruginosa. Our structure reveals that Ptx2 is an elongated, multi-domain protein that bears little resemblance to proteins of known function. Notably, the apparent flexibility of its domains suggests that Ptx2, like other membrane-depolarizing toxins, undergoes substantial conformational changes to facilitate membrane insertion. Guided by these predicted structural rearrangements, we used mutagenesis coupled with phenotypic assays to identify key features required for its toxic activity. Together, these findings provide the first molecular level insights into the structure and mechanism of VasX family effectors and expand our understanding of how these proteins contribute to interbacterial antagonism.

microbiology↗

bioRxiv2024

Structure of the F-tractin-F-actin complex

F-tractin is a short peptide widely used to visualize the actin cytoskeleton in live eukaryotic cells. Similar to other actin-binding probes, F-tractin alters actin organization and impairs cell migration when expressed at high levels. In addition, the probe has been reported to directly induce actin bundling. To elucidate the mechanism behind these effects, we determined the structure of the F-tractin-F-actin complex using electron cryo-microscopy. Our analysis revealed that the F-tractin peptide consists of a flexible N-terminal region and an amphipathic C-terminal helix. The N-terminal part is completely dispensable for F-actin binding but is responsible for the actin bundling effect. The C-terminal helical region interacts with a hydrophobic pocket formed by two neighboring actin subunits, a region identified as an interface for many other actin-binding polypeptides, including Lifeact, the most widely used actin-binding probe. Thus, rather than contrasting F-tractin and Lifeact, our data indicate that these peptides have analogous modes of interaction with F-actin. Our study dissects the structural elements of F-tractin and provides a mechanistic basis for the selection and future development of actin probes.

biochemistry↗